PMC 20201223 pmc.key 4919469 CC BY no 0 0 Investigation of the TOCA1-Cdc42 Interaction 10.1074/jbc.M116.724294 4919469 27129201 M116.724294 13875 26 actin CDC42 endocytosis nuclear magnetic resonance (NMR) protein-protein interaction BAR domain CIP4 FBP17 TOCA1 WASP Author's Choice—Final version free via Creative Commons CC-BY license. 13890 surname:Watson;given-names:Joanna R. surname:Fox;given-names:Helen M. surname:Nietlispach;given-names:Daniel surname:Gallop;given-names:Jennifer L. surname:Owen;given-names:Darerca surname:Mott;given-names:Helen R. TITLE HANDOVER OF Cdc42 TO THE ACTIN REGULATOR N-WASP IS FACILITATED BY DIFFERENTIAL BINDING AFFINITIES* front 291 2016 0 Investigation of the Interaction between Cdc42 and Its Effector TOCA1 0.8210846 protein cleaner0 2023-06-30T08:42:49Z PR: Cdc42 0.9986349 protein cleaner0 2023-06-30T08:43:34Z PR: TOCA1 ABSTRACT abstract 70 Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery. protein PR: cleaner0 2023-06-30T08:45:55Z Transducer of Cdc42-dependent actin assembly protein 1 0.9983543 protein cleaner0 2023-06-30T08:43:34Z PR: TOCA1 0.85511017 protein_type cleaner0 2023-07-03T10:12:30Z MESH: Rho family small G protein 0.74458474 protein cleaner0 2023-06-30T08:42:44Z PR: Cdc42 structure_element SO: cleaner0 2023-07-03T09:40:41Z F-BAR 0.9879572 structure_element cleaner0 2023-07-03T10:19:48Z SO: Src homology 3 0.99650854 structure_element cleaner0 2023-07-03T10:19:51Z SO: SH3 0.99472725 structure_element cleaner0 2023-07-03T10:19:55Z SO: G protein-binding homology region 1 0.9979664 structure_element cleaner0 2023-06-30T08:45:24Z SO: HR1 0.99841833 protein cleaner0 2023-06-30T08:43:34Z PR: TOCA1 0.955355 protein cleaner0 2023-06-30T08:42:49Z PR: Cdc42 protein_type MESH: melaniev@ebi.ac.uk 2023-07-21T09:53:05Z actin 0.886836 experimental_method cleaner0 2023-07-03T11:42:50Z MESH: solved 0.9950669 evidence cleaner0 2023-07-03T11:56:18Z DUMMY: structure 0.99860483 structure_element cleaner0 2023-06-30T08:45:22Z SO: HR1 0.9988238 protein cleaner0 2023-06-30T08:43:34Z PR: TOCA1 0.960793 evidence cleaner0 2023-07-03T11:56:21Z DUMMY: structural data 0.99884087 protein cleaner0 2023-06-30T08:43:34Z PR: TOCA1 0.9984865 structure_element cleaner0 2023-06-30T08:45:24Z SO: HR1 0.99869126 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 0.9985139 structure_element cleaner0 2023-06-30T08:45:24Z SO: HR1 structure_element SO: cleaner0 2023-06-30T08:45:24Z HR1 0.99875283 protein cleaner0 2023-07-03T10:01:15Z PR: TOCA 0.9985581 structure_element cleaner0 2023-06-30T08:45:24Z SO: HR1 0.99559635 protein cleaner0 2023-06-30T08:42:49Z PR: Cdc42 0.9930555 protein cleaner0 2023-06-30T08:42:49Z PR: Cdc42 0.8609198 site cleaner0 2023-07-03T08:16:02Z SO: G protein-binding regions 0.9987367 protein cleaner0 2023-06-30T08:43:34Z PR: TOCA1 protein_type MESH: cleaner0 2023-06-30T08:44:27Z Wiskott-Aldrich syndrome protein family 0.9749348 protein cleaner0 2023-06-30T08:44:49Z PR: N-WASP 0.998451 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 0.9961826 protein cleaner0 2023-06-30T08:42:49Z PR: Cdc42 0.9564533 protein cleaner0 2023-06-30T08:44:50Z PR: N-WASP 0.82657814 site cleaner0 2023-07-03T08:16:16Z SO: G protein-binding region 0.9965149 protein cleaner0 2023-06-30T08:42:49Z PR: Cdc42 0.98908406 experimental_method cleaner0 2023-07-03T11:42:54Z MESH: NMR 0.7975464 site cleaner0 2023-07-03T08:16:23Z SO: Cdc42-binding domain 0.969029 protein cleaner0 2023-06-30T08:44:50Z PR: N-WASP 0.9986004 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 0.9983797 structure_element cleaner0 2023-06-30T08:45:24Z SO: HR1 0.9954324 protein cleaner0 2023-06-30T08:42:49Z PR: Cdc42 protein PR: cleaner0 2023-06-30T08:44:50Z N-WASP 0.9983676 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 0.9985555 structure_element cleaner0 2023-06-30T08:45:24Z SO: HR1 0.9982083 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 0.99187285 protein cleaner0 2023-06-30T08:42:49Z PR: Cdc42 0.8817688 protein cleaner0 2023-06-30T08:42:49Z PR: Cdc42 0.7142731 evidence cleaner0 2023-07-03T11:56:24Z DUMMY: binding affinities 0.99870765 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 0.9540443 protein cleaner0 2023-06-30T08:44:50Z PR: N-WASP INTRO title_1 1594 Introduction INTRO paragraph 1607 The Ras superfamily of small GTPases comprises over 150 members that regulate a multitude of cellular processes in eukaryotes. The superfamily can be divided into five families based on structural and functional similarities: Ras, Rho, Rab, Arf, and Ran. All members share a well defined core structure of ∼20 kDa known as the G domain, which is responsible for guanine nucleotide binding. It is this guanine nucleotide binding that underlies their function as molecular switches, controlling a vast array of signaling pathways. These molecular switches cycle between active, GTP-bound, and inactive, GDP-bound, states with the help of auxiliary proteins. The guanine nucleotide exchange factors mediate formation of the active state by promoting the dissociation of GDP, allowing GTP to bind. The GTPase-activating proteins stimulate the rate of intrinsic GTP hydrolysis, mediating the return to the inactive state (reviewed in Ref.). 0.9878229 protein_type cleaner0 2023-07-03T10:12:35Z MESH: Ras superfamily 0.9795511 protein_type cleaner0 2023-07-03T10:12:38Z MESH: small GTPases 0.9944252 taxonomy_domain cleaner0 2023-07-03T10:42:09Z DUMMY: eukaryotes 0.99181974 protein_type cleaner0 2023-07-03T10:12:48Z MESH: Ras 0.98704046 protein_type cleaner0 2023-07-03T10:12:51Z MESH: Rho 0.990781 protein_type cleaner0 2023-07-03T10:12:53Z MESH: Rab 0.9855112 protein_type cleaner0 2023-07-03T10:12:55Z MESH: Arf 0.93829954 protein_type cleaner0 2023-07-03T10:12:57Z MESH: Ran 0.9947895 structure_element cleaner0 2023-07-03T10:20:05Z SO: G domain chemical CHEBI: cleaner0 2023-07-03T10:20:17Z guanine nucleotide 0.997357 protein_state cleaner0 2023-07-03T10:54:29Z DUMMY: active 0.99498653 protein_state cleaner0 2023-07-03T08:18:41Z DUMMY: GTP-bound 0.9957853 protein_state cleaner0 2023-07-03T13:25:57Z DUMMY: inactive 0.9956725 protein_state cleaner0 2023-07-03T08:18:46Z DUMMY: GDP-bound 0.9952165 protein_type cleaner0 2023-07-03T10:13:14Z MESH: guanine nucleotide exchange factors 0.9958912 protein_state cleaner0 2023-07-03T13:26:02Z DUMMY: active 0.9906346 chemical cleaner0 2023-07-03T08:18:13Z CHEBI: GDP 0.9917891 chemical cleaner0 2023-07-03T08:18:27Z CHEBI: GTP 0.9971633 protein_type cleaner0 2023-07-03T10:13:18Z MESH: GTPase-activating proteins chemical CHEBI: cleaner0 2023-07-03T08:18:28Z GTP 0.99240416 protein_state cleaner0 2023-07-03T13:26:05Z DUMMY: inactive INTRO paragraph 2545 The overall conformation of small G proteins in the active and inactive states is similar, but they differ significantly in two main regions known as switch I and switch II. These regions are responsible for “sensing” the nucleotide state, with the GTP-bound state showing greater rigidity and the GDP-bound state adopting a more relaxed conformation (reviewed in Ref.). In the active state, G proteins bind to an array of downstream effectors, through which they exert their extensive roles within the cell. The structures of more than 60 small G protein·effector complexes have been solved, and, not surprisingly, the switch regions have been implicated in a large proportion of the G protein-effector interactions (reviewed in Ref.). However, because each of the 150 members of the superfamily interacts with multiple effectors, there are still a huge number of known G protein-effector interactions that have not yet been studied structurally. 0.9710317 protein_type cleaner0 2023-07-03T10:13:22Z MESH: small G proteins 0.9971058 protein_state cleaner0 2023-07-03T13:27:31Z DUMMY: active 0.9968178 protein_state cleaner0 2023-07-03T13:27:34Z DUMMY: inactive 0.9972334 site cleaner0 2023-07-03T09:34:36Z SO: switch I 0.997305 site cleaner0 2023-07-03T09:26:23Z SO: switch II 0.9949434 protein_state cleaner0 2023-07-03T08:18:42Z DUMMY: GTP-bound 0.9952473 protein_state cleaner0 2023-07-03T08:18:47Z DUMMY: GDP-bound 0.99743396 protein_state cleaner0 2023-07-03T13:27:37Z DUMMY: active 0.986418 protein_type cleaner0 2023-07-03T08:23:00Z MESH: G proteins 0.9969451 evidence cleaner0 2023-07-03T11:56:29Z DUMMY: structures 0.685185 protein_type cleaner0 2023-07-03T08:23:06Z MESH: G protein 0.6945974 experimental_method cleaner0 2023-07-03T11:43:03Z MESH: solved 0.9977436 site cleaner0 2023-07-03T09:34:22Z SO: switch regions protein_type MESH: cleaner0 2023-07-03T08:23:07Z G protein protein_type MESH: cleaner0 2023-07-03T08:23:07Z G protein INTRO paragraph 3498 The Rho family comprises 20 members, of which three, RhoA, Rac1, and Cdc42, have been relatively well studied. The role of these three proteins in the coordination of the actin cytoskeleton has been examined extensively. RhoA acts to rearrange existing actin structures to form stress fibers, whereas Rac1 and Cdc42 promote de novo actin polymerization to form lamellipodia and filopodia, respectively. A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics. 0.830895 protein_type cleaner0 2023-07-03T10:13:32Z MESH: Rho family 0.7184562 protein cleaner0 2023-06-30T08:46:55Z PR: RhoA 0.7119027 protein cleaner0 2023-06-30T08:47:05Z PR: Rac1 0.78280514 protein cleaner0 2023-06-30T08:42:49Z PR: Cdc42 0.78088295 protein cleaner0 2023-06-30T08:46:55Z PR: RhoA 0.69492334 protein cleaner0 2023-06-30T08:47:06Z PR: Rac1 0.68728393 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 0.6052976 protein cleaner0 2023-06-30T08:46:55Z PR: RhoA 0.5806924 protein cleaner0 2023-06-30T08:47:06Z PR: Rac1 0.8666725 protein_type cleaner0 2023-07-03T10:13:59Z MESH: protein kinase C-related kinase 0.9031287 protein_type cleaner0 2023-07-03T10:14:03Z MESH: PRK 0.9225908 protein_type cleaner0 2023-07-03T10:14:05Z MESH: 6 0.8432732 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 protein_type MESH: cleaner0 2023-06-30T08:47:37Z Wiskott-Aldrich syndrome 0.6893535 protein_type cleaner0 2023-07-03T10:14:10Z MESH: WASP 0.600192 protein_type cleaner0 2023-07-03T08:19:56Z MESH: Cdc42-dependent actin assembly 0.56222975 protein_type cleaner0 2023-07-03T10:14:16Z MESH: TOCA INTRO paragraph 4256 Cdc42 effectors, TOCA1 and the ubiquitously expressed member of the WASP family, N-WASP, have been implicated in the regulation of actin polymerization downstream of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). N-WASP exists in an autoinhibited conformation, which is released upon PI(4,5)P2 and Cdc42 binding or by other factors, such as phosphorylation. Following their release, the C-terminal regions of N-WASP are free to interact with G-actin and a known nucleator of actin assembly, the Arp2/3 complex. The importance of TOCA1 in actin polymerization has been demonstrated in a range of in vitro and in vivo studies, but the exact role of TOCA1 in the many pathways involving actin assembly remains unclear. The most widely studied role of TOCA1 is in membrane invagination and endocytosis, although it has also been implicated in filopodia formation, neurite elongation, transcriptional reprogramming via nuclear actin, and interaction with ZO-1 at tight junctions. A role in cell motility and invasion has also been established. 0.88614994 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 0.9972018 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 0.87142515 protein_type cleaner0 2023-07-03T10:14:22Z MESH: WASP family 0.9864705 protein cleaner0 2023-06-30T08:44:50Z PR: N-WASP 0.73301053 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 0.9973161 chemical cleaner0 2023-07-03T10:42:15Z CHEBI: phosphatidylinositol 4,5-bisphosphate 0.9972431 chemical cleaner0 2023-07-03T09:41:52Z CHEBI: PI(4,5)P2 0.9875979 protein cleaner0 2023-06-30T08:44:50Z PR: N-WASP 0.8615777 protein_state cleaner0 2023-07-03T13:27:44Z DUMMY: autoinhibited conformation 0.9969345 chemical cleaner0 2023-07-03T09:41:52Z CHEBI: PI(4,5)P2 0.8908446 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 0.99487746 structure_element cleaner0 2023-07-03T10:20:45Z SO: C-terminal regions 0.98714715 protein cleaner0 2023-06-30T08:44:50Z PR: N-WASP 0.99644774 protein_type cleaner0 2023-07-03T13:19:34Z MESH: G-actin complex_assembly GO: cleaner0 2023-07-03T08:20:42Z Arp2/3 0.998018 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 0.99811685 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 0.9979778 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 protein_type MESH: melaniev@ebi.ac.uk 2023-07-21T09:48:20Z actin 0.9499348 protein cleaner0 2023-07-03T10:01:30Z PR: ZO-1 INTRO paragraph 5309 TOCA1 comprises an N-terminal F-BAR domain, a central homology region 1 (HR1) domain, and a C-terminal SH3 domain. The F-BAR domain is a known dimerization, membrane-binding, and membrane-deforming module found in a number of cell signaling proteins. The TOCA1 SH3 domain has many known binding partners, including N-WASP and dynamin. The HR1 domain has been directly implicated in the interaction between TOCA1 and Cdc42, representing the first Cdc42-HR1 domain interaction to be identified. 0.99863905 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 structure_element SO: cleaner0 2023-07-03T09:40:41Z F-BAR 0.9966971 structure_element cleaner0 2023-07-03T10:21:05Z SO: central homology region 1 0.9981489 structure_element cleaner0 2023-06-30T08:45:24Z SO: HR1 0.9985121 structure_element cleaner0 2023-07-03T10:21:09Z SO: SH3 structure_element SO: cleaner0 2023-07-03T09:40:41Z F-BAR 0.99870265 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 structure_element SO: cleaner0 2023-07-03T10:21:01Z SH3 0.9708707 protein cleaner0 2023-06-30T08:44:50Z PR: N-WASP 0.99252945 protein cleaner0 2023-07-03T10:02:37Z PR: dynamin 0.99865294 structure_element cleaner0 2023-06-30T08:45:24Z SO: HR1 0.9986656 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 0.52810615 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 0.53813136 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 0.48928082 structure_element cleaner0 2023-06-30T08:45:24Z SO: HR1 INTRO paragraph 5802 Other HR1 domains studied so far, including those from the PRK family, have been found to bind their cognate Rho family G protein-binding partner with high specificity and affinities in the nanomolar range. The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. Both of the G protein switch regions are involved in the interaction. The coiled-coil fold is shared by the HR1 domain of the TOCA family protein, CIP4, and, based on sequence homology, by TOCA1 itself. These HR1 domains, however, show specificity for Cdc42, rather than RhoA or Rac1. How different HR1 domain proteins distinguish their specific G protein partners remains only partially understood, and structural characterization of a novel G protein-HR1 domain interaction would add to the growing body of information pertaining to these protein complexes. Furthermore, the biological function of the interaction between TOCA1 and Cdc42 remains poorly understood, and so far there has been no biophysical or structural insight. structure_element SO: cleaner0 2023-06-30T08:45:24Z HR1 0.89815104 protein_type cleaner0 2023-07-03T10:14:49Z MESH: PRK family protein_type MESH: cleaner0 2023-07-03T08:23:07Z G protein 0.99019694 evidence cleaner0 2023-07-03T11:56:35Z DUMMY: affinities 0.99401087 evidence cleaner0 2023-07-03T11:56:38Z DUMMY: structures 0.9980007 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 0.99824715 structure_element cleaner0 2023-07-03T10:21:14Z SO: HR1a 0.94389236 protein_state cleaner0 2023-07-03T13:27:49Z DUMMY: complex with 0.9936864 protein cleaner0 2023-06-30T08:46:56Z PR: RhoA structure_element SO: cleaner0 2023-07-03T10:21:30Z HR1b 0.9635602 protein_state cleaner0 2023-07-03T13:27:52Z DUMMY: complex with 0.9928364 protein cleaner0 2023-06-30T08:47:06Z PR: Rac1 structure_element SO: cleaner0 2023-06-30T08:45:24Z HR1 0.99282736 structure_element cleaner0 2023-07-03T10:21:38Z SO: anti-parallel coiled-coil protein_type MESH: cleaner0 2023-07-03T08:23:07Z G protein 0.70623326 structure_element cleaner0 2023-07-03T10:21:41Z SO: helices site SO: cleaner0 2023-07-03T10:02:22Z G protein switch regions 0.996804 structure_element cleaner0 2023-07-03T10:21:44Z SO: coiled-coil fold structure_element SO: cleaner0 2023-06-30T08:45:24Z HR1 0.9743039 protein_type cleaner0 2023-07-03T10:14:55Z MESH: TOCA family protein 0.99742043 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 0.99869365 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 structure_element SO: cleaner0 2023-06-30T08:45:24Z HR1 0.9809748 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 0.97429186 protein cleaner0 2023-06-30T08:46:56Z PR: RhoA 0.97543836 protein cleaner0 2023-06-30T08:47:06Z PR: Rac1 structure_element SO: cleaner0 2023-06-30T08:45:24Z HR1 protein_type MESH: cleaner0 2023-07-03T08:23:07Z G protein protein_type MESH: cleaner0 2023-07-03T08:23:07Z G protein structure_element SO: cleaner0 2023-06-30T08:45:24Z HR1 0.99876165 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 0.9816049 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 INTRO paragraph 6973 The interactions of TOCA1 and N-WASP with Cdc42 as well as with each other have raised questions as to whether the two Cdc42 effectors can interact with a single molecule of Cdc42 simultaneously. There is some evidence for a ternary complex between Cdc42, N-WASP, and TOCA1, but there was no direct demonstration of simultaneous contacts between the two effectors and a single molecule of Cdc42. Nonetheless, the substantial difference between the structures of the G protein-binding regions of the two effectors is intriguing and implies that they bind to Cdc42 quite differently, providing motivation for investigating the possibility that Cdc42 can bind both effectors concurrently. WASP interacts with Cdc42 via a conserved, unstructured binding motif known as the Cdc42- and Rac-interactive binding region (CRIB), which forms an intermolecular β-sheet, expanding the anti-parallel β2 and β3 strands of Cdc42. In contrast, the TOCA family proteins are thought to interact via the HR1 domain, which may form a triple coiled-coil with switch II of Rac1, like the HR1b domain of PRK1. 0.99872357 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 0.98440695 protein cleaner0 2023-06-30T08:44:50Z PR: N-WASP 0.9968567 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 0.87137836 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 0.99684143 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 0.9507448 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 0.97352785 protein cleaner0 2023-06-30T08:44:50Z PR: N-WASP 0.998511 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 0.99682486 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 0.9932863 evidence cleaner0 2023-07-03T11:56:46Z DUMMY: structures 0.8868308 site cleaner0 2023-07-03T08:16:05Z SO: G protein-binding regions 0.99732095 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 0.9966552 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 0.9979085 protein_type cleaner0 2023-07-03T10:22:06Z MESH: WASP 0.9968953 protein cleaner0 2023-06-30T08:42:50Z PR: Cdc42 0.99366945 protein_state cleaner0 2023-07-03T13:28:04Z DUMMY: conserved 0.9845956 structure_element cleaner0 2023-07-03T10:21:52Z SO: unstructured binding motif structure_element SO: cleaner0 2023-07-03T08:24:50Z Cdc42- and Rac-interactive binding region 0.99738663 structure_element cleaner0 2023-07-03T10:22:31Z SO: CRIB 0.99092513 structure_element cleaner0 2023-07-03T10:22:34Z SO: intermolecular β-sheet 0.8364334 structure_element cleaner0 2023-07-03T10:22:37Z SO: β2 and β3 strands 0.9955338 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.9845709 protein_type cleaner0 2023-07-03T10:15:21Z MESH: TOCA family proteins structure_element SO: cleaner0 2023-06-30T08:45:24Z HR1 0.9933011 structure_element cleaner0 2023-07-03T10:22:40Z SO: triple coiled-coil 0.9933278 site cleaner0 2023-07-03T09:26:23Z SO: switch II 0.97717255 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 structure_element SO: cleaner0 2023-07-03T10:22:53Z HR1b 0.99831796 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 INTRO paragraph 8068 Here, we present the solution NMR structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We also present data pertaining to binding of the TOCA HR1 domain to Cdc42, which is the first biophysical description of an HR1 domain binding this particular Rho family small G protein. Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics. 0.9930488 experimental_method cleaner0 2023-07-03T11:43:09Z MESH: solution NMR 0.9892476 evidence cleaner0 2023-07-03T11:56:51Z DUMMY: structure structure_element SO: cleaner0 2023-06-30T08:45:24Z HR1 0.99888533 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 0.93522906 evidence cleaner0 2023-07-03T11:56:53Z DUMMY: structural data 0.99885 protein_type cleaner0 2023-07-03T10:22:23Z MESH: TOCA structure_element SO: cleaner0 2023-06-30T08:45:24Z HR1 0.986479 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.9983109 structure_element cleaner0 2023-06-30T08:45:25Z SO: HR1 0.9928767 protein_type cleaner0 2023-07-03T10:15:27Z MESH: Rho family small G protein 0.99433196 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.9940287 site cleaner0 2023-07-03T08:16:05Z SO: G protein-binding regions 0.9988475 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 0.96599275 protein cleaner0 2023-06-30T08:44:50Z PR: N-WASP protein_type MESH: cleaner0 2023-07-03T08:23:07Z G protein 0.9955519 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.94650984 protein cleaner0 2023-06-30T08:44:50Z PR: N-WASP 0.9988159 protein cleaner0 2023-06-30T08:43:35Z PR: TOCA1 protein_type MESH: melaniev@ebi.ac.uk 2023-07-21T09:54:37Z actin METHODS title_1 8667 Experimental Procedures METHODS title_4 8691 Expression Constructs METHODS paragraph 8713 The Xenopus tropicalis TOCA1 HR1 domain (residues 330–426 and N-terminally extended constructs as indicated) were amplified from cDNA (TOCA1 accession number NM_001005148) and cloned into pGEX-6P-1 (GE Healthcare) or pGEX-HisP. The HR1 domain of human CIP4 (residues 388–481) was amplified from IMAGE clone 3532036, the Xenopus laevis FBP17 HR1 domain (residues 385–486) from IMAGE clone 5514481, and the X. tropicalis N-WASP G protein-binding domain (GBD) (residues 197–255) from IMAGE clone 5379332, and all were cloned into pGEX-6P-1. The resulting constructs express the proteins as N-terminal GST fusions with a 3C protease-cleavable tag, with pGEX-HisP expressing an additional C-terminal His6 tag. Human Cdc42Δ7Q61L and full-length Cdc42 were cloned into pGEX-2T (GE Healthcare) and pGEX-6P-1, respectively. A C-terminally extended construct of TOCA1 comprising residues 330–545 was cloned into pMAT10-P.7 The resulting construct expresses TOCA1 330–545 as an N-terminal His-MBP fusion protein with a 3C protease-cleavable tag. Full-length X. tropicalis TOCA1, TOCA1 F-BAR (residues 1–287), and TOCA1 ΔSH3 (residues 1–480) were PCR-amplified from a cDNA clone (IMAGE 5157175) and cloned into pET-His6-SNAP using FseI and AscI sites that had been incorporated into the primers to create His-SNAP-TOCA1 proteins. METHODS title_4 10049 Protein Expression METHODS paragraph 10068 GST fusion proteins (HR1 domains and Cdc42) were expressed in E. coli BL21 cells (Invitrogen). Stationary cultures were diluted 1:10 and grown at 37 °C until an A600 of ∼0.8 was reached and then induced with 0.1 mm isopropyl-β-d-thiogalactopyranoside for 20 h at 20 °C. The GST-N-WASP GBD construct was expressed in E. coli BL21-CodonPlus®-RIL (Agilent Technologies). The proteins were purified using glutathione-agarose beads (Sigma) and eluted from the beads by cleavage of the GST tag with 3C protease (HR1 domains, N-WASP GBD, and full-length Cdc42Q61L) or thrombin (Novagen, Cdc42Δ7Q61L) prior to gel filtration on a 16/60 S75 column (GE Healthcare). His-MBP-HR1-SH3 was purified using nickel-nitrilotriacetic acid-agarose beads (Life Technologies) prior to cleavage with 3C protease and gel filtration. Full-length TOCA1, TOCA1 F-BAR, and TOCA1 ΔSH3 were expressed from pET-His6-SNAP in BL21 pLysS, grown at 37 °C until an A600 of ∼0.6 was reached, and induced with 0.3 mm isopropyl-β-d-thiogalactopyranoside overnight at 19 °C. Proteins were coupled to nickel-nitrilotriacetic acid-agarose (Qiagen), eluted using increasing concentrations of imidazole, and further purified by gel filtration using a 16/60 S200 column (GE Healthcare). All protein concentrations were determined by amino acid analysis (Protein and Nucleic Acid Chemistry Facility, Department of Biochemistry, University of Cambridge). METHODS title_4 11490 Nucleotide Exchange METHODS paragraph 11510 For NMR experiments, Cdc42 was nucleotide-exchanged for the non-hydrolyzable GTP analogue GMPPNP (Sigma) as described previously. For scintillation proximity assays (SPAs), Cdc42 was loaded with [3H]GTP using [8-3H]GTP (PerkinElmer Life Sciences), as described previously. The protein was confirmed as full-length using mass spectrometry (PNAC facility, Department of Biochemistry, University of Cambridge). METHODS title_4 11918 SPAs METHODS paragraph 11923 For direct assays, GST-PAK, GST-ACK, or His-tagged TOCA1 constructs were attached to a fluoromicrosphere via an anti-GST or anti-His antibody in the presence of Cdc42Δ7Q61L·[3H]GTP. Binding curves were fitted using a direct binding isotherm to obtain Kd values and their curve-fitting errors for the G protein-effector interactions. For competition assays, free ACK GBD, TOCA1 HR1, TOCA1 HR1SH3, or N-WASP GBD was titrated into a mixture of 30 nm Cdc42Δ7Q61L·[3H]GTP and 30 nm GST-ACK immobilized on a fluoromicrosphere as above. Data were fitted to competition binding isotherms to obtain Kd values and curve-fitting errors, as described previously. METHODS title_4 12582 NMR Spectroscopy METHODS paragraph 12599 The NMR experiments and resonance assignments of the HR1 domain are described. The NMR experiments were carried out with 0.9 mm 13C/15N-labeled HR1 domain in 20 mm sodium phosphate, pH 7.5, 150 mm NaCl, 5 mm MgCl2, 5 mm DTT, 10% D2O. Distance restraints were derived from a 15N-separated NOESY (100-ms mixing time) recorded on a Bruker DRX500 and a 13C-separated NOESY (100-ms mixing time) recorded on an Avance AV600. NMR data were processed using AZARA (W. Boucher, University of Cambridge) and analyzed using ANALYSIS. METHODS title_4 13121 Structure Calculation METHODS paragraph 13143 Structures were calculated iteratively using CNS version 1.0 interfaced to Aria version 2.3.1. The PROSLQ force field was used for non-bonded parameters. Backbone torsion angles were estimated from CA, CO, CB, N, and HA chemical shifts using TALOS-N. The “strong” φ and ψ restraints were included with an error of ±2 S.D. values of the averaged TALOS-N predictions. Dihedral angle predictions for residues 323–340 were weak, so no restraints were included for this region. METHODS title_4 13625 NMR Titrations METHODS paragraph 13640 All of the 15N and 13C HSQCs were recorded at 25 °C in 50 mm sodium phosphate, pH 5.5, 25 mm NaCl, 5 mm MgCl2, 5 mm DTT, 10% D2O on a Bruker DRX500. 15N-HR1 HSQC experiments were recorded on 0.2 mm 15N-HR1 domain with HR1/Cdc42·GMPPNP ratios of 1:0, 1:0.25, 1:0.5, 1:1, and 1:4. Experiments were recorded on 0.27 mm 15N-Cdc42·GMPPNP at Cdc42/HR1 ratios of 1:0, 1:0.25, 1:0.5, and 1:2.2. The 15N HSQC titrations with N-WASP were recorded on 0.6 mm 15N-HR1 domain or 0.15 mm 15N-Cdc42 at the ratios indicated in the figures. METHODS title_4 14166 Chemical Shift Mapping METHODS paragraph 14189 The chemical shift changes, δ, were calculated using the equation, where δ(1H) and δ(15N) are the chemical shift changes for the 1H and 15N dimensions, respectively. Residues that had disappeared were assigned a δ value larger than the maximum calculated δ for the data set, and residues that were too overlapped to be reliably assigned in the complex spectra were assigned δ = 0. The residues that had shifted more than the mean chemical shift change across the spectrum were classed as significant and were filtered for solvent accessibility using NACCESS. Residues with <50% solvent accessibility were considered to be buried and unavailable for binding. METHODS title_4 14872 Pyrene Actin Assays METHODS paragraph 14892 Pyrene actin assays were carried out as described previously. Xenopus high speed supernatant was used at 5 mg/ml and supplemented with 0.12 mg/ml pyrene actin as described previously. TOCA1 HR1 domain or N-WASP CRIB domain was added at the concentrations indicated. Liposomes were made, using methods described previously, from 60% phosphatidylcholine, 30% phosphatidylserine, and 10% PI(4,5)P2 to 2 mm final lipid concentration. All of the lipids used were natural brain or liver lipids from Avanti Polar Lipids. The assays were initiated by the addition of 5 μl of liposomes per 200 μl of reaction mix. RESULTS title_1 15499 Results RESULTS title_4 15507 Cdc42-TOCA1 Binding 0.5092524 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.9295319 protein cleaner0 2023-06-30T08:43:36Z PR: TOCA1 RESULTS paragraph 15527 TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42·GTPγS but not to Cdc42·GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42. The C. elegans TOCA1 orthologues also bind to Cdc42 via their consensus HR1 domain. The HR1 domains from the PRK family bind their G protein partners with a high affinity, exhibiting a range of submicromolar dissociation constants (Kd) as low as 26 nm. A Kd in the nanomolar range was therefore expected for the interaction of the TOCA1 HR1 domain with Cdc42. 0.9986526 protein cleaner0 2023-06-30T08:43:36Z PR: TOCA1 0.7888495 taxonomy_domain cleaner0 2023-07-03T09:39:16Z DUMMY: Xenopus 0.991585 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 complex_assembly GO: cleaner0 2023-07-03T08:32:07Z Cdc42·GTPγS complex_assembly GO: cleaner0 2023-07-03T08:32:28Z Cdc42·GDP 0.9952996 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.99546283 protein cleaner0 2023-06-30T08:46:56Z PR: RhoA 0.9461143 site cleaner0 2023-07-03T12:07:22Z SO: Rho family binding modules structure_element SO: cleaner0 2023-06-30T08:45:25Z HR1 0.9988255 protein cleaner0 2023-06-30T08:43:36Z PR: TOCA1 0.9971808 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.9944839 species cleaner0 2023-07-03T10:50:17Z MESH: C. elegans 0.9773745 protein cleaner0 2023-06-30T08:43:36Z PR: TOCA1 0.99759334 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 structure_element SO: cleaner0 2023-06-30T08:45:26Z HR1 structure_element SO: cleaner0 2023-06-30T08:45:26Z HR1 0.9276004 protein_type cleaner0 2023-07-03T10:15:34Z MESH: PRK family protein_type MESH: cleaner0 2023-07-03T08:23:07Z G protein 0.9576243 evidence cleaner0 2023-07-03T11:56:58Z DUMMY: dissociation constants 0.96872586 evidence cleaner0 2023-07-03T09:00:18Z DUMMY: Kd 0.9735395 evidence cleaner0 2023-07-03T09:00:18Z DUMMY: Kd 0.9988011 protein cleaner0 2023-06-30T08:43:36Z PR: TOCA1 0.9985483 structure_element cleaner0 2023-06-30T08:45:26Z SO: HR1 0.9972486 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 RESULTS paragraph 16196 We generated an X. tropicalis TOCA1 HR1 domain construct encompassing residues 330–426. This region comprises the complete HR1 domain based on secondary structure predictions and sequence alignments with another TOCA family member, CIP4, whose structure has been determined. The interaction between [3H]GTP·Cdc42 and a C-terminally His-tagged TOCA1 HR1 domain construct was investigated using SPA. The binding isotherm for the interaction is shown in Fig. 1A, together with the Cdc42-PAK interaction as a positive control. The binding of TOCA1 HR1 to Cdc42 was unexpectedly weak, with a Kd of >1 μm. It was not possible to estimate the Kd more accurately using direct SPA experiments, because saturation could not be reached due to nonspecific signal at higher protein concentrations. 0.99386984 species cleaner0 2023-07-03T10:50:22Z MESH: X. tropicalis 0.9987664 protein cleaner0 2023-06-30T08:43:36Z PR: TOCA1 0.9366215 structure_element cleaner0 2023-06-30T08:45:26Z SO: HR1 0.98613757 residue_range cleaner0 2023-07-03T10:50:33Z DUMMY: 330–426 structure_element SO: cleaner0 2023-06-30T08:45:26Z HR1 0.9026214 experimental_method cleaner0 2023-07-03T11:43:14Z MESH: sequence alignments 0.9625473 protein_type cleaner0 2023-07-03T10:15:39Z MESH: TOCA family 0.9986644 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 0.9560438 evidence cleaner0 2023-07-03T11:57:02Z DUMMY: structure complex_assembly GO: cleaner0 2023-07-03T08:32:59Z [3H]GTP·Cdc42 0.7794807 protein_state cleaner0 2023-07-03T10:16:04Z DUMMY: His-tagged 0.9986418 protein cleaner0 2023-06-30T08:43:36Z PR: TOCA1 0.9242694 structure_element cleaner0 2023-06-30T08:45:26Z SO: HR1 0.99000335 experimental_method cleaner0 2023-07-03T08:37:43Z MESH: SPA 0.99487644 evidence cleaner0 2023-07-03T11:57:06Z DUMMY: binding isotherm protein PR: cleaner0 2023-06-30T08:42:51Z Cdc42 0.59618574 protein cleaner0 2023-07-03T10:48:35Z PR: PAK 0.9986374 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 0.9962793 structure_element cleaner0 2023-06-30T08:45:26Z SO: HR1 0.9975297 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.9945845 evidence cleaner0 2023-07-03T09:00:18Z DUMMY: Kd 0.99462295 evidence cleaner0 2023-07-03T09:00:18Z DUMMY: Kd 0.988311 experimental_method cleaner0 2023-07-03T08:37:42Z MESH: SPA zbc0281646060001.jpg F1 FIG fig_caption 16985 The TOCA1 HR1-Cdc42 interaction is low affinity. A, curves derived from direct binding assays in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-PAK or HR1-His6 in SPAs. The SPA signal was corrected by subtraction of control data with no GST-PAK or HR1-His6. The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42Δ7Q61L·[3H]GTP or full-length Cdc42Q61L·[3H]GTP. The Kd values derived for the ACK GBD with Cdc42Δ7 and full-length Cdc42 were 0.032 ± 0.01 and 0.011 ± 0.01 μm, respectively. The Kd values derived for the TOCA1 HR1 with Cdc42Δ7 and full-length Cdc42 were 6.05 ± 1.96 and 5.39 ± 1.69 μm, respectively. 0.9968162 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 0.97470653 structure_element cleaner0 2023-06-30T08:45:26Z SO: HR1 protein PR: cleaner0 2023-06-30T08:42:51Z Cdc42 0.9902583 experimental_method cleaner0 2023-07-03T11:43:21Z MESH: direct binding assays complex_assembly GO: cleaner0 2023-07-03T08:34:49Z Cdc42Δ7Q61L·[3H]GTP 0.8137186 experimental_method cleaner0 2023-07-03T11:43:31Z MESH: incubated mutant MESH: cleaner0 2023-07-03T10:03:34Z GST-PAK 0.7914784 mutant cleaner0 2023-07-03T10:51:18Z MESH: HR1-His6 0.9534834 experimental_method cleaner0 2023-07-03T11:43:36Z MESH: SPAs experimental_method MESH: cleaner0 2023-07-03T08:37:43Z SPA mutant MESH: cleaner0 2023-07-03T10:03:58Z GST-PAK 0.9089138 mutant cleaner0 2023-07-03T10:51:21Z MESH: HR1-His6 0.67325974 evidence cleaner0 2023-07-03T11:57:20Z DUMMY: binding isotherm 0.9919743 evidence cleaner0 2023-07-03T09:00:18Z DUMMY: Kd experimental_method MESH: cleaner0 2023-07-03T08:39:34Z competition SPA 0.85910517 protein cleaner0 2023-07-03T09:29:57Z PR: ACK 0.9981895 structure_element cleaner0 2023-07-03T10:23:06Z SO: GBD 0.9983625 structure_element cleaner0 2023-06-30T08:45:26Z SO: HR1 0.8750449 experimental_method cleaner0 2023-07-03T11:43:40Z MESH: titrated mutant MESH: cleaner0 2023-07-03T08:42:00Z GST-ACK complex_assembly GO: cleaner0 2023-07-03T08:34:50Z Cdc42Δ7Q61L·[3H]GTP 0.9967753 protein_state cleaner0 2023-07-03T08:36:11Z DUMMY: full-length complex_assembly GO: cleaner0 2023-07-03T08:35:25Z Cdc42Q61L·[3H]GTP 0.99215734 evidence cleaner0 2023-07-03T09:00:18Z DUMMY: Kd 0.62932396 protein cleaner0 2023-07-03T09:29:58Z PR: ACK 0.9980578 structure_element cleaner0 2023-07-03T10:23:10Z SO: GBD 0.9941896 mutant cleaner0 2023-07-03T08:36:03Z MESH: Cdc42Δ7 0.9959187 protein_state cleaner0 2023-07-03T08:36:10Z DUMMY: full-length 0.99390113 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.9919451 evidence cleaner0 2023-07-03T09:00:18Z DUMMY: Kd 0.99818856 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 0.9983367 structure_element cleaner0 2023-06-30T08:45:26Z SO: HR1 0.9938573 mutant cleaner0 2023-07-03T08:36:04Z MESH: Cdc42Δ7 0.99614626 protein_state cleaner0 2023-07-03T08:36:11Z DUMMY: full-length 0.9942028 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 RESULTS paragraph 17901 It was possible that the low affinity observed was due to negative effects of immobilization of the HR1 domain, so other methods were employed, which utilized untagged proteins. Isothermal titration calorimetry was carried out, but no heat changes were observed at a range of concentrations and temperatures (data not shown), suggesting that the interaction is predominantly entropically driven. Other G protein-HR1 domain interactions have also failed to show heat changes in our hands.7 Infrared interferometry with immobilized Cdc42 was also attempted but was unsuccessful for both TOCA1 HR1 and for the positive control, ACK. 0.998464 structure_element cleaner0 2023-06-30T08:45:26Z SO: HR1 0.9960897 protein_state cleaner0 2023-07-03T13:28:12Z DUMMY: untagged 0.9961185 experimental_method cleaner0 2023-07-03T11:43:44Z MESH: Isothermal titration calorimetry protein_type MESH: cleaner0 2023-07-03T08:23:07Z G protein 0.49651623 structure_element cleaner0 2023-06-30T08:45:26Z SO: HR1 0.9960615 experimental_method cleaner0 2023-07-03T11:43:49Z MESH: Infrared interferometry 0.88655823 protein_state cleaner0 2023-07-03T13:28:15Z DUMMY: immobilized 0.891008 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.99855715 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 0.9980393 structure_element cleaner0 2023-06-30T08:45:26Z SO: HR1 0.9915769 protein cleaner0 2023-07-03T09:29:58Z PR: ACK RESULTS paragraph 18531 The affinity was therefore determined using competition SPAs. A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42, with radiolabeled [3H]GTP·Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined. Competition of GST-ACK GBD bound to [3H]GTP·Cdc42 by free ACK GBD was used as a control and to establish the value of background counts when Cdc42 is fully displaced. The data were fitted to a binding isotherm describing competition. Free ACK competed with itself with an affinity of 32 nm, similar to the value obtained by direct binding of 23 nm. The TOCA1 HR1 domain also fully competed with the GST-ACK but bound with an affinity of 6 μm (Fig. 1, B and C), in agreement with the low affinity observed in the direct binding experiments. 0.9201407 evidence cleaner0 2023-07-03T11:57:28Z DUMMY: affinity 0.9952146 experimental_method cleaner0 2023-07-03T11:43:55Z MESH: competition SPAs 0.77118975 experimental_method cleaner0 2023-07-03T11:47:18Z MESH: GST fusion 0.9981933 structure_element cleaner0 2023-07-03T10:23:23Z SO: GBD 0.99730754 protein cleaner0 2023-07-03T09:29:58Z PR: ACK 0.99683124 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 complex_assembly GO: cleaner0 2023-07-03T08:36:48Z [3H]GTP·Cdc42 experimental_method MESH: cleaner0 2023-07-03T11:46:22Z increasing concentrations 0.9938798 protein_state cleaner0 2023-07-03T13:28:19Z DUMMY: untagged 0.998295 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 structure_element SO: cleaner0 2023-06-30T08:45:26Z HR1 mutant MESH: cleaner0 2023-07-03T08:42:00Z GST-ACK 0.9519166 structure_element cleaner0 2023-07-03T10:23:41Z SO: GBD 0.9709375 protein_state cleaner0 2023-07-03T13:28:24Z DUMMY: bound to complex_assembly GO: cleaner0 2023-07-03T08:37:09Z [3H]GTP·Cdc42 0.99735993 protein_state cleaner0 2023-07-03T13:28:28Z DUMMY: free 0.99731404 protein cleaner0 2023-07-03T09:29:58Z PR: ACK 0.99670666 structure_element cleaner0 2023-07-03T10:23:26Z SO: GBD 0.9976126 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.68896246 evidence cleaner0 2023-07-03T11:58:31Z DUMMY: binding isotherm 0.9973116 protein_state cleaner0 2023-07-03T13:28:39Z DUMMY: Free 0.9951958 protein cleaner0 2023-07-03T09:29:58Z PR: ACK 0.8252186 evidence cleaner0 2023-07-03T11:57:32Z DUMMY: affinity experimental_method MESH: cleaner0 2023-07-03T11:46:35Z direct binding 0.9981353 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 0.9980393 structure_element cleaner0 2023-06-30T08:45:26Z SO: HR1 mutant MESH: cleaner0 2023-07-03T08:42:00Z GST-ACK 0.5593172 protein_state cleaner0 2023-07-03T13:28:44Z DUMMY: bound evidence DUMMY: cleaner0 2023-07-03T13:28:53Z affinity evidence DUMMY: cleaner0 2023-07-03T11:57:51Z affinity experimental_method MESH: cleaner0 2023-07-03T11:46:56Z direct binding experiments RESULTS paragraph 19358 The Cdc42 construct used in the binding assays has seven residues deleted from the C terminus to facilitate purification. These residues are not generally required for G protein-effector interactions, including the interaction between RhoA and the PRK1 HR1a domain. In contrast, the C terminus of Rac1 contains a polybasic sequence, which is crucial for Rac1 binding to the HR1b domain from PRK1. As the observed affinity between TOCA1 HR1 and Cdc42 was much lower than expected, we reasoned that the C terminus of Cdc42 might be necessary for a high affinity interaction. The binding experiments were repeated with full-length [3H]GTP·Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd ≈ 5 μm; Fig. 1C). Thus, the C-terminal region of Cdc42 is not required for maximal binding of TOCA1 HR1. 0.8992194 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.9828191 experimental_method cleaner0 2023-07-03T11:47:01Z MESH: binding assays 0.6498607 residue_range cleaner0 2023-07-03T10:50:48Z DUMMY: seven residues 0.8628736 experimental_method cleaner0 2023-07-03T11:47:21Z MESH: deleted protein_type MESH: cleaner0 2023-07-03T08:23:07Z G protein 0.99057966 protein cleaner0 2023-06-30T08:46:56Z PR: RhoA 0.9981152 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 0.9985483 structure_element cleaner0 2023-07-03T10:23:49Z SO: HR1a 0.98936355 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.5294273 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.99855596 structure_element cleaner0 2023-07-03T10:24:03Z SO: HR1b 0.9981103 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 0.9897843 evidence cleaner0 2023-07-03T11:57:57Z DUMMY: affinity 0.99851173 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 0.99845076 structure_element cleaner0 2023-06-30T08:45:26Z SO: HR1 0.97940904 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.8183707 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 evidence DUMMY: cleaner0 2023-07-03T11:58:15Z affinity 0.97323084 experimental_method cleaner0 2023-07-03T11:48:27Z MESH: binding experiments 0.9973202 protein_state cleaner0 2023-07-03T08:36:11Z DUMMY: full-length complex_assembly GO: cleaner0 2023-07-03T08:38:23Z [3H]GTP·Cdc42 0.984329 evidence cleaner0 2023-07-03T11:58:00Z DUMMY: affinity 0.99854726 structure_element cleaner0 2023-06-30T08:45:26Z SO: HR1 0.9974193 protein_state cleaner0 2023-07-03T08:36:11Z DUMMY: full-length 0.95086724 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.81067705 evidence cleaner0 2023-07-03T11:58:02Z DUMMY: affinity 0.99599564 protein_state cleaner0 2023-07-03T13:29:01Z DUMMY: truncated 0.87006044 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 evidence DUMMY: cleaner0 2023-07-03T09:00:18Z Kd structure_element SO: cleaner0 2023-07-03T10:24:26Z C-terminal region 0.6816385 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.99867505 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 0.998273 structure_element cleaner0 2023-06-30T08:45:26Z SO: HR1 RESULTS paragraph 20219 Another possible explanation for the low affinities observed was that the HR1 domain alone is not sufficient for maximal binding of the TOCA proteins to Cdc42 and that the other domains are required. Indeed, GST pull-downs performed with in vitro translated human TOCA1 fragments had suggested that residues N-terminal to the HR1 domain may be required to stabilize the HR1 domain structure. Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, β-PIX. TOCA1 dimerizes via its F-BAR domain, which could also affect Cdc42 binding, for example by presenting two HR1 domains for Cdc42 interactions. Various TOCA1 fragments (Fig. 2A) were therefore assessed for binding to full-length Cdc42 by direct SPA. The isolated F-BAR domain showed no binding to full-length Cdc42 (Fig. 2B). Full-length TOCA1 and ΔSH3 TOCA1 bound with micromolar affinity (Fig. 2B), in a similar manner to the isolated HR1 domain (Fig. 1A). The HR1-SH3 protein could not be purified to homogeneity as a fusion protein, so it was assayed in competition assays after cleavage of the His tag. This construct competed with GST-ACK GBD to give a similar affinity to the HR1 domain alone (Kd = 4.6 ± 4 μm; Fig. 2C). Taken together, these data suggest that the TOCA1 HR1 domain is sufficient for maximal binding and that this binding is of a relatively low affinity compared with many other Cdc42·effector complexes. 0.4559165 evidence cleaner0 2023-07-03T11:58:36Z DUMMY: affinities 0.9986058 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.9021469 protein_state cleaner0 2023-07-03T13:29:07Z DUMMY: alone protein_type MESH: cleaner0 2023-07-03T10:04:59Z TOCA proteins 0.99680364 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.9955291 experimental_method cleaner0 2023-07-03T11:48:33Z MESH: GST pull-downs 0.99461246 species cleaner0 2023-07-03T10:50:27Z MESH: human 0.9986971 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 structure_element SO: cleaner0 2023-06-30T08:45:27Z HR1 0.99834037 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.99793243 structure_element cleaner0 2023-07-03T10:24:32Z SO: BAR 0.99475163 structure_element cleaner0 2023-07-03T10:24:35Z SO: SH3 0.80330014 protein_type cleaner0 2023-07-03T08:23:02Z MESH: G proteins structure_element SO: cleaner0 2023-07-03T10:04:39Z BAR 0.9988439 protein cleaner0 2023-07-03T10:04:11Z PR: Arfaptin2 0.991657 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.9981688 protein cleaner0 2023-07-03T10:05:05Z PR: Arl1 0.9980222 structure_element cleaner0 2023-07-03T10:24:40Z SO: SH3 0.98025334 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.9829663 protein cleaner0 2023-07-03T10:05:39Z PR: guanine nucleotide exchange factor 0.9755714 protein cleaner0 2023-07-03T10:05:13Z PR: β-PIX 0.9987124 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 oligomeric_state DUMMY: cleaner0 2023-07-03T11:59:00Z dimer structure_element SO: cleaner0 2023-07-03T09:40:41Z F-BAR 0.7265375 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.99834585 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 protein PR: cleaner0 2023-06-30T08:42:51Z Cdc42 0.99605787 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 0.99578553 protein_state cleaner0 2023-07-03T08:36:11Z DUMMY: full-length 0.9968912 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.9564046 experimental_method cleaner0 2023-07-03T08:37:43Z MESH: SPA structure_element SO: cleaner0 2023-07-03T09:40:41Z F-BAR 0.99623495 protein_state cleaner0 2023-07-03T08:36:11Z DUMMY: full-length 0.99716526 protein cleaner0 2023-06-30T08:42:51Z PR: Cdc42 0.9969497 protein_state cleaner0 2023-07-03T08:36:11Z DUMMY: Full-length 0.998749 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 0.9977174 mutant cleaner0 2023-07-03T10:51:26Z MESH: ΔSH3 0.9916163 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 0.8478406 protein_state cleaner0 2023-07-03T13:29:13Z DUMMY: bound 0.9983576 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.6989486 mutant cleaner0 2023-07-03T10:26:50Z MESH: HR1-SH3 0.96284235 experimental_method cleaner0 2023-07-03T11:48:38Z MESH: competition assays mutant MESH: cleaner0 2023-07-03T08:42:00Z GST-ACK 0.5497209 structure_element cleaner0 2023-07-03T10:30:05Z SO: GBD structure_element SO: cleaner0 2023-06-30T08:45:27Z HR1 0.5491047 protein_state cleaner0 2023-07-03T13:29:17Z DUMMY: alone evidence DUMMY: cleaner0 2023-07-03T09:00:18Z Kd 0.99868995 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 0.99857676 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 protein PR: cleaner0 2023-06-30T08:42:52Z Cdc42 zbc0281646060002.jpg F2 FIG fig_caption 21812 The Cdc42-HR1 interaction is of low affinity in the context of full-length protein and in TOCA1 paralogues. A, diagram illustrating the TOCA1 constructs assayed for Cdc42 binding. Domain boundaries are derived from secondary structure predictions; B, binding curves derived from direct binding assays, in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-ACK or His-tagged TOCA1 constructs, as indicated, in SPAs. The SPA signal was corrected by subtraction of control data with no fusion protein. The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal. C–E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42Δ7Q61L·[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L·[3H]GTP. complex_assembly GO: cleaner0 2023-07-03T08:40:57Z Cdc42-HR1 0.9972544 protein_state cleaner0 2023-07-03T08:36:11Z DUMMY: full-length 0.6960263 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 0.9876616 protein cleaner0 2023-06-30T08:43:37Z PR: TOCA1 protein PR: cleaner0 2023-06-30T08:42:52Z Cdc42 0.9627325 evidence cleaner0 2023-07-03T11:59:08Z DUMMY: binding curves 0.97862166 experimental_method cleaner0 2023-07-03T11:48:56Z MESH: direct binding assays complex_assembly GO: cleaner0 2023-07-03T08:34:50Z Cdc42Δ7Q61L·[3H]GTP 0.8606415 experimental_method cleaner0 2023-07-03T11:49:01Z MESH: incubated mutant MESH: cleaner0 2023-07-03T08:41:54Z GST-ACK protein_state DUMMY: cleaner0 2023-07-03T10:27:27Z His-tagged 0.99304646 protein cleaner0 2023-06-30T08:43:38Z PR: TOCA1 0.8894759 experimental_method cleaner0 2023-07-03T11:49:04Z MESH: SPAs 0.575186 experimental_method cleaner0 2023-07-03T08:37:43Z MESH: SPA 0.90803355 evidence cleaner0 2023-07-03T11:59:15Z DUMMY: binding isotherm 0.9921481 evidence cleaner0 2023-07-03T09:00:18Z DUMMY: Kd 0.98921967 experimental_method cleaner0 2023-07-03T08:39:33Z MESH: competition SPA 0.99752015 protein cleaner0 2023-06-30T08:43:38Z PR: TOCA1 0.9798131 mutant cleaner0 2023-07-03T10:27:06Z MESH: HR1-SH3 0.9126827 experimental_method cleaner0 2023-07-03T11:49:08Z MESH: titrated mutant MESH: cleaner0 2023-07-03T08:42:00Z GST-ACK complex_assembly GO: cleaner0 2023-07-03T08:34:50Z Cdc42Δ7Q61L·[3H]GTP structure_element SO: cleaner0 2023-07-03T10:29:13Z HR1 protein PR: cleaner0 2023-07-03T10:29:21Z CIP4 structure_element SO: cleaner0 2023-07-03T10:29:37Z HR1 protein PR: cleaner0 2023-07-03T10:29:46Z FBP17 0.6758686 experimental_method cleaner0 2023-07-03T11:49:11Z MESH: titrated mutant MESH: cleaner0 2023-07-03T08:42:00Z GST-ACK complex_assembly GO: cleaner0 2023-07-03T08:40:09Z Cdc42FLQ61L·[3H]GTP RESULTS paragraph 22768 The low affinity of the TOCA1 HR1-Cdc42 interaction raised the question of whether the other known Cdc42-binding TOCA family proteins, FBP17 and CIP4, also bind weakly. The HR1 domains from FBP17 and CIP4 were purified and assayed for Cdc42 binding in competition SPAs, analogous to those carried out with the TOCA1 HR1 domain. The affinities of both the FBP17 and CIP4 HR1 domains were also in the low micromolar range (10 and 5 μm, respectively) (Fig. 2, D and E), suggesting that low affinity interactions with Cdc42 are a common feature within the TOCA family. 0.9988821 protein cleaner0 2023-06-30T08:43:38Z PR: TOCA1 0.9969554 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 protein PR: cleaner0 2023-06-30T08:42:52Z Cdc42 protein PR: cleaner0 2023-06-30T08:42:52Z Cdc42 0.92827624 protein_type cleaner0 2023-07-03T10:16:12Z MESH: TOCA family proteins 0.99891806 protein cleaner0 2023-07-03T09:39:11Z PR: FBP17 0.9988306 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 0.99804926 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.9988697 protein cleaner0 2023-07-03T09:39:11Z PR: FBP17 0.9987895 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 0.65796494 experimental_method cleaner0 2023-07-03T11:49:16Z MESH: purified protein PR: cleaner0 2023-06-30T08:42:52Z Cdc42 0.98995584 experimental_method cleaner0 2023-07-03T11:49:22Z MESH: competition SPAs 0.9988158 protein cleaner0 2023-06-30T08:43:38Z PR: TOCA1 0.99836105 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.98984015 evidence cleaner0 2023-07-03T11:59:19Z DUMMY: affinities 0.99884534 protein cleaner0 2023-07-03T09:39:11Z PR: FBP17 0.9987373 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 0.9981744 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.9975121 protein cleaner0 2023-06-30T08:42:52Z PR: Cdc42 0.9836099 protein_type cleaner0 2023-07-03T10:16:29Z MESH: TOCA family RESULTS title_4 23334 Structure of the TOCA1 HR1 Domain 0.98409134 evidence cleaner0 2023-07-03T11:59:27Z DUMMY: Structure 0.99884063 protein cleaner0 2023-06-30T08:43:38Z PR: TOCA1 0.9984864 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 RESULTS paragraph 23368 Because the TOCA1 HR1 domain was sufficient for maximal Cdc42-binding, we used this construct for structural studies. Initial experiments were performed with TOCA1 residues 324–426, but we observed that the N terminus was cleaved during purification to yield a new N terminus at residue 330 (data not shown). We therefore engineered a construct comprising residues 330–426 to produce the minimal, stable HR1 domain. Backbone and side chain resonances were assigned as described. 2,778 non-degenerate NOE restraints were used in initial structure calculations (1,791 unambiguous and 987 ambiguous), derived from three-dimensional 15N-separated NOESY and 13C-separated NOESY experiments. There were 1,845 unambiguous NOEs and 757 ambiguous NOEs after eight iterations. 100 structures were calculated in the final iteration; the 50 lowest energy structures were water-refined; and of these, the 35 lowest energy structures were analyzed. Table 1 indicates that the HR1 domain structure is well defined by the NMR data. 0.99864227 protein cleaner0 2023-06-30T08:43:38Z PR: TOCA1 0.99835 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 protein PR: cleaner0 2023-06-30T08:42:52Z Cdc42 0.9986461 protein cleaner0 2023-06-30T08:43:38Z PR: TOCA1 0.9822894 residue_range cleaner0 2023-07-03T10:50:54Z DUMMY: 324–426 0.6586194 residue_number cleaner0 2023-07-03T10:52:02Z DUMMY: 330 0.9837437 residue_range cleaner0 2023-07-03T10:50:59Z DUMMY: 330–426 0.99568766 protein_state cleaner0 2023-07-03T13:29:23Z DUMMY: minimal 0.8597201 protein_state cleaner0 2023-07-03T13:29:26Z DUMMY: stable 0.99823916 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.86151886 evidence cleaner0 2023-07-03T09:32:12Z DUMMY: NOE restraints 0.8851528 experimental_method cleaner0 2023-07-03T11:49:28Z MESH: structure calculations 0.9527896 experimental_method cleaner0 2023-07-03T11:49:40Z MESH: 15N-separated NOESY 0.9854063 experimental_method cleaner0 2023-07-03T11:49:43Z MESH: 13C-separated NOESY 0.45354816 evidence cleaner0 2023-07-03T11:59:34Z DUMMY: NOEs 0.42897236 evidence cleaner0 2023-07-03T11:59:37Z DUMMY: NOEs 0.9530915 evidence cleaner0 2023-07-03T11:59:40Z DUMMY: structures 0.5917383 experimental_method cleaner0 2023-07-03T11:49:50Z MESH: calculated 0.93783134 evidence cleaner0 2023-07-03T11:59:42Z DUMMY: structures 0.98379946 evidence cleaner0 2023-07-03T11:59:44Z DUMMY: structures 0.9981406 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.9937012 evidence cleaner0 2023-07-03T11:59:47Z DUMMY: structure 0.93590724 experimental_method cleaner0 2023-07-03T11:50:02Z MESH: NMR T1 TABLE table_caption 24388 Experimental restraints and structural statistics T1 TABLE table_footnote 24438 a <SA>, the average root mean square deviations for the ensemble ± S.D. evidence DUMMY: cleaner0 2023-07-03T12:00:51Z average root mean square deviations T1 TABLE table_footnote 24511 b <SA>c, values for the structure that is closest to the mean. 0.96417356 evidence cleaner0 2023-07-03T12:00:56Z DUMMY: structure RESULTS paragraph 24574 The structure closest to the mean is shown in Fig. 3A. The two α-helices of the HR1 domain interact to form an anti-parallel coiled-coil with a slight left-handed twist, reminiscent of the HR1 domains of CIP4 (PDB code 2KE4) and PRK1 (PDB codes 1CXZ and 1URF). A sequence alignment illustrating the secondary structure elements of the TOCA1 and CIP4 HR1 domains and the HR1a and HR1b domains from PRK1 is shown in Fig. 3B. 0.99404585 evidence cleaner0 2023-07-03T12:00:58Z DUMMY: structure 0.8651283 structure_element cleaner0 2023-07-03T10:30:14Z SO: α-helices 0.99835324 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.9731526 structure_element cleaner0 2023-07-03T10:30:19Z SO: anti-parallel coiled-coil 0.99825734 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.99867105 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 0.9985677 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 0.98215437 experimental_method cleaner0 2023-07-03T11:50:07Z MESH: sequence alignment 0.99869126 protein cleaner0 2023-06-30T08:43:38Z PR: TOCA1 0.9982533 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 0.9983543 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.9982765 structure_element cleaner0 2023-07-03T10:30:23Z SO: HR1a 0.9973948 structure_element cleaner0 2023-07-03T10:30:26Z SO: HR1b 0.9979417 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 zbc0281646060003.jpg F3 FIG fig_caption 25001 The structure of the TOCA1 HR1 domain. A, the backbone trace of the 35 lowest energy structures of the HR1 domain overlaid with the structure closest to the mean is shown alongside a schematic representation of the structure closest to the mean. Flexible regions at the N and C termini (residues 330–333 and 421–426) are omitted for clarity. B, a sequence alignment of the HR1 domains from TOCA1, CIP4, and PRK1. The secondary structure was deduced using Stride, based on the Ramachandran angles, and is indicated as follows: gray, turn; yellow, α-helix; blue, 310 helix; white, coil. C, a close-up of the N-terminal region of TOCA1 HR1, indicating some of the NOEs defining its position with respect to the two α-helices. Dotted lines, NOE restraints. D, a close-up of the interhelix loop region showing some of the contacts between the loop and helix 1. NOEs are indicated with dotted lines. All structural figures were generated using PyMOL. 0.9917618 evidence cleaner0 2023-07-03T12:01:03Z DUMMY: structure 0.99836546 protein cleaner0 2023-06-30T08:43:38Z PR: TOCA1 0.99838996 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.46982583 evidence cleaner0 2023-07-03T12:01:07Z DUMMY: trace 0.98632556 evidence cleaner0 2023-07-03T12:01:14Z DUMMY: structures 0.9983033 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.95813525 evidence cleaner0 2023-07-03T12:01:17Z DUMMY: structure 0.95095694 evidence cleaner0 2023-07-03T12:01:19Z DUMMY: structure 0.9852314 residue_range cleaner0 2023-07-03T10:51:04Z DUMMY: 330–333 0.9833619 residue_range cleaner0 2023-07-03T10:51:07Z DUMMY: 421–426 0.9557089 experimental_method cleaner0 2023-07-03T11:50:11Z MESH: sequence alignment structure_element SO: cleaner0 2023-06-30T08:45:27Z HR1 0.9984187 protein cleaner0 2023-06-30T08:43:38Z PR: TOCA1 0.9982509 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 0.9978508 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 0.8259468 experimental_method cleaner0 2023-07-03T11:50:20Z MESH: Stride 0.98253715 evidence cleaner0 2023-07-03T12:01:22Z DUMMY: Ramachandran angles 0.97842044 structure_element cleaner0 2023-07-03T10:30:31Z SO: α-helix 0.97030413 structure_element cleaner0 2023-07-03T10:30:34Z SO: 310 helix 0.99778396 protein cleaner0 2023-06-30T08:43:38Z PR: TOCA1 0.9985317 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 evidence DUMMY: cleaner0 2023-07-03T10:30:57Z NOEs 0.9908063 structure_element cleaner0 2023-07-03T10:30:37Z SO: α-helices evidence DUMMY: cleaner0 2023-07-03T09:32:13Z NOE restraints 0.9965214 structure_element cleaner0 2023-07-03T10:30:40Z SO: interhelix loop 0.99083734 structure_element cleaner0 2023-07-03T10:30:43Z SO: loop 0.9737114 structure_element cleaner0 2023-07-03T10:30:45Z SO: helix 1 evidence DUMMY: cleaner0 2023-07-03T10:31:06Z NOEs RESULTS paragraph 25954 In the HR1a domain of PRK1, a region N-terminal to helix 1 forms a short α-helix, which packs against both helices of the HR1 domain. This region of TOCA1 HR1 (residues 334–340) is well defined in the family of structures (Fig. 3A) but does not form an α-helix. It instead forms a series of turns, defined by NOE restraints observed between residues separated by one (residues 332–334, 333–335, etc.) or two (residues 337–340) residues in the sequence and the φ and ψ angles, assessed using Stride. These turns cause the chain to reverse direction, allowing the N-terminal segment (residues 334–340) to contact both helices of the HR1 domain. Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. These contacts are summarized in Fig. 3C. 0.9984731 structure_element cleaner0 2023-07-03T10:31:13Z SO: HR1a 0.997982 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 0.9921024 structure_element cleaner0 2023-07-03T10:31:16Z SO: helix 1 0.99745536 structure_element cleaner0 2023-07-03T10:31:18Z SO: short α-helix structure_element SO: cleaner0 2023-06-30T08:45:27Z HR1 0.99764353 protein cleaner0 2023-06-30T08:43:38Z PR: TOCA1 0.99843866 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.9797867 residue_range cleaner0 2023-07-03T10:31:57Z DUMMY: 334–340 0.9959418 evidence cleaner0 2023-07-03T12:01:31Z DUMMY: structures 0.9889684 structure_element cleaner0 2023-07-03T10:31:26Z SO: α-helix 0.9776318 evidence cleaner0 2023-07-03T09:32:13Z DUMMY: NOE restraints 0.9790006 residue_range cleaner0 2023-07-03T10:31:52Z DUMMY: 332–334 0.9800062 residue_range cleaner0 2023-07-03T10:31:54Z DUMMY: 333–335 0.97902375 residue_range cleaner0 2023-07-03T10:31:50Z DUMMY: 337–340 0.9578368 evidence cleaner0 2023-07-03T12:01:38Z DUMMY: φ and ψ angles 0.5262887 experimental_method cleaner0 2023-07-03T11:50:25Z MESH: Stride 0.9672632 residue_range cleaner0 2023-07-03T10:31:48Z DUMMY: 334–340 0.9985348 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.73902595 evidence cleaner0 2023-07-03T12:01:56Z DUMMY: NOEs 0.8344713 residue_name_number cleaner0 2023-07-03T10:52:15Z DUMMY: Leu-334 0.88838047 residue_name_number cleaner0 2023-07-03T10:52:18Z DUMMY: Glu-335 0.90254325 residue_name_number cleaner0 2023-07-03T10:52:20Z DUMMY: Asp-336 0.90361804 residue_name_number cleaner0 2023-07-03T10:52:22Z DUMMY: Trp-413 0.8858938 structure_element cleaner0 2023-07-03T10:31:31Z SO: helix 2 residue_name_number DUMMY: cleaner0 2023-07-03T08:47:33Z Leu-334 residue_name_number DUMMY: cleaner0 2023-07-03T08:47:33Z Lys-409 0.8401229 structure_element cleaner0 2023-07-03T10:31:34Z SO: helix 2 0.93479484 residue_name_number cleaner0 2023-07-03T10:52:29Z DUMMY: Phe-337 0.9307402 residue_name_number cleaner0 2023-07-03T10:52:32Z DUMMY: Ser-338 0.8267959 residue_name_number cleaner0 2023-07-03T10:52:34Z DUMMY: Arg-345 0.8390352 residue_name_number cleaner0 2023-07-03T10:52:37Z DUMMY: Arg-348 0.8806424 residue_name_number cleaner0 2023-07-03T10:52:39Z DUMMY: Leu-349 0.93881685 structure_element cleaner0 2023-07-03T10:31:37Z SO: helix 1 RESULTS paragraph 26850 The two α-helices of TOCA1 HR1 are separated by a long loop of 10 residues (residues 380–389) that contains two short 310 helices (residues 381–383 and 386–389). Interestingly, side chains of residues within the loop region point back toward helix 1; for example, there are numerous distinct NOEs between the side chains of Asn-380 and Met-383 of the loop region and Tyr-377 and Val-376 of helix 1 (Fig. 3D). The backbone NH and CHα groups of Gly-384 and Asp-385 also show NOEs with the side chain of Tyr-377. 0.982701 structure_element cleaner0 2023-07-03T10:31:39Z SO: α-helices 0.9983334 protein cleaner0 2023-06-30T08:43:38Z PR: TOCA1 0.99819154 structure_element cleaner0 2023-06-30T08:45:27Z SO: HR1 0.9698297 structure_element cleaner0 2023-07-03T10:31:42Z SO: loop 0.97348136 residue_range cleaner0 2023-07-03T10:31:45Z DUMMY: 380–389 0.9480012 structure_element cleaner0 2023-07-03T10:32:07Z SO: short 310 helices 0.97844666 residue_range cleaner0 2023-07-03T10:32:01Z DUMMY: 381–383 0.9779238 residue_range cleaner0 2023-07-03T10:31:59Z DUMMY: 386–389 0.9881967 structure_element cleaner0 2023-07-03T10:32:13Z SO: loop region 0.99350333 structure_element cleaner0 2023-07-03T10:32:15Z SO: helix 1 residue_name_number DUMMY: cleaner0 2023-07-03T08:47:33Z Asn-380 0.9134205 residue_name_number cleaner0 2023-07-03T10:52:43Z DUMMY: Met-383 0.9575105 structure_element cleaner0 2023-07-03T10:32:18Z SO: loop region 0.90971285 residue_name_number cleaner0 2023-07-03T10:52:46Z DUMMY: Tyr-377 0.8663984 residue_name_number cleaner0 2023-07-03T10:52:48Z DUMMY: Val-376 0.9936434 structure_element cleaner0 2023-07-03T10:32:20Z SO: helix 1 0.8596821 residue_name_number cleaner0 2023-07-03T10:52:50Z DUMMY: Gly-384 0.8184116 residue_name_number cleaner0 2023-07-03T10:52:52Z DUMMY: Asp-385 0.85514146 residue_name_number cleaner0 2023-07-03T10:52:55Z DUMMY: Tyr-377 RESULTS title_4 27368 Mapping the TOCA1 and Cdc42 Binding Interfaces 0.9871063 protein cleaner0 2023-06-30T08:43:38Z PR: TOCA1 0.9959323 site cleaner0 2023-07-03T08:44:28Z SO: Cdc42 Binding Interfaces RESULTS paragraph 27415 The HR1TOCA1-Cdc42 interface was investigated using NMR spectroscopy. A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42Δ7Q61L·GMPPNP to map the Cdc42-binding surface. A comparison of the 15N HSQC spectra of free HR1 and HR1 in the presence of excess Cdc42 shows that although some peaks were shifted, several were much broader in the complex, and a considerable subset had disappeared (Fig. 4A). This behavior cannot be explained by the increase in molecular mass (from 12 to 33 kDa) when Cdc42 binds and is more likely to be due to conformational exchange. This leads to broadening of the peaks so that they are not detectable. Overall chemical shift perturbations (CSPs) were calculated for each residue, whereas those that had disappeared were assigned a shift change of 0.2 (Fig. 4B). A peak that disappeared or had a CSP above the mean CSP for the spectrum was considered to be significantly affected. 0.9960294 site cleaner0 2023-07-03T08:44:31Z SO: HR1TOCA1-Cdc42 interface 0.98970586 experimental_method cleaner0 2023-07-03T11:50:30Z MESH: NMR spectroscopy 0.97077554 experimental_method cleaner0 2023-07-03T11:50:34Z MESH: 15N HSQC chemical CHEBI: cleaner0 2023-07-03T13:31:28Z 15N protein_state DUMMY: cleaner0 2023-07-03T13:31:19Z labeled 0.99717796 protein cleaner0 2023-06-30T08:43:38Z PR: TOCA1 0.9970541 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 protein_state DUMMY: cleaner0 2023-07-03T08:55:29Z presence of 0.74030936 experimental_method cleaner0 2023-07-03T11:51:00Z MESH: increasing concentrations 0.9769086 protein_state cleaner0 2023-07-03T09:35:21Z DUMMY: unlabeled 0.7965565 complex_assembly cleaner0 2023-07-03T08:48:08Z GO: Cdc42Δ7Q61L·GMPPNP 0.9976872 site cleaner0 2023-07-03T08:44:35Z SO: Cdc42-binding surface 0.91865736 experimental_method cleaner0 2023-07-03T11:51:06Z MESH: 15N HSQC 0.8572031 evidence cleaner0 2023-07-03T12:02:03Z DUMMY: spectra 0.9965006 protein_state cleaner0 2023-07-03T13:29:39Z DUMMY: free 0.9915951 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.9891768 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.6970707 protein_state cleaner0 2023-07-03T08:55:29Z DUMMY: presence of 0.99477273 protein cleaner0 2023-06-30T08:42:52Z PR: Cdc42 0.9363838 protein cleaner0 2023-06-30T08:42:52Z PR: Cdc42 0.932084 experimental_method melaniev@ebi.ac.uk 2023-07-06T15:22:48Z MESH: chemical shift perturbations 0.9159061 experimental_method melaniev@ebi.ac.uk 2023-07-06T15:22:48Z MESH: CSPs 0.9407353 experimental_method melaniev@ebi.ac.uk 2023-07-06T15:22:48Z MESH: CSP 0.8318418 experimental_method melaniev@ebi.ac.uk 2023-07-06T15:22:48Z MESH: CSP zbc0281646060004.jpg F4 FIG fig_caption 28418 Mapping the binding surface of Cdc42 onto the TOCA1 HR1 domain. A, the 15N HSQC of 200 μm TOCA1 HR1 domain is shown in the free form (black) and in the presence of a 4-fold molar excess of Cdc42Δ7Q61L·GMPPNP (red). Expansions of two regions are shown with peak assignments, showing backbone amides in fast or intermediate exchange. B, CSPs were calculated as described under “Experimental Procedures” and are shown for backbone and side chain NH groups. The mean CSP is marked with a red line. Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.2 but were excluded when calculating the mean CSP and are indicated with open bars. Those that were not traceable due to spectral overlap were assigned a CSP of zero and are marked with an asterisk below the bar. Residues with affected side chain CSPs derived from 13C HSQCs are marked with green asterisks above the bars. Secondary structure elements are shown below the graph. C, a schematic representation of the HR1 domain. Residues with significantly affected backbone or side chain chemical shifts when Cdc42 bound and that are buried are colored dark blue, whereas those that are solvent-accessible are colored yellow. Residues with significantly affected backbone and side chain groups that are solvent-accessible are colored red. A close-up of the binding region is shown, with affected side chain heavy atoms shown as sticks. D, the G protein-binding region is marked in red onto structures of the HR1 domains as indicated. 0.9903181 site cleaner0 2023-07-03T12:07:28Z SO: binding surface 0.84945166 protein cleaner0 2023-06-30T08:42:52Z PR: Cdc42 0.99849176 protein cleaner0 2023-06-30T08:43:39Z PR: TOCA1 0.9983016 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.76035815 experimental_method cleaner0 2023-07-03T11:51:13Z MESH: 15N HSQC 0.99810374 protein cleaner0 2023-06-30T08:43:39Z PR: TOCA1 0.9980843 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.97064435 protein_state cleaner0 2023-07-03T13:29:57Z DUMMY: free form protein_state DUMMY: cleaner0 2023-07-03T08:55:29Z presence of complex_assembly GO: cleaner0 2023-07-03T08:46:25Z Cdc42Δ7Q61L·GMPPNP 0.9844333 experimental_method melaniev@ebi.ac.uk 2023-07-06T15:22:48Z MESH: CSPs 0.8512195 experimental_method melaniev@ebi.ac.uk 2023-07-06T15:22:48Z MESH: CSP 0.9545903 protein_state cleaner0 2023-07-03T08:55:29Z DUMMY: presence of 0.9684184 protein cleaner0 2023-06-30T08:42:52Z PR: Cdc42 0.98246807 experimental_method melaniev@ebi.ac.uk 2023-07-06T15:22:48Z MESH: CSP 0.6897554 experimental_method melaniev@ebi.ac.uk 2023-07-06T15:22:48Z MESH: CSP 0.96462184 experimental_method melaniev@ebi.ac.uk 2023-07-06T15:22:48Z MESH: CSP 0.9118489 experimental_method melaniev@ebi.ac.uk 2023-07-06T15:22:48Z MESH: CSPs 0.8412275 experimental_method cleaner0 2023-07-03T11:51:17Z MESH: 13C HSQCs 0.9983872 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.96418285 protein_state cleaner0 2023-07-03T13:30:33Z DUMMY: Cdc42 bound 0.8325718 protein_state cleaner0 2023-07-03T13:30:37Z DUMMY: solvent-accessible 0.85676384 protein_state cleaner0 2023-07-03T13:30:48Z DUMMY: solvent-accessible 0.99203086 site cleaner0 2023-07-03T12:07:31Z SO: binding region 0.99327934 site cleaner0 2023-07-03T08:16:18Z SO: G protein-binding region 0.8936646 evidence cleaner0 2023-07-03T12:02:19Z DUMMY: structures structure_element SO: cleaner0 2023-06-30T08:45:28Z HR1 RESULTS paragraph 29929 15N HSQC shift mapping experiments report on changes to amide groups, which are mainly inaccessible because they are buried inside the helices and are involved in hydrogen bonds. Therefore, 13C HSQC and methyl-selective SOFAST-HMQC experiments were also recorded on 15N,13C-labeled TOCA1 HR1 to yield more information on side chain involvement. The affected CH groups underwent significant line broadening, similarly to the NH peaks. Side chains whose CH groups disappeared in the presence of Cdc42 are marked on the graph in Fig. 4B with green asterisks. 0.9546366 experimental_method cleaner0 2023-07-03T11:51:23Z MESH: 15N HSQC shift mapping 0.96178645 structure_element cleaner0 2023-07-03T10:32:26Z SO: helices bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:06Z hydrogen bonds 0.98837936 experimental_method cleaner0 2023-07-03T11:51:27Z MESH: 13C HSQC 0.99327534 experimental_method cleaner0 2023-07-03T11:51:30Z MESH: methyl-selective SOFAST-HMQC 0.3927528 chemical cleaner0 2023-07-03T13:19:45Z CHEBI: 15N 0.8210875 chemical cleaner0 2023-07-03T13:19:48Z CHEBI: 13C 0.5613502 protein_state cleaner0 2023-07-03T13:30:56Z DUMMY: labeled 0.9974596 protein cleaner0 2023-06-30T08:43:39Z PR: TOCA1 0.99560153 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.92838216 protein_state cleaner0 2023-07-03T08:55:29Z DUMMY: presence of 0.90058 protein cleaner0 2023-06-30T08:42:52Z PR: Cdc42 RESULTS paragraph 30485 TOCA1 residues whose signals were affected by Cdc42 binding were mapped onto the structure of TOCA1 HR1 (Fig. 4C). The changes were localized to one end of the coiled-coil, and the binding site appeared to include residues from both α-helices and the loop region that joins them. Residues outside of this region were not significantly affected, indicating that there was no widespread conformational change. The residues in the interhelical loop and helix 1 that contact each other (Fig. 3D) show shift changes in their backbone NH and side chains in the presence of Cdc42. For example, the side chain of Asn-380 and the backbones of Val-376 and Tyr-377 were significantly affected but are all buried in the free TOCA1 HR1 structure, indicating that local conformational changes in the loop may facilitate complex formation. The chemical shift mapping data indicate that the G protein-binding region of the TOCA1 HR1 domain is broadly similar to that of the CIP4 and PRK1 HR1 domains (Figs. 3B and 4D). 0.99861026 protein cleaner0 2023-06-30T08:43:39Z PR: TOCA1 0.94039685 protein cleaner0 2023-06-30T08:42:52Z PR: Cdc42 0.9960568 evidence cleaner0 2023-07-03T12:02:23Z DUMMY: structure 0.99862313 protein cleaner0 2023-06-30T08:43:39Z PR: TOCA1 0.99835455 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.99796206 structure_element cleaner0 2023-07-03T10:32:31Z SO: coiled-coil 0.9978348 site cleaner0 2023-07-03T12:07:36Z SO: binding site 0.99615437 structure_element cleaner0 2023-07-03T10:32:34Z SO: α-helices 0.74406147 structure_element cleaner0 2023-07-03T10:32:36Z SO: loop region 0.9945371 structure_element cleaner0 2023-07-03T10:32:38Z SO: interhelical loop 0.9971713 structure_element cleaner0 2023-07-03T10:32:41Z SO: helix 1 0.9901208 protein_state cleaner0 2023-07-03T08:55:29Z DUMMY: presence of 0.98446804 protein cleaner0 2023-06-30T08:42:52Z PR: Cdc42 residue_name_number DUMMY: cleaner0 2023-07-03T08:47:08Z Asn-380 0.90919566 residue_name_number cleaner0 2023-07-03T10:53:00Z DUMMY: Val-376 0.9255726 residue_name_number cleaner0 2023-07-03T10:53:03Z DUMMY: Tyr-377 0.99773324 protein_state cleaner0 2023-07-03T13:31:34Z DUMMY: free 0.99823344 protein cleaner0 2023-06-30T08:43:39Z PR: TOCA1 0.9959603 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.99719596 evidence cleaner0 2023-07-03T12:02:26Z DUMMY: structure 0.9835715 structure_element cleaner0 2023-07-03T10:32:50Z SO: loop 0.947562 experimental_method cleaner0 2023-07-03T11:51:33Z MESH: chemical shift mapping 0.9967858 site cleaner0 2023-07-03T08:16:18Z SO: G protein-binding region 0.99871266 protein cleaner0 2023-06-30T08:43:39Z PR: TOCA1 structure_element SO: cleaner0 2023-06-30T08:45:28Z HR1 0.99850225 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 0.9983553 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 0.998538 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 RESULTS paragraph 31492 The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42Δ7Q61L·GMPPNP or 15N/13C -Cdc42Δ7Q61L·GMPPNP in the presence of unlabeled HR1 domain. The overall CSP was calculated for each residue. As was the case when labeled HR1 was observed, several peaks were shifted in the complex, but many disappeared, indicating exchange on an unfavorable, millisecond time scale (Fig. 5A). Detailed side chain data could not be obtained for all residues due to spectral overlap, but constant time 13C HSQC and methyl-selective SOFAST-HMQC experiments provided further information on certain well resolved side chains (marked with green asterisks in Fig. 5B). 0.94884044 experimental_method cleaner0 2023-07-03T11:51:39Z MESH: 15N 0.83301735 experimental_method cleaner0 2023-07-03T11:51:41Z MESH: 13C NMR 0.67671585 chemical cleaner0 2023-07-03T13:19:52Z CHEBI: 15N complex_assembly GO: cleaner0 2023-07-03T08:48:08Z Cdc42Δ7Q61L·GMPPNP 0.7961572 chemical cleaner0 2023-07-03T13:19:55Z CHEBI: 15N 0.81509006 chemical cleaner0 2023-07-03T13:19:58Z CHEBI: 13C complex_assembly GO: cleaner0 2023-07-03T08:48:08Z Cdc42Δ7Q61L·GMPPNP 0.66658735 protein_state cleaner0 2023-07-03T08:55:29Z DUMMY: presence of 0.99382895 protein_state cleaner0 2023-07-03T09:35:21Z DUMMY: unlabeled 0.99755055 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.9804678 experimental_method melaniev@ebi.ac.uk 2023-07-06T15:22:48Z MESH: CSP 0.99102825 protein_state cleaner0 2023-07-03T13:31:42Z DUMMY: labeled 0.9974668 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.98695385 experimental_method cleaner0 2023-07-03T11:52:04Z MESH: constant time 13C HSQC 0.99371284 experimental_method cleaner0 2023-07-03T11:52:07Z MESH: methyl-selective SOFAST-HMQC zbc0281646060005.jpg F5 FIG fig_caption 32166 Mapping the binding surface of the HR1 domain onto Cdc42. A, the 15N HSQC of Cdc42Δ7Q61L·GMPPNP is shown in its free form (black) and in the presence of excess TOCA1 HR1 domain (1:2.2, red). Expansions of two regions are shown, with most peaks in fast or intermediate exchange. B, CSPs are shown for backbone NH groups. The red line indicates the mean CSP, plus one S.D. Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.1 and are indicated with open bars. Those that were not traceable due to overlap are marked with an asterisk. Residues with disappeared peaks in 13C HSQC experiments are marked on the chart with green asterisks. Secondary structure elements are indicated below the graph. C, the residues with significantly affected backbone and side chain groups are highlighted on an NMR structure of free Cdc42Δ7Q61L·GMPPNP; those that are buried are colored dark blue, whereas those that are solvent-accessible are colored red. Residues with either side chain or backbone groups affected are colored blue if buried and yellow if solvent-accessible. Residues without information from shift mapping are colored gray. The flexible switch regions are circled. 0.9814966 site cleaner0 2023-07-03T12:07:43Z SO: binding surface 0.99824286 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.96610725 protein cleaner0 2023-06-30T08:42:52Z PR: Cdc42 0.84818137 experimental_method cleaner0 2023-07-03T11:52:11Z MESH: 15N HSQC 0.78093916 complex_assembly cleaner0 2023-07-03T08:48:07Z GO: Cdc42Δ7Q61L·GMPPNP 0.983827 protein_state cleaner0 2023-07-03T13:31:48Z DUMMY: free form 0.9001876 protein_state cleaner0 2023-07-03T08:55:30Z DUMMY: presence of 0.9961718 protein cleaner0 2023-06-30T08:43:39Z PR: TOCA1 0.99764293 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.98125 experimental_method melaniev@ebi.ac.uk 2023-07-06T15:22:48Z MESH: CSPs 0.9674589 experimental_method melaniev@ebi.ac.uk 2023-07-06T15:22:48Z MESH: CSP 0.9740281 protein_state cleaner0 2023-07-03T08:55:30Z DUMMY: presence of 0.5233301 protein cleaner0 2023-06-30T08:42:52Z PR: Cdc42 0.98254895 experimental_method melaniev@ebi.ac.uk 2023-07-06T15:22:48Z MESH: CSP 0.87693703 experimental_method cleaner0 2023-07-03T11:52:16Z MESH: 13C HSQC 0.8213444 experimental_method cleaner0 2023-07-03T11:52:19Z MESH: NMR 0.99046105 evidence cleaner0 2023-07-03T12:02:32Z DUMMY: structure 0.99755764 protein_state cleaner0 2023-07-03T13:31:53Z DUMMY: free 0.82752824 complex_assembly cleaner0 2023-07-03T08:48:08Z GO: Cdc42Δ7Q61L·GMPPNP protein_state DUMMY: cleaner0 2023-07-03T13:32:16Z solvent-accessible protein_state DUMMY: cleaner0 2023-07-03T13:32:34Z solvent-accessible experimental_method MESH: cleaner0 2023-07-03T11:53:19Z shift mapping 0.71962774 protein_state cleaner0 2023-07-03T13:32:38Z DUMMY: flexible 0.99757636 site cleaner0 2023-07-03T09:34:22Z SO: switch regions RESULTS paragraph 33364 As many of the peaks disappeared, the mean chemical shift change was relatively low, so a threshold of the mean plus one S.D. value was used to define a significant CSP. Residues that disappeared were also classed as significantly affected. Parts of the switch regions (Fig. 5, B and C) are invisible in NMR spectra recorded on free Cdc42 due to conformational exchange. These switch regions become visible in Cdc42 and other small G protein·effector complexes due to a decrease in conformational freedom upon complex formation. The switch regions of Cdc42 did not, however, become visible in the presence of the TOCA1 HR1 domain. Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain. This suggests that the switch regions are not rigidified in the HR1 complex and are still in conformational exchange. Nevertheless, mapping of the affected residues onto the NMR structure of free Cdc42Δ7Q61L·GMPPNP (Fig. 5C)8 shows that, although they are relatively widespread compared with changes in the HR1 domain, in general, they are on the face of the protein that includes the switches. Although the binding interface may be overestimated, this suggests that the switch regions are involved in binding to TOCA1. 0.8175822 evidence cleaner0 2023-07-03T12:02:35Z DUMMY: mean chemical shift change experimental_method MESH: melaniev@ebi.ac.uk 2023-07-06T15:22:48Z CSP 0.9972377 site cleaner0 2023-07-03T09:34:22Z SO: switch regions 0.8591063 experimental_method cleaner0 2023-07-03T11:52:32Z MESH: NMR 0.8200651 evidence cleaner0 2023-07-03T12:02:50Z DUMMY: spectra 0.99719834 protein_state cleaner0 2023-07-03T13:32:45Z DUMMY: free 0.9924085 protein cleaner0 2023-06-30T08:42:52Z PR: Cdc42 0.9964552 site cleaner0 2023-07-03T09:34:23Z SO: switch regions 0.9869178 protein cleaner0 2023-06-30T08:42:52Z PR: Cdc42 protein_type MESH: cleaner0 2023-07-03T08:23:08Z G protein 0.996436 site cleaner0 2023-07-03T09:34:23Z SO: switch regions 0.9908557 protein cleaner0 2023-06-30T08:42:52Z PR: Cdc42 0.9667624 protein_state cleaner0 2023-07-03T08:55:30Z DUMMY: presence of 0.9985393 protein cleaner0 2023-06-30T08:43:39Z PR: TOCA1 structure_element SO: cleaner0 2023-06-30T08:45:28Z HR1 0.78033185 residue_name_number cleaner0 2023-07-03T10:53:08Z DUMMY: Ser-30 0.99427783 site cleaner0 2023-07-03T09:34:35Z SO: switch I 0.81245255 residue_name_number cleaner0 2023-07-03T10:53:11Z DUMMY: Arg-66 0.8686878 residue_name_number cleaner0 2023-07-03T10:53:13Z DUMMY: Arg-68 0.85998726 residue_name_number cleaner0 2023-07-03T10:53:15Z DUMMY: Leu-70 0.83775693 residue_name_number cleaner0 2023-07-03T10:53:17Z DUMMY: Ser-71 0.99408966 site cleaner0 2023-07-03T09:26:23Z SO: switch II 0.99761605 protein_state cleaner0 2023-07-03T13:32:49Z DUMMY: free 0.9889713 protein cleaner0 2023-06-30T08:42:52Z PR: Cdc42 0.9672233 protein_state cleaner0 2023-07-03T08:55:30Z DUMMY: presence of 0.9982116 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.99595547 site cleaner0 2023-07-03T09:34:23Z SO: switch regions structure_element SO: cleaner0 2023-06-30T08:45:28Z HR1 0.7635831 experimental_method cleaner0 2023-07-03T12:03:00Z MESH: NMR 0.9886608 evidence cleaner0 2023-07-03T12:03:05Z DUMMY: structure 0.99748886 protein_state cleaner0 2023-07-03T13:32:52Z DUMMY: free 0.711201 complex_assembly cleaner0 2023-07-03T08:48:08Z GO: Cdc42Δ7Q61L·GMPPNP structure_element SO: cleaner0 2023-06-30T08:45:28Z HR1 0.99592566 site cleaner0 2023-07-03T09:34:44Z SO: switches 0.99736357 site cleaner0 2023-07-03T12:07:49Z SO: binding interface 0.99577093 site cleaner0 2023-07-03T09:34:21Z SO: switch regions 0.99841404 protein cleaner0 2023-06-30T08:43:39Z PR: TOCA1 RESULTS title_4 34673 Modeling the Cdc42·TOCA1 HR1 Complex 0.9399527 complex_assembly cleaner0 2023-07-03T10:48:47Z GO: Cdc42·TOCA1 HR1 RESULTS paragraph 34711 The Cdc42·HR1TOCA1 complex was not amenable to full structural analysis due to the weak interaction and the extensive exchange broadening seen in the NMR experiments. HADDOCK was therefore used to perform rigid body docking based on the structures of free HR1 domain and Cdc42 and ambiguous interaction restraints derived from the titration experiments described above. Residues with significantly affected resonances and more than 50% solvent accessibility were defined as active. Passive residues were defined automatically as those neighboring active residues. 0.8960567 complex_assembly cleaner0 2023-07-03T10:48:51Z GO: Cdc42·HR1TOCA1 0.9907706 experimental_method cleaner0 2023-07-03T11:52:37Z MESH: NMR 0.98815465 experimental_method cleaner0 2023-07-03T08:50:25Z MESH: HADDOCK experimental_method MESH: melaniev@ebi.ac.uk 2023-07-21T09:49:54Z rigid body docking 0.9960586 evidence cleaner0 2023-07-03T12:03:11Z DUMMY: structures 0.9974323 protein_state cleaner0 2023-07-03T13:32:56Z DUMMY: free 0.99764234 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.93101466 protein cleaner0 2023-06-30T08:42:53Z PR: Cdc42 0.70206016 experimental_method cleaner0 2023-07-03T11:52:44Z MESH: titration experiments RESULTS paragraph 35276 The orientation of the HR1 domain with respect to Cdc42 cannot be definitively concluded in the absence of unambiguous distance restraints; hence, HADDOCK produced a set of models in which the HR1 domain contacts the same surface on Cdc42 but is in various orientations with respect to Cdc42. The cluster with the lowest root mean square deviation from the lowest energy structure is assumed to be the best model. By these criteria, in the best model, the HR1 domain is in a similar orientation to the HR1a domain of PRK1 bound to RhoA and the HR1b domain bound to Rac1. A representative model from this cluster is shown in Fig. 6A alongside the Rac1-HR1b structure (PDB code 2RMK) in Fig. 6B. 0.9981976 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.99376076 protein cleaner0 2023-06-30T08:42:53Z PR: Cdc42 0.98814255 experimental_method cleaner0 2023-07-03T08:50:25Z MESH: HADDOCK 0.99816895 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.9891117 protein cleaner0 2023-06-30T08:42:53Z PR: Cdc42 0.9906749 protein cleaner0 2023-06-30T08:42:53Z PR: Cdc42 0.97768056 evidence cleaner0 2023-07-03T12:03:20Z DUMMY: root mean square deviation 0.9844689 evidence cleaner0 2023-07-03T12:03:23Z DUMMY: structure 0.9981712 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.9973526 structure_element cleaner0 2023-07-03T10:32:55Z SO: HR1a 0.99825805 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 0.9890354 protein_state cleaner0 2023-07-03T13:33:26Z DUMMY: bound to 0.93857586 protein cleaner0 2023-06-30T08:46:56Z PR: RhoA 0.997436 structure_element cleaner0 2023-07-03T10:34:33Z SO: HR1b 0.98779166 protein_state cleaner0 2023-07-03T13:33:30Z DUMMY: bound to 0.71836126 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 complex_assembly GO: cleaner0 2023-07-03T10:33:52Z Rac1-HR1b 0.992141 evidence cleaner0 2023-07-03T12:03:25Z DUMMY: structure zbc0281646060006.jpg F6 FIG fig_caption 35970 Model of Cdc42·HR1 complex. A, a representative model of the Cdc42·HR1 complex from the cluster closest to the lowest energy model produced using HADDOCK. Residues of Cdc42 that are affected in the presence of the HR1 domain but are not in close proximity to it are colored in red and labeled. B, structure of Rac1 in complex with the HR1b domain of PRK1 (PDB code 2RMK). C, sequence alignment of RhoA, Cdc42 and Rac1. Contact residues of RhoA and Rac1 to PRK1 HR1a and HR1b, respectively, are colored cyan. Residues of Cdc42 that disappear or show chemical shift changes in the presence of TOCA1 are colored cyan if also identified as contacts in RhoA and Rac1 and yellow if they are not. Residues equivalent to Rac1 and RhoA contact sites but that are invisible in free Cdc42 are gray. D, regions of interest of the Cdc42·HR1 domain model. The four lowest energy structures in the chosen HADDOCK cluster are shown overlaid, with the residues of interest shown as sticks and labeled. Cdc42 is shown in cyan, and TOCA1 is shown in purple. 0.9912746 complex_assembly cleaner0 2023-07-03T10:48:56Z GO: Cdc42·HR1 0.9905351 complex_assembly cleaner0 2023-07-03T10:48:59Z GO: Cdc42·HR1 0.9757337 experimental_method cleaner0 2023-07-03T08:50:24Z MESH: HADDOCK 0.77116495 protein cleaner0 2023-06-30T08:42:53Z PR: Cdc42 protein_state DUMMY: cleaner0 2023-07-03T08:55:30Z presence of 0.9980514 structure_element cleaner0 2023-06-30T08:45:28Z SO: HR1 0.93390775 evidence cleaner0 2023-07-03T12:03:31Z DUMMY: structure 0.68168455 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.8599246 protein_state cleaner0 2023-07-03T13:33:34Z DUMMY: in complex with 0.99765766 structure_element cleaner0 2023-07-03T10:34:37Z SO: HR1b 0.9963977 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 0.9856179 experimental_method cleaner0 2023-07-03T11:52:49Z MESH: sequence alignment 0.8277353 protein cleaner0 2023-06-30T08:46:56Z PR: RhoA 0.7358657 protein cleaner0 2023-06-30T08:42:53Z PR: Cdc42 0.86080074 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.77970403 protein cleaner0 2023-06-30T08:46:56Z PR: RhoA 0.77326703 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.99702734 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 0.996442 structure_element cleaner0 2023-07-03T10:34:40Z SO: HR1a 0.99660885 structure_element cleaner0 2023-07-03T10:34:43Z SO: HR1b 0.5916415 protein cleaner0 2023-06-30T08:42:53Z PR: Cdc42 0.95339227 protein_state cleaner0 2023-07-03T08:55:30Z DUMMY: presence of 0.99708956 protein cleaner0 2023-06-30T08:43:39Z PR: TOCA1 0.874548 protein cleaner0 2023-06-30T08:46:56Z PR: RhoA 0.84730613 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.61742294 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.4232686 protein cleaner0 2023-06-30T08:46:56Z PR: RhoA 0.9848926 site cleaner0 2023-07-03T12:08:00Z SO: contact sites 0.9961755 protein_state cleaner0 2023-07-03T13:33:39Z DUMMY: free 0.8306941 protein cleaner0 2023-06-30T08:42:53Z PR: Cdc42 complex_assembly GO: cleaner0 2023-07-03T08:50:14Z Cdc42·HR1 0.94688374 evidence cleaner0 2023-07-03T12:03:38Z DUMMY: structures experimental_method MESH: cleaner0 2023-07-03T08:50:25Z HADDOCK 0.96888226 protein cleaner0 2023-06-30T08:42:53Z PR: Cdc42 0.99732983 protein cleaner0 2023-06-30T08:43:39Z PR: TOCA1 RESULTS paragraph 37012 A sequence alignment of RhoA, Cdc42, and Rac1 is shown in Fig. 6C. The RhoA and Rac1 contact residues in the switch regions are invisible in the spectra of Cdc42, but they are generally conserved between all three G proteins. Several Cdc42 residues identified by chemical shift mapping are not in close contact in the Cdc42·TOCA1 model (Fig. 6A). Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. Lys-16Cdc42 is unlikely to be a contact residue because it is involved in nucleotide binding, but the others may represent specific Cdc42-TOCA1 contacts. In the model, these side chains are involved in direct contacts (Fig. 6D). 0.9871194 experimental_method cleaner0 2023-07-03T11:52:53Z MESH: sequence alignment 0.9734803 protein cleaner0 2023-06-30T08:46:57Z PR: RhoA 0.97490567 protein cleaner0 2023-06-30T08:42:53Z PR: Cdc42 0.9736101 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.8214345 protein cleaner0 2023-06-30T08:46:57Z PR: RhoA 0.81268275 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.9973299 site cleaner0 2023-07-03T09:34:23Z SO: switch regions 0.98021424 evidence cleaner0 2023-07-03T12:03:41Z DUMMY: spectra 0.95324767 protein cleaner0 2023-06-30T08:42:53Z PR: Cdc42 0.9873949 protein_state cleaner0 2023-07-03T13:33:46Z DUMMY: conserved protein_type MESH: cleaner0 2023-07-03T08:23:02Z G proteins 0.33471435 protein cleaner0 2023-06-30T08:42:53Z PR: Cdc42 0.9574032 experimental_method cleaner0 2023-07-03T11:52:56Z MESH: chemical shift mapping 0.9928748 complex_assembly cleaner0 2023-07-03T08:50:51Z GO: Cdc42·TOCA1 residue_name_number DUMMY: cleaner0 2023-07-03T08:51:40Z Thr-24 protein PR: cleaner0 2023-07-03T08:57:58Z Cdc42 residue_name_number DUMMY: cleaner0 2023-07-03T08:52:03Z Leu-160 protein PR: cleaner0 2023-07-03T08:58:02Z Cdc42 residue_name_number DUMMY: cleaner0 2023-07-03T08:52:24Z Lys-163 protein PR: cleaner0 2023-07-03T08:58:05Z Cdc42 0.99660486 site cleaner0 2023-07-03T09:34:36Z SO: switch I 0.9976216 site cleaner0 2023-07-03T10:34:56Z SO: switch residue_name_number DUMMY: cleaner0 2023-07-03T08:52:50Z Glu-95 protein PR: cleaner0 2023-07-03T08:58:08Z Cdc42 residue_name_number DUMMY: cleaner0 2023-07-03T08:53:11Z Lys-96 protein PR: cleaner0 2023-07-03T08:58:11Z Cdc42 0.9833968 structure_element cleaner0 2023-07-03T10:34:59Z SO: helix 0.99711096 site cleaner0 2023-07-03T09:26:23Z SO: switch II 0.9944163 complex_assembly cleaner0 2023-07-03T08:50:52Z GO: Cdc42·TOCA1 0.9544345 protein cleaner0 2023-06-30T08:46:57Z PR: RhoA 0.9498586 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 residue_name_number DUMMY: cleaner0 2023-07-03T08:53:33Z Gln-2 protein PR: cleaner0 2023-07-03T08:58:14Z Cdc42 residue_name_number DUMMY: cleaner0 2023-07-03T08:53:53Z Lys-16 protein PR: cleaner0 2023-07-03T08:58:17Z Cdc42 residue_name_number DUMMY: cleaner0 2023-07-03T08:54:15Z Thr-52 protein PR: cleaner0 2023-07-03T08:58:20Z Cdc42 residue_name_number DUMMY: cleaner0 2023-07-03T08:54:36Z Arg-68 protein PR: cleaner0 2023-07-03T08:58:23Z Cdc42 residue_name_number DUMMY: cleaner0 2023-07-03T08:54:56Z Lys-16 protein PR: cleaner0 2023-07-03T08:58:28Z Cdc42 0.9658783 complex_assembly cleaner0 2023-07-03T10:49:05Z GO: Cdc42-TOCA1 RESULTS title_4 38047 Competition between N-WASP and TOCA1 0.97834396 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.9982578 protein cleaner0 2023-06-30T08:43:40Z PR: TOCA1 RESULTS paragraph 38084 From the known interactions and effects of the proteins in biological systems, it has been suggested that TOCA1 and N-WASP could bind Cdc42 simultaneously. Studies in CHO cells indicated that a Cdc42·N-WASP·TOCA1 complex existed because FRET was observed between RFP-TOCA1 and GFP-N-WASP, and the efficiency was decreased when an N-WASP mutant was used that no longer binds Cdc42. An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP would interfere sterically with the TOCA1 HR1. A basic region in WASP including three lysines (residues 230–232), N-terminal to the core CRIB, has been implicated in an electrostatic steering mechanism, and these residues would be free to bind in the presence of TOCA1 HR1 (Fig. 7A). 0.99870217 protein cleaner0 2023-06-30T08:43:40Z PR: TOCA1 0.97666854 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.9955902 protein cleaner0 2023-06-30T08:42:53Z PR: Cdc42 0.9863807 complex_assembly cleaner0 2023-07-03T10:49:16Z GO: Cdc42·N-WASP·TOCA1 0.4309258 evidence cleaner0 2023-07-03T12:03:47Z DUMMY: FRET 0.21124144 chemical cleaner0 2023-07-03T13:20:15Z CHEBI: RFP 0.99814546 protein cleaner0 2023-06-30T08:43:40Z PR: TOCA1 0.2754772 chemical cleaner0 2023-07-03T13:20:18Z CHEBI: GFP 0.94308394 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.9076988 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.6556517 protein_state cleaner0 2023-07-03T10:51:40Z DUMMY: mutant 0.9952695 protein cleaner0 2023-06-30T08:42:53Z PR: Cdc42 0.9898616 experimental_method cleaner0 2023-07-03T11:53:25Z MESH: overlay 0.69386333 experimental_method cleaner0 2023-07-03T08:50:26Z MESH: HADDOCK 0.8598288 evidence cleaner0 2023-07-03T12:03:53Z DUMMY: model 0.7695202 complex_assembly cleaner0 2023-07-03T10:49:19Z GO: Cdc42·HR1TOCA1 0.993002 evidence cleaner0 2023-07-03T12:03:50Z DUMMY: structure 0.9959825 protein cleaner0 2023-06-30T08:42:54Z PR: Cdc42 protein_state DUMMY: cleaner0 2023-07-03T13:34:09Z in complex with 0.9987808 structure_element cleaner0 2023-07-03T10:35:03Z SO: GBD 0.9786554 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.9971476 protein cleaner0 2023-07-03T10:05:52Z PR: WASP 0.9967649 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.9980094 site cleaner0 2023-07-03T12:08:05Z SO: GBD binding sites 0.96977437 protein_state cleaner0 2023-07-03T08:55:27Z DUMMY: presence of 0.9988593 protein cleaner0 2023-06-30T08:43:40Z PR: TOCA1 0.99852246 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.9982327 structure_element cleaner0 2023-07-03T10:35:07Z SO: CRIB 0.99749184 protein cleaner0 2023-07-03T10:07:14Z PR: WASP 0.99687827 protein cleaner0 2023-06-30T08:42:54Z PR: Cdc42 0.9986425 structure_element cleaner0 2023-07-03T10:35:11Z SO: CRIB 0.9971403 protein cleaner0 2023-07-03T10:07:21Z PR: WASP 0.9988255 protein cleaner0 2023-06-30T08:43:40Z PR: TOCA1 0.998401 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.99724376 protein cleaner0 2023-07-03T10:07:25Z PR: WASP 0.98834026 residue_name cleaner0 2023-07-03T10:07:28Z SO: lysines 0.96353865 residue_range cleaner0 2023-07-03T10:07:31Z DUMMY: 230–232 0.9983254 structure_element cleaner0 2023-07-03T10:35:14Z SO: CRIB 0.9870566 protein_state cleaner0 2023-07-03T08:55:30Z DUMMY: presence of 0.9988324 protein cleaner0 2023-06-30T08:43:40Z PR: TOCA1 0.99829537 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 zbc0281646060007.jpg F7 FIG fig_caption 39243 The N-WASP GBD displaces the TOCA1 HR1 domain. A, the model of the Cdc42·TOCA1 HR1 domain complex overlaid with the Cdc42-WASP structure. Cdc42 is shown in green, and TOCA1 is shown in purple. The core CRIB region of WASP is shown in red, whereas its basic region is shown in orange and the C-terminal region required for maximal affinity is shown in cyan. A semitransparent surface representation of Cdc42 and WASP is shown overlaid with the schematic. B, competition SPA experiments carried out with indicated concentrations of the N-WASP GBD construct titrated into 30 nm GST-ACK or GST-WASP GBD and 30 nm Cdc42Δ7Q61L·[3H]GTP. C, Selected regions of the 15N HSQC of 145 μm Cdc42Δ7Q61L·GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD. D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP. 0.95112664 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.997769 structure_element cleaner0 2023-07-03T10:35:19Z SO: GBD 0.99819857 protein cleaner0 2023-06-30T08:43:40Z PR: TOCA1 0.99853647 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.9385584 complex_assembly cleaner0 2023-07-03T08:50:52Z GO: Cdc42·TOCA1 structure_element SO: cleaner0 2023-06-30T08:45:29Z HR1 0.97159547 complex_assembly cleaner0 2023-07-03T10:49:25Z GO: Cdc42-WASP 0.98695135 evidence cleaner0 2023-07-03T12:04:19Z DUMMY: structure 0.74240917 protein cleaner0 2023-06-30T08:42:54Z PR: Cdc42 0.99751484 protein cleaner0 2023-06-30T08:43:40Z PR: TOCA1 structure_element SO: cleaner0 2023-07-03T10:07:59Z CRIB 0.97892684 protein cleaner0 2023-07-03T10:08:06Z PR: WASP 0.70855325 protein cleaner0 2023-06-30T08:42:54Z PR: Cdc42 0.98284966 protein cleaner0 2023-07-03T10:08:28Z PR: WASP 0.9904779 experimental_method cleaner0 2023-07-03T08:39:34Z MESH: competition SPA 0.8967762 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.9722796 structure_element cleaner0 2023-07-03T10:35:24Z SO: GBD 0.9190314 experimental_method cleaner0 2023-07-03T11:53:33Z MESH: titrated mutant MESH: cleaner0 2023-07-03T08:42:00Z GST-ACK mutant MESH: cleaner0 2023-07-03T08:57:47Z GST-WASP 0.9404615 structure_element cleaner0 2023-07-03T10:35:29Z SO: GBD complex_assembly GO: cleaner0 2023-07-03T08:34:50Z Cdc42Δ7Q61L·[3H]GTP 0.9067199 experimental_method cleaner0 2023-07-03T12:04:44Z MESH: 15N HSQC complex_assembly GO: cleaner0 2023-07-03T08:48:08Z Cdc42Δ7Q61L·GMPPNP 0.9981357 protein cleaner0 2023-06-30T08:43:40Z PR: TOCA1 0.99832815 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.94294506 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.9961972 structure_element cleaner0 2023-07-03T10:35:34Z SO: GBD 0.99766076 protein cleaner0 2023-07-03T10:08:35Z PR: TOCA 0.99845433 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.94999 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.9978635 structure_element cleaner0 2023-07-03T10:35:37Z SO: GBD 0.9153466 experimental_method cleaner0 2023-07-03T12:04:29Z MESH: 15N HSQC 0.99819165 protein cleaner0 2023-06-30T08:43:40Z PR: TOCA1 0.9979571 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-07-06T15:23:26Z in complex with 0.99709153 protein cleaner0 2023-06-30T08:42:54Z PR: Cdc42 0.9785906 protein_state cleaner0 2023-07-03T13:35:00Z DUMMY: absence 0.6804149 protein_state cleaner0 2023-07-03T08:55:30Z DUMMY: presence of 0.9550614 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.99571025 structure_element cleaner0 2023-07-03T10:35:40Z SO: GBD 0.9975139 protein cleaner0 2023-06-30T08:42:54Z PR: Cdc42 0.9984224 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.96989036 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP RESULTS paragraph 40284 An N-WASP GBD construct was produced, and its affinity for Cdc42 was measured by competition SPA (Fig. 7B). The Kd that was determined (37 nm) is consistent with the previously reported affinity. Unlabeled N-WASP GBD was titrated into 15N-Cdc42Δ7Q61L·GMPPNP, and the backbone NH groups were monitored using HSQCs (Fig. 7C). Unlabeled HR1TOCA1 was then added to the Cdc42·N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1. These experiments were recorded at sufficiently high protein concentrations (145 μm Cdc42, 145 μm N-WASP GBD, 725 μm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd ≈ 5 μm, N-WASP Kd = 37 nm). A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). 0.9396019 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.66169256 structure_element cleaner0 2023-07-03T10:35:45Z SO: GBD 0.9258649 evidence cleaner0 2023-07-03T12:04:52Z DUMMY: affinity 0.99747723 protein cleaner0 2023-06-30T08:42:54Z PR: Cdc42 0.989219 experimental_method cleaner0 2023-07-03T08:39:34Z MESH: competition SPA 0.9949738 evidence cleaner0 2023-07-03T09:00:18Z DUMMY: Kd 0.95968497 evidence cleaner0 2023-07-03T12:04:58Z DUMMY: affinity 0.9111927 protein_state cleaner0 2023-07-03T09:35:21Z DUMMY: Unlabeled 0.9536869 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.8714688 structure_element cleaner0 2023-07-03T10:36:05Z SO: GBD 0.89591146 experimental_method cleaner0 2023-07-03T11:53:37Z MESH: titrated 0.8920911 chemical cleaner0 2023-07-03T13:20:28Z CHEBI: 15N complex_assembly GO: cleaner0 2023-07-03T08:48:08Z Cdc42Δ7Q61L·GMPPNP 0.9824735 experimental_method cleaner0 2023-07-03T11:53:42Z MESH: HSQCs 0.9628933 protein_state cleaner0 2023-07-03T09:35:20Z DUMMY: Unlabeled structure_element SO: cleaner0 2023-07-03T08:59:26Z HR1 protein PR: cleaner0 2023-07-03T08:59:34Z TOCA1 0.9708315 complex_assembly cleaner0 2023-07-03T10:49:31Z GO: Cdc42·N-WASP 0.95585173 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.9966275 structure_element cleaner0 2023-07-03T10:36:29Z SO: GBD protein_state DUMMY: cleaner0 2023-07-03T08:55:30Z presence of structure_element SO: cleaner0 2023-07-03T08:59:50Z HR1 protein PR: cleaner0 2023-07-03T08:59:57Z TOCA1 0.9978908 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.94267553 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.46139213 structure_element cleaner0 2023-07-03T10:36:16Z SO: GBD 0.99755055 protein cleaner0 2023-06-30T08:43:40Z PR: TOCA1 0.9964689 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 evidence DUMMY: cleaner0 2023-07-03T09:00:11Z Kd 0.99711514 protein cleaner0 2023-06-30T08:43:40Z PR: TOCA1 evidence DUMMY: cleaner0 2023-07-03T09:00:18Z Kd 0.96482223 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP evidence DUMMY: cleaner0 2023-07-03T09:00:18Z Kd 0.97246563 experimental_method cleaner0 2023-07-03T11:53:47Z MESH: HSQC 0.96047896 chemical cleaner0 2023-07-03T13:20:32Z CHEBI: 15N 0.9969433 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.97844476 protein_state cleaner0 2023-07-03T13:35:08Z DUMMY: alone protein_state DUMMY: cleaner0 2023-07-03T08:55:30Z presence of 0.99561125 protein cleaner0 2023-06-30T08:43:40Z PR: TOCA1 0.98355776 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.9025988 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.94289047 structure_element cleaner0 2023-07-03T10:36:25Z SO: GBD 0.97607386 evidence cleaner0 2023-07-03T12:05:01Z DUMMY: spectra 0.77785087 protein_state cleaner0 2023-07-03T08:55:30Z DUMMY: presence of 0.9605486 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP protein_state DUMMY: cleaner0 2023-07-03T08:55:30Z presence of 0.95731276 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.9966145 protein cleaner0 2023-06-30T08:43:40Z PR: TOCA1 0.98916936 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 RESULTS paragraph 41289 Furthermore, 15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42Δ7Q61L·GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD. The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). When in fast exchange, the NMR signal represents a population-weighted average between free and bound states, so the intermediate spectrum indicates that the population comprises a mixture of free and bound HR1 domain. Hence, a third, intermediate state that includes all three proteins is unlikely. Again, the experiments were recorded on protein samples far in excess of the individual Kd values (600 μm each protein). These data indicate that the HR1 domain is displaced from Cdc42 by N-WASP and that a ternary complex comprising TOCA1 HR1, N-WASP GBD, and Cdc42 is not formed. Taken together, the data in Fig. 7, C and D, indicate unidirectional competition for Cdc42 binding in which the N-WASP GBD displaces TOCA1 HR1 but not vice versa. 0.9332237 chemical cleaner0 2023-07-03T13:23:00Z CHEBI: 15N 0.9982925 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.9974394 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 protein_state DUMMY: cleaner0 2023-07-03T08:55:30Z presence of 0.95294714 protein_state cleaner0 2023-07-03T09:35:21Z DUMMY: unlabeled complex_assembly GO: cleaner0 2023-07-03T08:48:08Z Cdc42Δ7Q61L·GMPPNP 0.8684736 protein_state cleaner0 2023-07-03T09:35:21Z DUMMY: unlabeled 0.97671705 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.9954027 structure_element cleaner0 2023-07-03T10:36:33Z SO: GBD 0.9841208 evidence cleaner0 2023-07-03T12:05:17Z DUMMY: spectrum 0.9683415 protein cleaner0 2023-06-30T08:44:51Z PR: N-WASP 0.99813795 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.99704844 protein_state cleaner0 2023-07-03T13:35:25Z DUMMY: free 0.9981317 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.9801952 evidence cleaner0 2023-07-03T12:05:19Z DUMMY: spectrum protein_state DUMMY: cleaner0 2023-07-03T08:55:30Z presence of 0.9631322 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.99390113 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.84866285 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.99701947 protein_state cleaner0 2023-07-03T13:35:32Z DUMMY: free 0.9362432 protein_state cleaner0 2023-07-03T13:35:35Z DUMMY: complex 0.8787579 evidence cleaner0 2023-07-03T12:05:22Z DUMMY: spectra 0.94687283 experimental_method cleaner0 2023-07-03T11:53:49Z MESH: NMR 0.99748236 protein_state cleaner0 2023-07-03T13:35:46Z DUMMY: free 0.98963296 protein_state cleaner0 2023-07-03T13:35:49Z DUMMY: bound 0.8823185 evidence cleaner0 2023-07-03T12:05:25Z DUMMY: spectrum 0.9976744 protein_state cleaner0 2023-07-03T13:35:53Z DUMMY: free 0.99598557 protein_state cleaner0 2023-07-03T13:35:56Z DUMMY: bound 0.9983581 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.71245635 evidence cleaner0 2023-07-03T09:00:18Z DUMMY: Kd 0.9983242 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.99732745 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.9735255 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.99852884 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.997738 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.971786 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.9977214 structure_element cleaner0 2023-07-03T10:36:55Z SO: GBD 0.9973015 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.9388516 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.9646563 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.9982608 structure_element cleaner0 2023-07-03T10:36:57Z SO: GBD 0.9985617 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.99745125 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 RESULTS paragraph 42436 To extend these studies to a more complex system and to assess the ability of TOCA1 HR1 to compete with full-length N-WASP, pyrene actin assays were employed. These assays, described in detail elsewhere, were carried out using pyrene actin-supplemented Xenopus extracts into which exogenous TOCA1 HR1 domain or N-WASP GBD was added, to assess their effects on actin polymerization. Actin polymerization in all cases was initiated by the addition of PI(4,5)P2-containing liposomes. Actin polymerization triggered by the addition of PI(4,5)P2-containing liposomes has previously been shown to depend on TOCA1 and N-WASP. Endogenous N-WASP is present at ∼100 nm in Xenopus extracts, whereas TOCA1 is present at a 10-fold lower concentration than N-WASP. 0.9985815 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.9963374 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.9972822 protein_state cleaner0 2023-07-03T08:36:11Z DUMMY: full-length 0.98861784 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.9900012 experimental_method cleaner0 2023-07-03T11:53:55Z MESH: pyrene actin assays 0.7194145 chemical cleaner0 2023-07-03T13:22:25Z CHEBI: pyrene actin 0.71539295 taxonomy_domain cleaner0 2023-07-03T09:39:16Z DUMMY: Xenopus 0.99879575 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.9967769 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.9877314 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.9883178 structure_element cleaner0 2023-07-03T10:37:03Z SO: GBD protein_type MESH: cleaner0 2023-07-03T13:23:19Z actin protein_type MESH: cleaner0 2023-07-03T13:23:10Z Actin 0.99500054 chemical cleaner0 2023-07-03T09:41:52Z CHEBI: PI(4,5)P2 protein_type MESH: cleaner0 2023-07-03T13:21:12Z Actin 0.9945503 chemical cleaner0 2023-07-03T09:41:52Z CHEBI: PI(4,5)P2 0.99870133 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.9839406 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.9804876 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.51483136 taxonomy_domain cleaner0 2023-07-03T09:39:16Z DUMMY: Xenopus 0.9981664 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.9842655 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP RESULTS paragraph 43189 The addition of the isolated N-WASP GBD significantly inhibited the polymerization of actin at concentrations as low as 100 nm and completely abolished polymerization at higher concentrations (Fig. 8). The GBD presumably acts as a dominant negative, sequestering endogenous Cdc42 and preventing endogenous full-length N-WASP from binding and becoming activated. The addition of the TOCA1 HR1 domain to 100 μm had no significant effect on the rate of actin polymerization or maximum fluorescence. This is consistent with endogenous N-WASP, activated by other components of the assay, outcompeting the TOCA1 HR1 domain for Cdc42 binding. experimental_method MESH: cleaner0 2023-07-03T11:55:01Z addition 0.99054986 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.99848133 structure_element cleaner0 2023-07-03T10:37:08Z SO: GBD 0.97212505 protein_type cleaner0 2023-07-03T13:22:34Z MESH: actin 0.9984763 structure_element cleaner0 2023-07-03T10:37:11Z SO: GBD 0.99606305 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.5580302 protein_state cleaner0 2023-07-03T13:36:05Z DUMMY: endogenous 0.99722224 protein_state cleaner0 2023-07-03T08:36:11Z DUMMY: full-length 0.9901004 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.90093863 experimental_method cleaner0 2023-07-03T11:55:06Z MESH: addition 0.99855965 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.99839646 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 protein_type MESH: cleaner0 2023-07-03T13:22:44Z actin 0.93018484 evidence cleaner0 2023-07-03T12:05:31Z DUMMY: maximum fluorescence 0.9937324 protein_state cleaner0 2023-07-03T13:36:15Z DUMMY: endogenous 0.98900014 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.9986438 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.998454 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.99387544 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 zbc0281646060008.jpg F8 FIG fig_caption 43826 Actin polymerization downstream of Cdc42·N-WASP·TOCA1 is inhibited by excess N-WASP GBD but not by the TOCA1 HR1 domain. Fluorescence curves show actin polymerization in the presence of increasing concentrations of N-WASP GBD or TOCA1 HR1 domain as indicated. Maximal rates of actin polymerization derived from the linear region of the curves are represented in bar charts below. Error bars, S.E. complex_assembly GO: cleaner0 2023-07-03T09:01:02Z Cdc42·N-WASP·TOCA1 0.5454533 protein_state cleaner0 2023-07-03T13:36:33Z DUMMY: inhibited 0.9489117 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.9776324 structure_element cleaner0 2023-07-03T10:37:17Z SO: GBD 0.9981376 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.9981976 structure_element cleaner0 2023-06-30T08:45:29Z SO: HR1 0.9425819 evidence cleaner0 2023-07-03T12:05:34Z DUMMY: Fluorescence curves protein_state DUMMY: cleaner0 2023-07-03T08:55:30Z presence of 0.7197472 experimental_method cleaner0 2023-07-03T11:55:10Z MESH: increasing concentrations 0.9465313 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.9019326 structure_element cleaner0 2023-07-03T10:37:21Z SO: GBD 0.9979225 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.9975677 structure_element cleaner0 2023-06-30T08:45:30Z SO: HR1 DISCUSS title_1 44225 Discussion DISCUSS title_4 44236 The Cdc42-TOCA1 Interaction protein PR: cleaner0 2023-07-03T13:36:53Z Cdc42 protein PR: cleaner0 2023-07-03T13:36:59Z TOCA1 DISCUSS paragraph 44264 The TOCA1 HR1 domain alone is sufficient for Cdc42 binding in vitro, yet the affinity of the TOCA1 HR1 domain for Cdc42 is remarkably low (Kd ≈ 5 μm). This is over 100 times lower than that of the N-WASP GBD (Kd = 37 nm) and considerably lower than other known G protein-HR1 domain interactions. The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction. A single binding interface on both the HR1 domain and Cdc42 can be concluded from the data presented here. Furthermore, the interfaces are comparable with those of other G protein-HR1 interactions (Fig. 4), and the lowest energy model produced in rigid body docking resembles previously studied G protein·HR1 complexes (Fig. 6). It seems, therefore, that the interaction, despite its relatively low affinity, is specific and sterically similar to other HR1 domain-G protein interactions. 0.99874794 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 0.9605068 protein_state cleaner0 2023-07-03T13:37:03Z DUMMY: alone 0.9922667 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.98681575 evidence cleaner0 2023-07-03T12:05:40Z DUMMY: affinity 0.9987087 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 0.996276 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.9067749 evidence cleaner0 2023-07-03T09:00:18Z DUMMY: Kd 0.95897865 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.9980869 structure_element cleaner0 2023-07-03T10:37:27Z SO: GBD evidence DUMMY: cleaner0 2023-07-03T09:00:18Z Kd protein_type MESH: cleaner0 2023-07-03T08:23:08Z G protein 0.41392598 structure_element cleaner0 2023-06-30T08:45:30Z SO: HR1 0.91059816 structure_element cleaner0 2023-07-03T10:37:40Z SO: C-terminal region 0.6926602 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.99878424 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.9494978 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.99840575 structure_element cleaner0 2023-07-03T10:37:44Z SO: HR1b 0.9986798 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 0.9982907 protein cleaner0 2023-07-03T09:35:52Z PR: PRK1 0.98214185 structure_element cleaner0 2023-07-03T10:37:47Z SO: HR1a protein PR: cleaner0 2023-06-30T08:46:57Z RhoA 0.9923845 site cleaner0 2023-07-03T12:08:10Z SO: binding interface 0.9986285 structure_element cleaner0 2023-06-30T08:45:30Z SO: HR1 0.9925438 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.96070576 site cleaner0 2023-07-03T12:08:13Z SO: interfaces protein_type MESH: cleaner0 2023-07-03T08:23:08Z G protein structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 evidence DUMMY: cleaner0 2023-07-03T12:05:58Z model 0.9632737 experimental_method cleaner0 2023-07-03T11:55:15Z MESH: rigid body docking complex_assembly GO: cleaner0 2023-07-03T09:02:19Z G protein·HR1 0.62017566 structure_element cleaner0 2023-06-30T08:45:30Z SO: HR1 protein_type MESH: cleaner0 2023-07-03T08:23:08Z G protein DISCUSS paragraph 45296 The TOCA1 HR1 domain is a left-handed coiled-coil comparable with other known HR1 domains. A short region N-terminal to the coiled-coil exhibits a series of turns and contacts residues of both helices of the coiled-coil (Fig. 3). The corresponding sequence in CIP4 also includes a series of turns but is flexible, whereas in the HR1a domain of PRK1, the equivalent region adopts an α-helical structure that packs against the coiled-coil. The contacts between the N-terminal region and the coiled-coil are predominantly hydrophobic in both cases, but sequence-specific contacts do not appear to be conserved. This region is distant from the G protein-binding interface of the HR1 domains, so the structural differences may relate to the structure and regulation of these domains rather than their G protein interactions. 0.9981933 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.9984641 structure_element cleaner0 2023-06-30T08:45:30Z SO: HR1 0.99416256 structure_element cleaner0 2023-07-03T10:37:52Z SO: coiled-coil structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 0.99510807 structure_element cleaner0 2023-07-03T10:37:57Z SO: coiled-coil 0.9957609 structure_element cleaner0 2023-07-03T10:37:59Z SO: coiled-coil 0.99836725 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 0.99846256 structure_element cleaner0 2023-07-03T10:38:02Z SO: HR1a 0.99815816 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 0.8583672 structure_element cleaner0 2023-07-03T10:38:04Z SO: α-helical structure 0.99529916 structure_element cleaner0 2023-07-03T10:38:06Z SO: coiled-coil bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:06Z contacts 0.992884 structure_element cleaner0 2023-07-03T10:38:09Z SO: coiled-coil bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:06Z hydrophobic 0.9960044 site cleaner0 2023-07-03T12:08:18Z SO: G protein-binding interface structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 protein_type MESH: cleaner0 2023-07-03T08:23:08Z G protein DISCUSS paragraph 46120 The interhelical loops of TOCA1 and CIP4 differ from the same region in the HR1 domains of PRK1 in that they are longer and contain two short stretches of 310-helix. This region lies within the G protein-binding surface of all of the HR1 domains (Fig. 4D). TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1. The structural features shared by TOCA1 and CIP4 may therefore be related to Cdc42 binding specificity and the low affinities. In free TOCA1, the side chains of the interhelical region make extensive contacts with residues in helix 1. Many of these residues are significantly affected in the presence of Cdc42, so it is likely that the conformation of this loop is altered in the Cdc42 complex. These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42. 0.99714184 structure_element cleaner0 2023-07-03T10:38:13Z SO: interhelical loops 0.9987884 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.99845755 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 0.9985311 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 0.9948606 structure_element cleaner0 2023-07-03T10:38:17Z SO: 310-helix 0.99405587 site cleaner0 2023-07-03T12:08:21Z SO: G protein-binding surface structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 0.9987643 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.99849117 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 0.99687356 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.9984357 structure_element cleaner0 2023-07-03T10:38:21Z SO: HR1a 0.9983943 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 0.9965754 protein cleaner0 2023-06-30T08:46:57Z PR: RhoA 0.99643517 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.99843675 structure_element cleaner0 2023-07-03T10:38:25Z SO: HR1b 0.9964896 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.9988292 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.9986552 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 protein PR: cleaner0 2023-06-30T08:42:55Z Cdc42 0.9972121 protein_state cleaner0 2023-07-03T13:37:08Z DUMMY: free 0.99870944 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.9975205 structure_element cleaner0 2023-07-03T10:38:29Z SO: interhelical region 0.996721 structure_element cleaner0 2023-07-03T10:38:32Z SO: helix 1 0.99045527 protein_state cleaner0 2023-07-03T08:55:30Z DUMMY: presence of 0.9958423 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.6028027 structure_element cleaner0 2023-07-03T10:38:34Z SO: loop 0.9048001 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.9841298 experimental_method cleaner0 2023-07-03T11:55:20Z MESH: mutation 0.9799066 residue_name_number cleaner0 2023-07-03T10:53:22Z DUMMY: Met383 0.87663805 residue_name_number cleaner0 2023-07-03T10:53:24Z DUMMY: Gly384 0.95014256 residue_name_number cleaner0 2023-07-03T10:53:27Z DUMMY: Asp385 0.98716545 residue_name_number cleaner0 2023-07-03T10:53:32Z DUMMY: Ile383 0.96805173 residue_name_number cleaner0 2023-07-03T10:53:34Z DUMMY: Ser384 0.9810523 residue_name_number cleaner0 2023-07-03T10:53:36Z DUMMY: Thr385 0.9977755 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.9953963 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 DISCUSS paragraph 47071 The lowest energy model produced by HADDOCK using ambiguous interaction restraints from the titration data resembled the NMR structures of RhoA and Rac1 in complex with their HR1 domain partners. Some speculative conclusions can be made based on this model. For example, Phe-56Cdc42, which is not visible in free Cdc42 or Cdc42·HR1TOCA1, is close to the TOCA1 HR1 (Fig. 6A). Phe-56Cdc42, which is a Trp in both Rac1 and RhoA (Fig. 6C), is thought to pack behind switch I when Cdc42 interacts with ACK, maintaining the switch in a binding-competent orientation. This residue has also been identified as important for Cdc42-WASP binding. Phe-56Cdc42 is therefore likely to be involved in the Cdc42-TOCA1 interaction, probably by stabilizing the position of switch I. evidence DUMMY: cleaner0 2023-07-03T12:06:21Z model 0.9877753 experimental_method cleaner0 2023-07-03T08:50:26Z MESH: HADDOCK 0.34142372 evidence cleaner0 2023-07-03T12:06:26Z DUMMY: titration 0.99043894 experimental_method cleaner0 2023-07-03T11:55:24Z MESH: NMR 0.9946361 evidence cleaner0 2023-07-03T12:06:29Z DUMMY: structures 0.7614925 protein cleaner0 2023-06-30T08:46:57Z PR: RhoA 0.8085813 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.9361132 protein_state cleaner0 2023-07-03T13:37:13Z DUMMY: in complex with structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 residue_name_number DUMMY: cleaner0 2023-07-03T09:03:45Z Phe-56 protein PR: cleaner0 2023-07-03T09:03:54Z Cdc42 0.9960108 protein_state cleaner0 2023-07-03T13:37:16Z DUMMY: free 0.5524952 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 complex_assembly GO: cleaner0 2023-07-03T09:04:18Z Cdc42·HR1TOCA1 0.99825543 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.99869174 structure_element cleaner0 2023-06-30T08:45:30Z SO: HR1 residue_name_number DUMMY: cleaner0 2023-07-03T09:36:28Z Phe-56 protein PR: cleaner0 2023-07-03T09:36:37Z Cdc42 0.9886572 residue_name cleaner0 2023-07-03T10:52:09Z SO: Trp 0.6558546 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.692283 protein cleaner0 2023-06-30T08:46:57Z PR: RhoA 0.9735813 site cleaner0 2023-07-03T09:34:36Z SO: switch I 0.9794703 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.98919964 protein cleaner0 2023-07-03T09:29:56Z PR: ACK protein PR: cleaner0 2023-06-30T08:42:55Z Cdc42 protein PR: cleaner0 2023-07-03T09:05:04Z WASP residue_name_number DUMMY: cleaner0 2023-07-03T09:04:42Z Phe-56 protein PR: cleaner0 2023-07-03T09:04:49Z Cdc42 0.53643703 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.5590447 protein cleaner0 2023-06-30T08:43:41Z PR: TOCA1 0.9758419 site cleaner0 2023-07-03T09:34:36Z SO: switch I DISCUSS paragraph 47837 Some residues that are affected in the Cdc42·HR1TOCA1 complex but do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) may contact HR1TOCA1 directly (Fig. 6D). Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil. Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1. N52T is one of a combination of seven residues found to confer ACK binding on Rac1 and so may represent a specific Cdc42-effector contact residue. The position equivalent to Lys-372TOCA1 in PRK1 is Glu-58HR1a or Gln-151HR1b. Thr-52Cdc42-Lys-372TOCA1 may therefore represent a specific Cdc42-HR1TOCA1 contact. Arg-68Cdc42 of switch II is positioned close to Glu-395TOCA1 (Fig. 6D), suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain. The equivalent Arg in Rac1 and RhoA is pointing away from the HR1 domains of PRK1. The importance of this residue in the Cdc42-TOCA1 interaction remains unclear, although its mutation reduces binding to RhoGAP, suggesting that it can be involved in Cdc42 interactions. 0.99348646 complex_assembly cleaner0 2023-07-03T10:49:42Z GO: Cdc42·HR1TOCA1 0.9682967 protein cleaner0 2023-06-30T08:46:58Z PR: RhoA 0.9662049 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 structure_element SO: cleaner0 2023-07-03T09:28:38Z HR1 protein PR: cleaner0 2023-07-03T09:28:46Z TOCA1 residue_name_number DUMMY: cleaner0 2023-07-03T10:53:56Z Gln-2 protein PR: cleaner0 2023-07-03T10:54:06Z Cdc42 0.9904862 complex_assembly cleaner0 2023-07-03T10:49:46Z GO: Cdc42·ACK residue_name_number DUMMY: cleaner0 2023-07-03T09:20:31Z Val-376 protein PR: cleaner0 2023-07-03T09:20:40Z TOCA1 residue_name_number DUMMY: cleaner0 2023-07-03T09:20:57Z Asn-380 protein PR: cleaner0 2023-07-03T09:21:05Z TOCA1 0.99275684 structure_element cleaner0 2023-07-03T10:39:34Z SO: interhelical loop 0.98493385 structure_element cleaner0 2023-07-03T10:39:37Z SO: first helix 0.99854326 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.994997 structure_element cleaner0 2023-07-03T10:39:39Z SO: coiled-coil residue_name_number DUMMY: cleaner0 2023-07-03T09:21:25Z Thr-52 protein PR: cleaner0 2023-07-03T09:21:34Z Cdc42 protein PR: cleaner0 2023-07-03T09:29:58Z ACK structure_element SO: cleaner0 2023-07-03T09:29:01Z HR1 protein PR: cleaner0 2023-07-03T09:29:11Z TOCA1 0.9896097 structure_element cleaner0 2023-07-03T10:39:45Z SO: helix 1 residue_name_number DUMMY: cleaner0 2023-07-03T09:21:51Z Lys-372 protein PR: cleaner0 2023-07-03T09:21:58Z TOCA1 0.9880695 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 residue_name_number DUMMY: cleaner0 2023-07-03T09:22:18Z Asn-52 protein PR: cleaner0 2023-07-03T09:22:29Z Rac1 0.9608026 mutant cleaner0 2023-07-03T10:51:46Z MESH: N52T protein PR: cleaner0 2023-07-03T09:29:58Z ACK 0.9936155 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 protein PR: cleaner0 2023-06-30T08:42:55Z Cdc42 residue_name_number DUMMY: cleaner0 2023-07-03T09:22:49Z Lys-372 protein PR: cleaner0 2023-07-03T09:22:58Z TOCA1 0.99796826 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 residue_name_number DUMMY: cleaner0 2023-07-03T09:23:18Z Glu-58 structure_element SO: cleaner0 2023-07-03T09:23:30Z HR1a residue_name_number DUMMY: cleaner0 2023-07-03T09:23:51Z Gln-151 structure_element SO: cleaner0 2023-07-03T09:24:02Z HR1b residue_name_number DUMMY: cleaner0 2023-07-03T09:24:18Z Thr-52 protein PR: cleaner0 2023-07-03T09:24:32Z Cdc42 residue_name_number DUMMY: cleaner0 2023-07-03T09:24:48Z Lys-372 protein PR: cleaner0 2023-07-03T09:24:55Z TOCA1 0.8249155 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 structure_element SO: cleaner0 2023-07-03T09:29:27Z HR1 protein PR: cleaner0 2023-07-03T09:29:37Z TOCA1 residue_name_number DUMMY: cleaner0 2023-07-03T09:25:15Z Arg-68 protein PR: cleaner0 2023-07-03T09:25:24Z Cdc42 0.98761934 site cleaner0 2023-07-03T09:26:14Z SO: switch II residue_name_number DUMMY: cleaner0 2023-07-03T09:25:43Z Glu-395 protein PR: cleaner0 2023-07-03T09:25:50Z TOCA1 0.9952789 site cleaner0 2023-07-03T09:26:23Z SO: switch II 0.9441022 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 0.9952605 structure_element cleaner0 2023-07-03T10:39:51Z SO: helix 2 structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 0.9854915 residue_name cleaner0 2023-07-03T10:16:56Z SO: Arg 0.98867166 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.9793021 protein cleaner0 2023-06-30T08:46:58Z PR: RhoA structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 0.99827313 protein cleaner0 2023-07-03T09:35:53Z PR: PRK1 protein PR: cleaner0 2023-07-03T09:27:10Z Cdc42 protein PR: cleaner0 2023-07-03T09:27:19Z TOCA1 0.5818127 experimental_method cleaner0 2023-07-03T11:55:42Z MESH: mutation 0.45419523 protein cleaner0 2023-07-03T10:16:46Z PR: RhoGAP protein PR: cleaner0 2023-06-30T08:42:55Z Cdc42 DISCUSS paragraph 49213 The solution structure of the TOCA1 HR1 domain presented here, along with the model of the HR1TOCA1·Cdc42 complex is consistent with a conserved mode of binding across the known HR1 domain-Rho family interactions, despite their differing affinities. The weak binding prevented detailed structural and thermodynamic studies of the complex. Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding. 0.8974091 evidence cleaner0 2023-07-03T12:06:34Z DUMMY: solution structure 0.99868196 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.99838424 structure_element cleaner0 2023-06-30T08:45:30Z SO: HR1 complex_assembly GO: cleaner0 2023-07-03T09:27:40Z HR1TOCA1·Cdc42 0.9820432 structure_element cleaner0 2023-06-30T08:45:30Z SO: HR1 0.92416924 experimental_method cleaner0 2023-07-03T11:55:47Z MESH: structural and thermodynamic studies 0.97563076 experimental_method cleaner0 2023-07-03T11:55:50Z MESH: structural studies 0.9985752 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.99842036 structure_element cleaner0 2023-06-30T08:45:30Z SO: HR1 0.97121984 experimental_method cleaner0 2023-07-03T11:55:52Z MESH: chemical shift mapping 0.9524198 protein cleaner0 2023-06-30T08:42:55Z PR: Cdc42 structure_element SO: cleaner0 2023-07-03T10:09:15Z HR1 protein PR: cleaner0 2023-07-03T10:09:21Z TOCA1 0.9924744 protein cleaner0 2023-06-30T08:46:58Z PR: RhoA 0.98837924 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 structure_element SO: cleaner0 2023-07-03T10:08:54Z HR1 protein PR: cleaner0 2023-07-03T10:09:01Z PRK1 DISCUSS paragraph 49760 We have previously postulated that the inherent flexibility of HR1 domains contributes to their ability to bind to different Rho family G proteins, with Rho-binding HR1 domains displaying increased flexibility, reflected in their lower melting temperatures (Tm) and Rac binders being more rigid. The Tm of the TOCA1 HR1 domain is 61.9 °C (data not shown), which is the highest Tm that we have measured for an HR1 domain thus far. As such, the ability of the TOCA1 HR1 domain to bind to Cdc42 (a close relative of Rac1 rather than RhoA) fits this trend. An investigation into the local motions, particularly in the G protein-binding regions, may offer further insight into the differential specificities and affinities of G protein-HR1 domain interactions. structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 0.8696185 protein_type cleaner0 2023-07-03T10:17:13Z MESH: Rho family G proteins structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 0.9898145 evidence cleaner0 2023-07-03T12:06:39Z DUMMY: melting temperatures 0.9740339 evidence cleaner0 2023-07-03T12:06:42Z DUMMY: Tm protein_type MESH: cleaner0 2023-07-03T13:37:43Z Rac 0.9928907 evidence cleaner0 2023-07-03T12:06:45Z DUMMY: Tm 0.9961492 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 0.98497283 evidence cleaner0 2023-07-03T12:06:47Z DUMMY: Tm 0.9982868 structure_element cleaner0 2023-06-30T08:45:30Z SO: HR1 0.99699247 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 0.6991982 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.9253086 protein cleaner0 2023-06-30T08:47:07Z PR: Rac1 0.8978462 protein cleaner0 2023-06-30T08:46:58Z PR: RhoA 0.9958286 site cleaner0 2023-07-03T08:16:05Z SO: G protein-binding regions protein_type MESH: cleaner0 2023-07-03T08:23:08Z G protein structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 DISCUSS title_4 50517 Significance of a Weak, Transient Interaction DISCUSS paragraph 50563 The low affinity of the Cdc42-HR1TOCA1 interaction is consistent with a tightly spatially and temporally regulated pathway, requiring combinatorial signals leading to a series of coincident weak interactions that elicit full activation. The HR1 domains from other TOCA family members, CIP4 and FBP17, also bind at low micromolar affinities to Cdc42, so the low affinity interaction appears to be commonplace among this family of HR1 domain proteins, in contrast to the PRK family. Weak, transient protein-protein interactions are functionally significant in several systems; for example, the binding of adaptor proteins to protein cargo during the formation of clathrin-coated vesicles in endocytosis involves multiple interactions of micromolar affinity. protein PR: cleaner0 2023-06-30T08:42:56Z Cdc42 structure_element SO: cleaner0 2023-07-03T09:38:29Z HR1 protein PR: cleaner0 2023-07-03T09:38:37Z TOCA1 structure_element SO: cleaner0 2023-06-30T08:45:30Z HR1 0.90049523 protein_type cleaner0 2023-07-03T09:38:55Z MESH: TOCA family members 0.9987978 protein cleaner0 2023-07-03T09:39:04Z PR: CIP4 0.9988563 protein cleaner0 2023-07-03T09:39:10Z PR: FBP17 0.61347103 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.9585526 protein_type cleaner0 2023-07-03T09:38:57Z MESH: HR1 domain proteins 0.84394807 protein_type cleaner0 2023-07-03T09:39:00Z MESH: PRK family DISCUSS paragraph 51319 The low affinity of the HR1TOCA1-Cdc42 interaction in the context of the physiological concentration of TOCA1 in Xenopus extracts (∼10 nm) suggests that binding between TOCA1 and Cdc42 is likely to occur in vivo only when TOCA1 is at high local concentrations and membrane-localized and therefore in close proximity to activated Cdc42. Evidence suggests that the TOCA family of proteins are recruited to the membrane via an interaction between their F-BAR domain and specific signaling lipids. For example, electrostatic interactions between the F-BAR domain and the membrane are required for TOCA1 recruitment to membrane vesicles and tubules, and TOCA1-dependent actin polymerization is known to depend specifically on PI(4,5)P2. Furthermore, the isolated F-BAR domain of FBP17 has been shown to induce membrane tubulation of brain liposomes and BAR domain proteins that promote tubulation cluster on membranes at high densities. Once at the membrane, high local concentrations of TOCA1 could exceed the Kd of F-BAR dimerization (likely to be comparable with that of the FCHo2 F-BAR domain (2.5 μm)) and that of the Cdc42-HR1TOCA1 interaction. Cdc42-HR1TOCA1 binding would then be favorable, as long as coincident activation of Cdc42 had occurred, leading to stabilization of TOCA1 at the membrane and downstream activation of N-WASP. 0.4805834 structure_element cleaner0 2023-07-03T09:19:55Z SO: HR1TOCA1 protein PR: cleaner0 2023-06-30T08:42:56Z Cdc42 0.99852484 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.8006676 taxonomy_domain cleaner0 2023-07-03T09:39:15Z DUMMY: Xenopus 0.99829346 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.9548792 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.9982255 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.99125063 protein_state cleaner0 2023-07-03T13:38:08Z DUMMY: activated 0.986712 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.7707484 protein_type cleaner0 2023-07-03T10:17:19Z MESH: TOCA family structure_element SO: cleaner0 2023-07-03T09:40:40Z F-BAR bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:06Z electrostatic interactions structure_element SO: cleaner0 2023-07-03T09:40:41Z F-BAR 0.99763405 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.48940825 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.9973473 chemical cleaner0 2023-07-03T09:41:52Z CHEBI: PI(4,5)P2 0.48511603 experimental_method cleaner0 2023-07-03T11:55:58Z MESH: isolated structure_element SO: cleaner0 2023-07-03T09:40:41Z F-BAR 0.99882823 protein cleaner0 2023-07-03T09:39:11Z PR: FBP17 structure_element SO: cleaner0 2023-07-03T09:41:27Z BAR 0.99782467 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.8462519 evidence cleaner0 2023-07-03T09:00:18Z DUMMY: Kd structure_element SO: cleaner0 2023-07-03T09:40:41Z F-BAR oligomeric_state DUMMY: cleaner0 2023-07-03T10:10:53Z dimer 0.9982218 protein cleaner0 2023-07-03T10:09:37Z PR: FCHo2 structure_element SO: cleaner0 2023-07-03T09:40:41Z F-BAR protein PR: cleaner0 2023-06-30T08:42:56Z Cdc42 structure_element SO: cleaner0 2023-07-03T09:39:45Z HR1 protein PR: cleaner0 2023-07-03T09:39:53Z TOCA1 protein PR: cleaner0 2023-06-30T08:42:56Z Cdc42 structure_element SO: cleaner0 2023-07-03T09:40:10Z HR1 protein PR: cleaner0 2023-07-03T09:40:18Z TOCA1 0.97662354 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.99832636 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.9216368 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP DISCUSS paragraph 52659 It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced. The unfolded, high affinity state of WASP is represented by a short peptide, the GBD, which binds with a low nanomolar affinity to Cdc42. In contrast, the best estimate of the affinity of full-length WASP for Cdc42 is low micromolar. In the inactive state of WASP, the actin- and Arp2/3-binding VCA domain contacts the GBD, competing for Cdc42 binding. The high affinity of Cdc42 for the unfolded, active form pushes the equilibrium in favor of (N-)WASP activation. Binding of PI(4,5)P2 to the basic region just N-terminal to the GBD further favors the active conformation. A substantial body of data has illuminated the complex regulation of WASP/N-WASP proteins, and current evidence suggests that these allosteric activation mechanisms and oligomerization combine to regulate WASP activity, allowing the synchronization and integration of multiple potential activation signals (reviewed in Ref.). Our data are easily reconciled with this model. 0.9956033 protein_type cleaner0 2023-07-03T10:18:34Z MESH: WASP 0.9431486 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.9977189 protein_state cleaner0 2023-07-03T09:43:40Z DUMMY: folded 0.99728596 protein_state cleaner0 2023-07-03T13:38:13Z DUMMY: inactive 0.9969168 protein_state cleaner0 2023-07-03T09:43:31Z DUMMY: unfolded 0.9972771 protein_state cleaner0 2023-07-03T13:38:17Z DUMMY: active 0.96331924 evidence cleaner0 2023-07-03T12:06:55Z DUMMY: affinity 0.997189 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.99731463 protein_state cleaner0 2023-07-03T09:43:31Z DUMMY: unfolded 0.8940232 protein_type cleaner0 2023-07-03T10:17:30Z MESH: WASP 0.9970182 protein_state cleaner0 2023-07-03T09:43:31Z DUMMY: unfolded 0.9939663 protein_type cleaner0 2023-07-03T10:18:44Z MESH: WASP 0.40008605 chemical cleaner0 2023-07-03T13:25:51Z CHEBI: peptide 0.9983557 structure_element cleaner0 2023-07-03T10:40:03Z SO: GBD 0.99671304 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.96908647 evidence cleaner0 2023-07-03T12:06:58Z DUMMY: affinity 0.99755406 protein_state cleaner0 2023-07-03T08:36:11Z DUMMY: full-length 0.99496436 protein_type cleaner0 2023-07-03T10:19:01Z MESH: WASP 0.99721116 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.9978344 protein_state cleaner0 2023-07-03T13:38:21Z DUMMY: inactive 0.9945903 protein_type cleaner0 2023-07-03T10:18:53Z MESH: WASP complex_assembly GO: cleaner0 2023-07-03T10:10:09Z Arp2/3 structure_element SO: cleaner0 2023-07-03T10:40:19Z VCA 0.99843746 structure_element cleaner0 2023-07-03T10:40:22Z SO: GBD 0.995122 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.9971827 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.9971264 protein_state cleaner0 2023-07-03T09:43:31Z DUMMY: unfolded 0.99629635 protein_state cleaner0 2023-07-03T13:38:25Z DUMMY: active protein PR: cleaner0 2023-07-03T09:42:29Z (N-)WASP 0.99715185 chemical cleaner0 2023-07-03T09:41:51Z CHEBI: PI(4,5)P2 0.9984573 structure_element cleaner0 2023-07-03T10:40:24Z SO: GBD 0.99588937 protein_state cleaner0 2023-07-03T13:38:28Z DUMMY: active 0.9502268 protein_type cleaner0 2023-07-03T09:42:01Z MESH: WASP/N-WASP proteins protein_type MESH: cleaner0 2023-07-03T10:19:10Z WASP DISCUSS paragraph 53811 We envisage that TOCA1 is first recruited to the appropriate membrane in response to PI(4,5)P2 via its F-BAR domain, where the local increase in concentration favors F-BAR-mediated dimerization of TOCA1. Cdc42 is activated in response to co-incident signals and can then bind to TOCA1, further stabilizing TOCA1 at the membrane. TOCA1 can then recruit N-WASP via an interaction between its SH3 domain and the N-WASP proline-rich region. The recruitment of N-WASP alone and of the N-WASP·WIP complex by TOCA1 and FBP17 has been demonstrated. WIP inhibits the activation of N-WASP by Cdc42, an effect that is reversed by TOCA1. It may therefore be envisaged that WIP and TOCA1 exert opposing allosteric effects on N-WASP, with TOCA1 favoring the unfolded, active conformation of N-WASP and increasing its affinity for Cdc42. TOCA1 may also activate N-WASP by effective oligomerization because clustering of TOCA1 at the membrane following coincident interactions with PI(4,5)P2 and Cdc42 would in turn lead to clustering of N-WASP, in addition to pushing the equilibrium toward the unfolded, active state. 0.99835396 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.99706554 chemical cleaner0 2023-07-03T09:41:52Z CHEBI: PI(4,5)P2 structure_element SO: cleaner0 2023-07-03T09:40:41Z F-BAR 0.9961631 structure_element cleaner0 2023-07-03T09:40:41Z SO: F-BAR oligomeric_state DUMMY: cleaner0 2023-07-03T10:11:07Z dimer 0.99844754 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.9230027 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.9984131 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.99826837 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.9982101 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.96607906 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP structure_element SO: cleaner0 2023-07-03T09:43:10Z SH3 0.9393429 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.996428 structure_element cleaner0 2023-07-03T10:40:30Z SO: proline-rich region 0.97201604 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.54663813 protein_state cleaner0 2023-07-03T13:38:32Z DUMMY: alone 0.9858632 complex_assembly cleaner0 2023-07-03T10:49:52Z GO: N-WASP·WIP 0.9984554 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.9982558 protein cleaner0 2023-07-03T09:39:11Z PR: FBP17 0.99213284 protein cleaner0 2023-07-03T10:11:26Z PR: WIP 0.9729282 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.9949456 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.9963007 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.9971215 protein cleaner0 2023-07-03T10:11:30Z PR: WIP 0.99797446 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.9702464 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.9983481 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.99529207 protein_state cleaner0 2023-07-03T09:43:31Z DUMMY: unfolded 0.9926866 protein_state cleaner0 2023-07-03T13:38:37Z DUMMY: active 0.96953297 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.9952303 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.9981888 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.97341126 protein cleaner0 2023-06-30T08:44:52Z PR: N-WASP 0.9983552 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.99681854 chemical cleaner0 2023-07-03T09:41:52Z CHEBI: PI(4,5)P2 0.9909847 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.94290686 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP 0.99534625 protein_state cleaner0 2023-07-03T09:43:31Z DUMMY: unfolded 0.99433976 protein_state cleaner0 2023-07-03T13:38:40Z DUMMY: active DISCUSS paragraph 54916 In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. Our binding data suggest that TOCA1 HR1 binding is not allosterically regulated, and our NMR data, along with the high stability of TOCA1 HR1, suggest that there is no widespread conformational change in the presence of Cdc42. As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. A combination of allosteric activation by PI(4,5)P2, activated Cdc42 and TOCA1, and oligomeric activation implemented by TOCA1 would lead to full activation of N-WASP and downstream actin polymerization. 0.9974664 protein_state cleaner0 2023-07-03T08:36:11Z DUMMY: full-length 0.9987618 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.9171439 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP 0.84216446 evidence cleaner0 2023-07-03T12:07:03Z DUMMY: affinities 0.99756587 protein_state cleaner0 2023-07-03T13:38:44Z DUMMY: active 0.98924595 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.9969163 protein_state cleaner0 2023-07-03T09:43:30Z DUMMY: unfolded protein_state DUMMY: cleaner0 2023-07-03T13:39:05Z active 0.9685903 evidence cleaner0 2023-07-03T12:07:06Z DUMMY: affinity 0.93593836 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP 0.99408674 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.94578844 evidence cleaner0 2023-07-03T12:07:10Z DUMMY: binding data 0.9986324 protein cleaner0 2023-06-30T08:43:42Z PR: TOCA1 0.96670634 structure_element cleaner0 2023-06-30T08:45:30Z SO: HR1 0.977499 experimental_method cleaner0 2023-07-03T11:56:07Z MESH: NMR 0.9285208 protein_state cleaner0 2023-07-03T13:39:16Z DUMMY: stability 0.998789 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.9984781 structure_element cleaner0 2023-06-30T08:45:30Z SO: HR1 0.9854224 protein_state cleaner0 2023-07-03T08:55:30Z DUMMY: presence of 0.9940201 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.9972457 protein_state cleaner0 2023-07-03T08:36:11Z DUMMY: full-length 0.998825 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.4800219 protein_state cleaner0 2023-07-03T13:39:23Z DUMMY: isolated 0.9981285 structure_element cleaner0 2023-06-30T08:45:31Z SO: HR1 0.99625045 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 protein PR: cleaner0 2023-06-30T08:44:53Z N-WASP protein PR: cleaner0 2023-06-30T08:42:56Z Cdc42 0.91420364 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP 0.9985499 structure_element cleaner0 2023-07-03T10:40:36Z SO: GBD 0.9988022 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.9985341 structure_element cleaner0 2023-06-30T08:45:31Z SO: HR1 0.99631435 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.9966834 chemical cleaner0 2023-07-03T09:41:52Z CHEBI: PI(4,5)P2 0.9967855 protein_state cleaner0 2023-07-03T13:39:29Z DUMMY: activated 0.9756092 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.9987207 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.99872017 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.75998104 protein_state cleaner0 2023-07-03T13:39:37Z DUMMY: full activation 0.93770427 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP DISCUSS paragraph 55754 In such an array of molecules localized to a discrete region of the membrane, it is plausible that WASP could bind to a second Cdc42 molecule rather than displacing TOCA1 from its cognate Cdc42. Our NMR and affinity data, however, are consistent with displacement of the TOCA1 HR1 by the N-WASP GBD. Furthermore, TOCA1 is required for Cdc42-mediated activation of N-WASP·WIP, implying that it may not be possible for Cdc42 to bind and activate N-WASP prior to TOCA1-Cdc42 binding. The commonly used MGD → IST (Cdc42-binding deficient) mutant of TOCA1 has a reduced ability to activate the N-WASP·WIP complex, further indicating the importance of the Cdc42-HR1TOCA1 interaction prior to downstream activation of N-WASP. 0.99237895 protein cleaner0 2023-07-03T10:11:38Z PR: WASP 0.9970381 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.99847573 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.9959544 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.8592806 experimental_method cleaner0 2023-07-03T11:56:11Z MESH: NMR 0.70388633 evidence cleaner0 2023-07-03T12:07:14Z DUMMY: affinity data 0.9987017 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.9983523 structure_element cleaner0 2023-06-30T08:45:31Z SO: HR1 0.9263594 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP 0.99852484 structure_element cleaner0 2023-07-03T10:40:40Z SO: GBD 0.99824226 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.835499 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.95640403 complex_assembly cleaner0 2023-07-03T10:49:55Z GO: N-WASP·WIP 0.9964194 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.9571094 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP 0.9974758 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.8687983 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.99370867 mutant cleaner0 2023-07-03T10:51:53Z MESH: MGD → IST 0.9943775 protein_state cleaner0 2023-07-03T13:39:42Z DUMMY: Cdc42-binding deficient 0.9986313 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.9150546 complex_assembly cleaner0 2023-07-03T10:49:57Z GO: N-WASP·WIP 0.7008063 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 structure_element SO: cleaner0 2023-07-03T09:44:33Z HR1 protein PR: cleaner0 2023-07-03T09:44:40Z TOCA1 0.95238847 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP DISCUSS paragraph 56477 In light of this, we favor an “effector handover” scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule. Potentially, the TOCA1-Cdc42 interaction functions to position N-WASP and Cdc42 such that they are poised to interact with high affinity. The concomitant release of TOCA1 from Cdc42 while still bound to N-WASP presumably enhances the ability of TOCA1 to further activate N-WASP·WIP-induced actin polymerization. There is an advantage to such an effector handover, in that N-WASP would only be robustly recruited when F-BAR domains are already present. Hence, actin polymerization cannot occur until F-BAR domains are poised for membrane distortion. 0.9968759 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.98500466 protein cleaner0 2023-06-30T08:42:56Z PR: Cdc42 0.91776663 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP 0.8839886 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP 0.9966192 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.8415559 protein_state cleaner0 2023-07-03T13:40:00Z DUMMY: bound 0.9226735 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.9524078 protein_state cleaner0 2023-07-03T13:40:03Z DUMMY: fully activated 0.9962457 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.7135997 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 protein PR: cleaner0 2023-06-30T08:42:57Z Cdc42 0.94013363 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP 0.98841375 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.9968265 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.7197061 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 protein_state DUMMY: cleaner0 2023-07-03T13:40:28Z bound to 0.9319956 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP 0.9976781 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 complex_assembly GO: cleaner0 2023-07-03T09:45:24Z N-WASP·WIP 0.9111567 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP structure_element SO: cleaner0 2023-07-03T09:40:41Z F-BAR structure_element SO: cleaner0 2023-07-03T09:40:41Z F-BAR DISCUSS paragraph 57283 Our model of the Cdc42·HR1TOCA1 complex indicates a mechanism by which such a handover could take place (Fig. 9) because it shows that the effector binding sites only partially overlap on Cdc42. The lysine residues thought to be involved in an electrostatic steering mechanism in WASP-Cdc42 binding are conserved in N-WASP and would be able to interact with Cdc42 even when the TOCA1 HR1 domain is already bound. It has been postulated that the initial interactions between this basic region and Cdc42 could stabilize the active conformation of WASP, leading to high affinity binding between the core CRIB and Cdc42. The region C-terminal to the core CRIB, required for maximal affinity binding, would then fully displace the TOCA1 HR1. 0.8920825 complex_assembly cleaner0 2023-07-03T10:50:04Z GO: Cdc42·HR1TOCA1 0.99800205 site cleaner0 2023-07-03T12:08:45Z SO: effector binding sites 0.99696857 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.98572934 residue_name cleaner0 2023-07-03T10:40:48Z SO: lysine protein PR: cleaner0 2023-07-03T10:12:04Z WASP protein PR: cleaner0 2023-06-30T08:42:57Z Cdc42 0.98391724 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP 0.9976672 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.998718 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.99860877 structure_element cleaner0 2023-06-30T08:45:31Z SO: HR1 0.9362426 protein_state cleaner0 2023-07-03T13:40:40Z DUMMY: bound 0.99707055 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.99639326 protein_state cleaner0 2023-07-03T13:40:42Z DUMMY: active 0.9975865 protein cleaner0 2023-07-03T10:11:54Z PR: WASP 0.99788386 structure_element cleaner0 2023-07-03T10:40:51Z SO: CRIB 0.9972886 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.9981187 structure_element cleaner0 2023-07-03T10:40:54Z SO: CRIB 0.9987233 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.99854016 structure_element cleaner0 2023-06-30T08:45:31Z SO: HR1 zbc0281646060009.jpg F9 FIG fig_caption 58021 A simplified model of the early stages of Cdc42·N-WASP·TOCA1-dependent actin polymerization. Step 1, TOCA1 is recruited to the membrane via its F-BAR domain and/or Cdc42 interactions. F-BAR oligomerization is expected to occur following membrane binding, but a single monomer is shown for clarity. Step 2, N-WASP exists in an inactive, folded conformation. The TOCA1 SH3 domain interacts with N-WASP, causing an activatory allosteric effect. The HR1TOCA1-Cdc42 and SH3TOCA1-N-WASP interactions position Cdc42 and N-WASP for binding. Step 3, electrostatic interactions between Cdc42 and the basic region upstream of the CRIB initiate Cdc42·N-WASP binding. Step 4, the core CRIB binds with high affinity while the region C-terminal to the CRIB displaces the TOCA1 HR1 domain and increases the affinity of the N-WASP-Cdc42 interaction further. The VCA domain is released for downstream interactions, and actin polymerization proceeds. WH1, WASP homology 1 domain; PP, proline-rich region; VCA, verprolin homology, cofilin homology, acidic region. complex_assembly GO: cleaner0 2023-07-03T09:45:49Z Cdc42·N-WASP·TOCA1 0.99840397 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 structure_element SO: cleaner0 2023-07-03T09:40:41Z F-BAR 0.93579847 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.9775112 structure_element cleaner0 2023-07-03T09:40:41Z SO: F-BAR 0.9951125 oligomeric_state cleaner0 2023-07-03T11:42:11Z DUMMY: monomer 0.947602 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP 0.99736327 protein_state cleaner0 2023-07-03T13:40:54Z DUMMY: inactive 0.994083 protein_state cleaner0 2023-07-03T09:43:41Z DUMMY: folded 0.9987005 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 structure_element SO: cleaner0 2023-07-03T10:41:09Z SH3 0.9564964 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP structure_element SO: cleaner0 2023-07-03T09:46:33Z HR1 protein PR: cleaner0 2023-07-03T09:46:41Z TOCA1 0.62519926 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 structure_element SO: cleaner0 2023-07-03T09:46:55Z SH3 protein PR: cleaner0 2023-07-03T09:47:03Z TOCA1 protein PR: cleaner0 2023-06-30T08:44:53Z N-WASP 0.8279639 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.91391045 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:06Z electrostatic interactions 0.9968832 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.99876785 structure_element cleaner0 2023-07-03T10:41:14Z SO: CRIB complex_assembly GO: cleaner0 2023-07-03T09:46:15Z Cdc42·N-WASP 0.99859744 structure_element cleaner0 2023-07-03T10:41:20Z SO: CRIB 0.9987232 structure_element cleaner0 2023-07-03T10:41:18Z SO: CRIB 0.9986406 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.99863064 structure_element cleaner0 2023-06-30T08:45:31Z SO: HR1 0.57654375 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP protein PR: cleaner0 2023-06-30T08:42:57Z Cdc42 0.9983918 structure_element cleaner0 2023-07-03T10:41:23Z SO: VCA 0.9984913 structure_element cleaner0 2023-07-03T10:41:26Z SO: WH1 0.99590874 structure_element cleaner0 2023-07-03T10:41:28Z SO: WASP homology 1 domain 0.99861336 structure_element cleaner0 2023-07-03T10:41:30Z SO: PP 0.9971271 structure_element cleaner0 2023-07-03T10:41:32Z SO: proline-rich region 0.99818355 structure_element cleaner0 2023-07-03T10:41:35Z SO: VCA 0.9958004 structure_element cleaner0 2023-07-03T10:41:37Z SO: verprolin homology, cofilin homology, acidic region DISCUSS paragraph 59068 In conclusion, the data presented here show that the TOCA1 HR1 domain is sufficient for Cdc42 binding in vitro and that the interaction is of micromolar affinity, lower than that of other G protein-HR1 domain interactions. The analogous HR1 domains from other TOCA1 family members, FBP17 and CIP4, also exhibit micromolar affinity for Cdc42. A role for the TOCA1-, FBP17-, and CIP4-Cdc42 interactions in the recruitment of these proteins to the membrane therefore appears unlikely. Instead, our findings agree with earlier suggestions that the F-BAR domain is responsible for membrane recruitment. The role of the Cdc42-TOCA1 interaction remains somewhat elusive, but it may serve to position activated Cdc42 and N-WASP to allow full activation of N-WASP and as such serve to couple F-BAR-mediated membrane deformation with N-WASP activation. We envisage a complex interplay of equilibria between free and bound, active and inactive Cdc42, TOCA family, and WASP family proteins, facilitating a tightly spatially and temporally regulated pathway requiring numerous simultaneous events in order to achieve appropriate and robust activation of the downstream pathway. Our data are therefore easily reconciled with the dynamic instability models described in relation to the formation of endocytic vesicles and with the current data pertaining to the complex activation of WASP/N-WASP pathways by allosteric and oligomeric effects. 0.99871993 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 structure_element SO: cleaner0 2023-06-30T08:45:31Z HR1 0.93090767 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 protein_type MESH: cleaner0 2023-07-03T08:23:08Z G protein structure_element SO: cleaner0 2023-06-30T08:45:31Z HR1 structure_element SO: cleaner0 2023-06-30T08:45:31Z HR1 0.95327765 protein_type cleaner0 2023-07-03T10:19:18Z MESH: TOCA1 family 0.99848145 protein cleaner0 2023-07-03T09:39:11Z PR: FBP17 0.9982632 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 0.99616635 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.94887155 protein cleaner0 2023-06-30T08:43:43Z PR: TOCA1 0.9608345 protein cleaner0 2023-07-03T09:39:11Z PR: FBP17 0.9575774 protein cleaner0 2023-07-03T09:39:06Z PR: CIP4 0.6942188 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 structure_element SO: cleaner0 2023-07-03T09:40:41Z F-BAR protein PR: cleaner0 2023-06-30T08:42:57Z Cdc42 protein PR: cleaner0 2023-06-30T08:43:43Z TOCA1 0.99709356 protein_state cleaner0 2023-07-03T13:41:05Z DUMMY: activated 0.5552928 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.935388 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP 0.98660195 protein_state cleaner0 2023-07-03T13:41:09Z DUMMY: full activation 0.9621456 protein cleaner0 2023-06-30T08:44:53Z PR: N-WASP 0.9905291 structure_element cleaner0 2023-07-03T09:40:41Z SO: F-BAR 0.9283731 protein cleaner0 2023-06-30T08:44:54Z PR: N-WASP 0.99688065 protein_state cleaner0 2023-07-03T13:41:12Z DUMMY: free 0.9517934 protein_state cleaner0 2023-07-03T13:41:14Z DUMMY: bound 0.9954417 protein_state cleaner0 2023-07-03T13:41:17Z DUMMY: active 0.9683954 protein_state cleaner0 2023-07-03T13:41:21Z DUMMY: inactive 0.6535516 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.84625226 protein_type cleaner0 2023-07-03T10:19:26Z MESH: TOCA family 0.8193172 protein_type cleaner0 2023-07-03T10:19:29Z MESH: WASP 0.8569883 protein_type cleaner0 2023-07-03T10:19:32Z MESH: WASP protein PR: cleaner0 2023-07-03T10:19:42Z N-WASP DISCUSS paragraph 60496 It is clear from the data presented here that TOCA1 and N-WASP do not bind Cdc42 simultaneously and that N-WASP is likely to outcompete TOCA1 for Cdc42 binding. We therefore postulate an effector handover mechanism based on current evidence surrounding WASP/N-WASP activation and our model of the Cdc42·HR1TOCA1 complex. The displacement of the TOCA1 HR1 domain from Cdc42 by N-WASP may represent a unidirectional step in the pathway of Cdc42·N-WASP·TOCA1-dependent actin assembly. 0.9985322 protein cleaner0 2023-06-30T08:43:44Z PR: TOCA1 0.945636 protein cleaner0 2023-06-30T08:44:54Z PR: N-WASP 0.9955771 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.9167795 protein cleaner0 2023-06-30T08:44:54Z PR: N-WASP 0.99846774 protein cleaner0 2023-06-30T08:43:44Z PR: TOCA1 0.9865458 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.90799123 protein cleaner0 2023-07-03T10:12:21Z PR: WASP 0.7165899 protein cleaner0 2023-06-30T08:44:54Z PR: N-WASP 0.9785838 complex_assembly cleaner0 2023-07-03T10:50:09Z GO: Cdc42·HR1TOCA1 0.99869764 protein cleaner0 2023-06-30T08:43:44Z PR: TOCA1 0.99861073 structure_element cleaner0 2023-06-30T08:45:31Z SO: HR1 0.9908448 protein cleaner0 2023-06-30T08:42:57Z PR: Cdc42 0.928042 protein cleaner0 2023-06-30T08:44:54Z PR: N-WASP complex_assembly GO: cleaner0 2023-07-03T09:49:21Z Cdc42·N-WASP·TOCA1 AUTH_CONT title_1 60981 Author Contributions AUTH_CONT paragraph 61002 J. R. W. generated constructs and proteins, set up NMR experiments, analyzed NMR data, and performed binding experiments; D. N. set up NMR experiments; H. M. F. generated longer TOCA clones and proteins; J. L. G. supervised the pyrene actin assays; D. O. supervised the protein binding assays; and H. R. M. performed NMR experiments and analyzed NMR data. J. R. W., D. O., and H. R. M. wrote the paper with input from all authors. AUTH_CONT footnote 61433 The authors declare that they have no conflicts of interest with the contents of this article. AUTH_CONT footnote 61528 The atomic coordinates and structure factors (code 5FRG) have been deposited in the Protein Data Bank (http://wwpdb.org/). AUTH_CONT footnote 61651 D. Owen, unpublished data. AUTH_CONT footnote 61678 H. R. Mott and D. Owen, unpublished data. AUTH_CONT footnote 61720 PRK AUTH_CONT footnote 61724 protein kinase C related kinase AUTH_CONT footnote 61756 WASP AUTH_CONT footnote 61761 Wiskott-Aldrich syndrome protein AUTH_CONT footnote 61794 TOCA AUTH_CONT footnote 61799 transducer of Cdc42-dependent actin assembly protein AUTH_CONT footnote 61852 N-WASP AUTH_CONT footnote 61859 neural Wiskott-Aldrich syndrome protein AUTH_CONT footnote 61899 PI(4,5)P2 AUTH_CONT footnote 61909 phosphatidylinositol 4,5-bisphosphate AUTH_CONT footnote 61947 HR1 AUTH_CONT footnote 61951 homology region 1 AUTH_CONT footnote 61969 F-BAR AUTH_CONT footnote 61975 Fes/CIP4 homology BAR AUTH_CONT footnote 61997 SH3 AUTH_CONT footnote 62001 Src homology 3 AUTH_CONT footnote 62016 CRIB AUTH_CONT footnote 62021 Cdc42- and Rac-interactive binding AUTH_CONT footnote 62056 CIP4 AUTH_CONT footnote 62061 Cdc42-interacting protein 4 AUTH_CONT footnote 62089 MBP AUTH_CONT footnote 62093 maltose-binding protein AUTH_CONT footnote 62117 GBD AUTH_CONT footnote 62121 G protein binding domain AUTH_CONT footnote 62146 SPA AUTH_CONT footnote 62150 scintillation proximity assay AUTH_CONT footnote 62180 PAK AUTH_CONT footnote 62184 p21-activated kinase AUTH_CONT footnote 62205 ACK AUTH_CONT footnote 62209 activated Cdc42-associated kinase AUTH_CONT footnote 62243 HSQC AUTH_CONT footnote 62248 heteronuclear single quantum correlation AUTH_CONT footnote 62289 GMPPNP AUTH_CONT footnote 62296 guanosine 5′-[β,γ-imido] triphosphate AUTH_CONT footnote 62341 GTPγS AUTH_CONT footnote 62351 guanosine 5′-3-O-(thio)triphosphate AUTH_CONT footnote 62389 GBD AUTH_CONT footnote 62393 G protein-binding domain AUTH_CONT footnote 62418 CSP AUTH_CONT footnote 62422 chemical shift perturbation AUTH_CONT footnote 62450 PDB AUTH_CONT footnote 62454 Protein Data Bank. 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