PMC 20201215 pmc.key 4774019 CC BY no 0 0 10.1186/s12915-016-0236-7 4774019 26934976 236 14 Innate immunity IRG proteins GTPase Dynamin superfamily Dimerization Oligomerization Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. surname:Schulte;given-names:Kathrin surname:Pawlowski;given-names:Nikolaus surname:Faelber;given-names:Katja surname:Fröhlich;given-names:Chris surname:Howard;given-names:Jonathan surname:Daumke;given-names:Oliver surname:Howard;given-names:Jonathan surname:Daumke;given-names:Oliver TITLE Keywords front 14 2016 0 The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion 0.99856925 protein_type cleaner0 2023-07-27T10:33:20Z MESH: immunity-related GTPase 0.99922526 protein cleaner0 2023-07-27T10:33:25Z PR: Irga6 0.99804074 oligomeric_state cleaner0 2023-07-27T10:33:31Z DUMMY: dimerizes 0.998421 protein_state cleaner0 2023-07-27T10:33:35Z DUMMY: parallel head-to-head ABSTRACT abstract_title_1 79 Background ABSTRACT abstract 90 The immunity-related GTPases (IRGs) constitute a powerful cell-autonomous resistance system against several intracellular pathogens. Irga6 is a dynamin-like protein that oligomerizes at the parasitophorous vacuolar membrane (PVM) of Toxoplasma gondii leading to its vesiculation. Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization. 0.99871874 protein_type cleaner0 2023-07-27T10:37:44Z MESH: immunity-related GTPases 0.99926287 protein_type cleaner0 2023-07-27T10:33:42Z MESH: IRGs 0.9991574 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9987838 protein_type cleaner0 2023-07-27T10:33:48Z MESH: dynamin-like protein 0.99836946 species cleaner0 2023-07-27T10:33:54Z MESH: Toxoplasma gondii 0.99855673 experimental_method cleaner0 2023-07-27T10:34:13Z MESH: biochemical analysis 0.9992793 structure_element cleaner0 2023-07-27T10:34:08Z SO: GTPase domains 0.99919015 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.99237704 oligomeric_state cleaner0 2023-07-27T12:30:12Z DUMMY: dimerize protein_state DUMMY: cleaner0 2023-07-27T12:29:53Z antiparallel ABSTRACT abstract_title_1 526 Results ABSTRACT abstract 534 We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog. Contrary to the previous model, the structure shows that the GTPase domains dimerize in a parallel fashion. The nucleotides in the center of the interface participate in dimerization by forming symmetric contacts with each other and with the switch I region of the opposing Irga6 molecule. The latter contact appears to activate GTP hydrolysis by stabilizing the position of the catalytic glutamate 106 in switch I close to the active site. Further dimerization contacts involve switch II, the G4 helix and the trans stabilizing loop. experimental_method MESH: cleaner0 2023-07-27T12:41:13Z determined 0.9973475 evidence cleaner0 2023-07-27T12:40:59Z DUMMY: crystal structure 0.9970346 protein_state cleaner0 2023-07-27T10:34:19Z DUMMY: oligomerization-impaired 0.99841 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9990128 protein_state cleaner0 2023-07-27T10:34:25Z DUMMY: mutant 0.99903274 protein_state cleaner0 2023-07-27T10:34:29Z DUMMY: bound to chemical CHEBI: cleaner0 2023-07-27T10:35:02Z GTP 0.99806577 evidence cleaner0 2023-07-27T12:41:19Z DUMMY: structure 0.99867094 structure_element cleaner0 2023-07-27T10:34:08Z SO: GTPase domains 0.99238014 oligomeric_state cleaner0 2023-07-27T12:30:12Z DUMMY: dimerize protein_state DUMMY: cleaner0 2023-07-27T12:09:49Z parallel 0.9541489 chemical cleaner0 2023-07-27T12:37:37Z CHEBI: nucleotides 0.9987698 site cleaner0 2023-07-27T12:37:40Z SO: interface site SO: cleaner0 2023-07-27T10:35:34Z switch I 0.9991636 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.99857926 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.9832497 protein_state cleaner0 2023-07-27T10:35:43Z DUMMY: catalytic 0.83116907 residue_name_number cleaner0 2023-07-27T10:35:49Z DUMMY: glutamate 106 0.9981953 site cleaner0 2023-07-27T10:35:35Z SO: switch I 0.99897873 site cleaner0 2023-07-27T12:37:45Z SO: active site 0.9981766 site cleaner0 2023-07-27T10:35:38Z SO: switch II 0.9988177 structure_element cleaner0 2023-07-27T10:50:58Z SO: G4 helix structure_element SO: cleaner0 2023-07-27T10:44:28Z trans stabilizing loop ABSTRACT abstract_title_1 1189 Conclusions ABSTRACT abstract 1201 The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members. This study contributes important insights into the assembly and catalytic mechanisms of IRG proteins as prerequisite to understand their anti-microbial action. 0.99903893 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9976412 evidence cleaner0 2023-07-27T12:41:35Z DUMMY: structure protein_state DUMMY: cleaner0 2023-07-27T12:09:49Z parallel structure_element SO: cleaner0 2023-07-27T10:36:19Z GTPase domain 0.9474066 oligomeric_state cleaner0 2023-07-27T10:37:33Z DUMMY: dimer 0.99884295 protein_type cleaner0 2023-07-27T10:36:29Z MESH: dynamin protein_type MESH: cleaner0 2023-07-27T10:36:51Z septin protein_type MESH: cleaner0 2023-07-27T10:37:26Z IRG ABSTRACT abstract_title_1 1508 Electronic supplementary material ABSTRACT abstract 1542 The online version of this article (doi:10.1186/s12915-016-0236-7) contains supplementary material, which is available to authorized users. INTRO title_1 1682 Background INTRO paragraph 1693 Immunity-related GTPases (IRGs) comprise a family of dynamin-related cell-autonomous resistance proteins targeting intracellular pathogens, such as Mycobacterium tuberculosis, Mycobacterium avium, Listeria monocytogenes, Trypanosoma cruzi, and Toxoplasma gondii. In mice, the 23 IRG members are induced by interferons, whereas the single human homologue is constitutively expressed in some tissues, especially in testis. In non-infected cells, most IRGs are largely cytosolic. However, members of a small sub-family with regulatory function associate with specific intracellular membranes, with one member favoring the endoplasmic reticulum and others the Golgi membrane and the endolysosomal system. Infection by certain intracellular pathogens initiates the redistribution of several effector members to the parasitophorous vacuole, followed by its disruption. In this way, IRGs contribute to the release of the pathogen into the cytoplasm and its subsequent destruction. 0.9982124 protein_type cleaner0 2023-07-27T10:37:43Z MESH: Immunity-related GTPases 0.9992719 protein_type cleaner0 2023-07-27T10:33:43Z MESH: IRGs 0.9983795 protein_type cleaner0 2023-07-27T10:37:51Z MESH: dynamin-related cell-autonomous resistance proteins 0.997863 species cleaner0 2023-07-27T10:38:06Z MESH: Mycobacterium tuberculosis 0.99792004 species cleaner0 2023-07-27T10:38:11Z MESH: Mycobacterium avium 0.99799746 species cleaner0 2023-07-27T10:38:15Z MESH: Listeria monocytogenes 0.99820083 species cleaner0 2023-07-27T10:38:18Z MESH: Trypanosoma cruzi 0.9983006 species cleaner0 2023-07-27T10:33:54Z MESH: Toxoplasma gondii 0.7319738 taxonomy_domain cleaner0 2023-07-27T10:38:34Z DUMMY: mice protein_type MESH: cleaner0 2023-07-27T10:37:26Z IRG 0.77819955 protein_type cleaner0 2023-07-27T10:38:00Z MESH: interferons 0.9984085 species cleaner0 2023-07-27T10:38:38Z MESH: human 0.99926263 protein_type cleaner0 2023-07-27T10:33:43Z MESH: IRGs 0.99927527 protein_type cleaner0 2023-07-27T10:33:43Z MESH: IRGs INTRO paragraph 2667 Irga6, one of the effector IRG proteins, localizes to the intact parasitophorous vacuole membrane (PVM) and, after disruption of the PVM, is found associated with vesicular accumulations, presumably derived from the PVM. A myristoylation site at Gly2 is necessary for the recruitment to the PVM but not for the weak constitutive binding to the ER membrane. An internally oriented antibody epitope on helix A between positions 20 and 24 was demonstrated to be accessible in the GTP-, but not in the GDP-bound state. This indicates large-scale structural changes upon GTP binding that probably include exposure of the myristoyl group, enhancing binding to the PVM. Biochemical studies indicated that Irga6 hydrolyses GTP in a cooperative manner and forms GTP-dependent oligomers in vitro and in vivo. 0.9990559 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 protein_type MESH: cleaner0 2023-07-27T10:37:26Z IRG 0.9987713 site cleaner0 2023-07-27T10:39:19Z SO: myristoylation site 0.9995802 residue_name_number cleaner0 2023-07-27T10:39:25Z DUMMY: Gly2 0.9992757 structure_element cleaner0 2023-07-27T12:33:53Z SO: helix A residue_range DUMMY: cleaner0 2023-07-27T10:39:50Z 20 and 24 0.9980969 protein_state cleaner0 2023-07-27T10:40:06Z DUMMY: GTP-, 0.9988313 protein_state cleaner0 2023-07-27T10:40:09Z DUMMY: GDP-bound 0.9982768 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.9988266 experimental_method cleaner0 2023-07-27T10:40:21Z MESH: Biochemical studies 0.9991855 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9988405 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.99385804 protein_state cleaner0 2023-07-27T10:40:16Z DUMMY: GTP-dependent 0.9974528 oligomeric_state cleaner0 2023-07-27T10:40:32Z DUMMY: oligomers INTRO paragraph 3466 Crystal structures of Irga6 in various nucleotide-loaded states revealed the basic architecture of IRG proteins, including a GTPase domain and a composite helical domain. These studies additionally showed a dimerization interface in the nucleotide-free protein as well as in all nucleotide-bound states. It involves a GTPase domain surface, which is located at the opposite side of the nucleotide, and an interface in the helical domain, with a water-filled gap between the two contact surfaces. Mutagenesis of the contact surfaces suggests that this "backside" interface is not required for GTP-dependent oligomerization or cooperative hydrolysis, despite an earlier suggestion to the contrary. 0.99883634 evidence cleaner0 2023-07-27T12:41:41Z DUMMY: Crystal structures 0.99927765 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9988356 protein_state cleaner0 2023-07-27T10:41:38Z DUMMY: nucleotide-loaded protein_type MESH: cleaner0 2023-07-27T10:37:26Z IRG 0.998955 structure_element cleaner0 2023-07-27T10:36:20Z SO: GTPase domain structure_element SO: cleaner0 2023-07-27T10:41:04Z composite helical domain 0.9988985 site cleaner0 2023-07-27T10:41:23Z SO: dimerization interface 0.99874526 protein_state cleaner0 2023-07-27T10:41:29Z DUMMY: nucleotide-free 0.9989639 protein_state cleaner0 2023-07-27T10:41:33Z DUMMY: nucleotide-bound 0.99890107 site cleaner0 2023-07-27T10:41:20Z SO: GTPase domain surface 0.9985129 site cleaner0 2023-07-27T12:37:52Z SO: interface 0.99901116 structure_element cleaner0 2023-07-27T10:41:51Z SO: helical domain 0.992031 chemical cleaner0 2023-07-27T12:40:24Z CHEBI: water 0.9884542 site cleaner0 2023-07-27T12:37:57Z SO: contact surfaces 0.9971976 experimental_method cleaner0 2023-07-27T12:43:20Z MESH: Mutagenesis 0.99702406 site cleaner0 2023-07-27T12:38:00Z SO: contact surfaces 0.99781394 site cleaner0 2023-07-27T12:38:10Z SO: backside 0.98933053 site cleaner0 2023-07-27T12:38:13Z SO: interface 0.99357283 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP INTRO paragraph 4162 Extensive biochemical studies suggested that GTP-induced oligomerization of Irga6 requires an interface in the GTPase domain across the nucleotide-binding site. Recent structural studies indicated that a 'G interface' is typical of dynamin superfamily members, such as dynamin, MxA, the guanylate binding protein-1 (GBP-1), atlastin and the bacterial dynamin-like proteins (BDLP). For several of these proteins, formation of the G interface was shown to trigger GTP hydrolysis by inducing rearrangements of catalytic residues in cis. In dynamin, the G interface includes residues in the phosphate binding loop, the two switch regions, the 'trans stabilizing loop' and the 'G4 loop'. For Irga6, it was demonstrated that besides residues in the switch I and switch II regions, the 3'-OH group of the ribose participates in this interface. Since the signal recognition particle GTPase and its homologous receptor (called FfH and FtsY in bacteria) also employ the 3'-OH ribose group to dimerize in an anti-parallel orientation therefore activating its GTPase, an analogous dimerization model was proposed for Irga6. However, the crystal structure of Irga6 in the presence of the non-hydrolyzable GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP) showed only subtle differences relative to the apo or GDP-bound protein and did not reveal a new dimer interface associated with the GTPase domain. This structure was obtained by soaking GMPPNP in nucleotide-free crystals of Irga6, an approach which may have interfered with nucleotide-induced domain rearrangements. 0.99814224 experimental_method cleaner0 2023-07-27T12:43:23Z MESH: biochemical studies 0.99528426 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.9993851 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9989949 site cleaner0 2023-07-27T12:38:17Z SO: interface 0.9989977 structure_element cleaner0 2023-07-27T10:36:20Z SO: GTPase domain 0.9990971 site cleaner0 2023-07-27T12:38:21Z SO: nucleotide-binding site 0.998345 experimental_method cleaner0 2023-07-27T12:43:26Z MESH: structural studies 0.9990305 site cleaner0 2023-07-27T10:42:59Z SO: G interface protein_type MESH: cleaner0 2023-07-27T12:26:37Z dynamin protein_type MESH: cleaner0 2023-07-27T10:42:32Z dynamin 0.9992785 protein cleaner0 2023-07-27T10:42:39Z PR: MxA 0.9988429 protein cleaner0 2023-07-27T10:42:42Z PR: guanylate binding protein-1 0.99911195 protein cleaner0 2023-07-27T10:42:47Z PR: GBP-1 0.9993549 protein_type cleaner0 2023-07-27T12:13:53Z MESH: atlastin 0.99868435 taxonomy_domain cleaner0 2023-07-27T10:42:55Z DUMMY: bacterial 0.99672365 protein_type cleaner0 2023-07-27T10:44:43Z MESH: dynamin-like proteins 0.9980044 protein_type cleaner0 2023-07-27T10:44:54Z MESH: BDLP 0.99910605 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.9984066 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.9871726 protein_type cleaner0 2023-07-27T10:36:30Z MESH: dynamin 0.9990924 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.998894 structure_element cleaner0 2023-07-27T10:44:20Z SO: phosphate binding loop 0.99662805 site cleaner0 2023-07-27T12:38:44Z SO: switch regions 0.99888474 structure_element cleaner0 2023-07-27T10:44:27Z SO: trans stabilizing loop 0.9989444 structure_element cleaner0 2023-07-27T10:44:31Z SO: G4 loop 0.9993806 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9983735 site cleaner0 2023-07-27T10:35:35Z SO: switch I site SO: cleaner0 2023-07-27T10:35:39Z switch II 0.99814296 site cleaner0 2023-07-27T12:38:48Z SO: interface 0.65104276 protein_type cleaner0 2023-07-27T12:27:47Z MESH: signal recognition particle GTPase 0.8848597 protein_type cleaner0 2023-07-27T10:45:16Z MESH: receptor 0.9990214 protein cleaner0 2023-07-27T10:45:07Z PR: FfH 0.99926084 protein cleaner0 2023-07-27T10:45:11Z PR: FtsY 0.99863774 taxonomy_domain cleaner0 2023-07-27T10:44:50Z DUMMY: bacteria oligomeric_state DUMMY: cleaner0 2023-07-27T12:30:12Z dimerize protein_state DUMMY: cleaner0 2023-07-27T12:10:00Z anti-parallel 0.9983564 protein_type cleaner0 2023-07-27T10:45:14Z MESH: GTPase 0.99937195 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.99760354 evidence cleaner0 2023-07-27T12:41:55Z DUMMY: crystal structure 0.9993901 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.98288035 protein_state cleaner0 2023-07-27T10:44:05Z DUMMY: presence of 0.99829143 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.99908435 chemical cleaner0 2023-07-27T10:43:21Z CHEBI: 5'-guanylyl imidodiphosphate 0.99934644 chemical cleaner0 2023-07-27T10:43:26Z CHEBI: GMPPNP 0.9992712 protein_state cleaner0 2023-07-27T10:44:00Z DUMMY: apo 0.99909383 protein_state cleaner0 2023-07-27T10:44:10Z DUMMY: GDP-bound 0.99903345 site cleaner0 2023-07-27T10:43:18Z SO: dimer interface 0.99882746 structure_element cleaner0 2023-07-27T10:36:20Z SO: GTPase domain 0.99785787 evidence cleaner0 2023-07-27T12:41:57Z DUMMY: structure 0.9982498 experimental_method cleaner0 2023-07-27T12:43:33Z MESH: soaking 0.99927694 chemical cleaner0 2023-07-27T10:43:26Z CHEBI: GMPPNP 0.99860454 protein_state cleaner0 2023-07-27T10:41:30Z DUMMY: nucleotide-free 0.9980411 evidence cleaner0 2023-07-27T12:42:00Z DUMMY: crystals 0.99938655 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 INTRO paragraph 5722 To clarify the dimerization mode via the G interface, we determined the GMPPNP-bound crystal structure of a non-oligomerizing Irga6 variant. The structure revealed that Irga6 can dimerize via the G interface in a parallel head-to-head fashion. This dimerization mode explains previously published biochemical data, and shows in particular how the 3'-OH group of the ribose participates in the assembly. Our data suggest that a parallel dimerization mode may be a unifying feature in all dynamin and septin superfamily proteins. 0.9990747 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.9935353 experimental_method cleaner0 2023-07-27T12:43:37Z MESH: determined 0.9989519 protein_state cleaner0 2023-07-27T10:45:26Z DUMMY: GMPPNP-bound 0.99767303 evidence cleaner0 2023-07-27T12:41:50Z DUMMY: crystal structure 0.9989877 protein_state cleaner0 2023-07-27T10:45:32Z DUMMY: non-oligomerizing 0.97425914 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9017479 protein_state cleaner0 2023-07-27T12:30:56Z DUMMY: variant 0.998187 evidence cleaner0 2023-07-27T12:42:03Z DUMMY: structure 0.9992094 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9518729 oligomeric_state cleaner0 2023-07-27T12:30:12Z DUMMY: dimerize 0.99904406 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.9986 protein_state cleaner0 2023-07-27T10:33:35Z DUMMY: parallel head-to-head protein_state DUMMY: cleaner0 2023-07-27T12:09:49Z parallel 0.99881124 protein_type cleaner0 2023-07-27T10:36:30Z MESH: dynamin protein_type MESH: cleaner0 2023-07-27T10:36:52Z septin RESULTS title_1 6250 Results RESULTS paragraph 6258 Previous results indicated that Irga6 mutations in a loosely defined surface region (the "secondary patch"), which is distant from the G-interface and only slightly overlapping with the backside interface (see below), individually reduced GTP-dependent oligomerization. A combination of four of these mutations (R31E, K32E, K176E, and K246E) essentially eliminated GTP-dependent assembly (Additional file 1: Figure S1) and allowed crystallization of Irga6 in the presence of GMPPNP. Crystals diffracted to 3.2 Å resolution and displayed one exceptionally long unit cell axis of 1289 Å (Additional file 1: Table S1). The structure was solved by molecular replacement and refined to Rwork/Rfree of 29.7 %/31.7 % (Additional file 1: Table S2). The asymmetric unit contained seven Irga6 molecules that were arranged in a helical pattern along the long cell axis (Additional file 1: Figure S2). 0.99924433 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.7618282 experimental_method cleaner0 2023-07-27T12:43:45Z MESH: mutations 0.72960615 site cleaner0 2023-07-27T12:38:53Z SO: surface region 0.98579633 site cleaner0 2023-07-27T10:48:51Z SO: secondary patch 0.99895334 site cleaner0 2023-07-27T12:38:58Z SO: G-interface 0.9989779 site cleaner0 2023-07-27T10:46:44Z SO: backside interface 0.99561846 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP experimental_method MESH: cleaner0 2023-07-27T10:46:21Z combination of four of these mutations 0.9989084 mutant cleaner0 2023-07-27T10:46:24Z MESH: R31E 0.99891293 mutant cleaner0 2023-07-27T10:46:29Z MESH: K32E 0.9988226 mutant cleaner0 2023-07-27T10:46:33Z MESH: K176E 0.99895966 mutant cleaner0 2023-07-27T10:46:37Z MESH: K246E 0.530567 protein_state cleaner0 2023-07-27T12:31:20Z DUMMY: eliminated 0.96393704 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.9978302 experimental_method cleaner0 2023-07-27T12:43:50Z MESH: crystallization 0.9992912 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9966885 protein_state cleaner0 2023-07-27T10:44:05Z DUMMY: presence of 0.99935204 chemical cleaner0 2023-07-27T10:43:26Z CHEBI: GMPPNP 0.9900343 evidence cleaner0 2023-07-27T12:42:08Z DUMMY: Crystals 0.98809695 evidence cleaner0 2023-07-27T12:42:11Z DUMMY: structure 0.9985469 experimental_method cleaner0 2023-07-27T12:43:54Z MESH: molecular replacement 0.9984352 evidence cleaner0 2023-07-27T12:42:19Z DUMMY: Rwork 0.7509895 evidence cleaner0 2023-07-27T12:42:21Z DUMMY: Rfree 0.99925333 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 12915_2016_236_Fig1_HTML.jpg Fig1 FIG fig_caption 7150 Structure of the Irga6 dimer. a Schematic view of the domain architecture of mouse Irga6. The first and last amino acids of each domain are indicated. b Ribbon-type representation of the Irga6 dimer. In the left molecule, domains are colored according to the domain architecture, the right molecule is colored in grey. The nucleotide and Mg2+ ion (green) are shown in sphere representation. The GTPase domain dimer is boxed. The dotted line indicates a 2-fold axis. Secondary structure was numbered according to ref.. c Top view on the GTPase domain dimer. d Magnification of the contact sites. Dotted lines indicate interactions. e Superposition of different switch I conformations in the asymmetric unit; the same colors as in Additional file 1: Figure S2 are used for the switch I regions of the individual subunits. Switch I residues of subunit A (yellow) involved in ribose binding are labelled and shown in stick representation. Irga6 immunity-related GTPase 6 evidence DUMMY: cleaner0 2023-07-27T12:44:12Z Structure 0.99921584 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9988256 oligomeric_state cleaner0 2023-07-27T10:37:33Z DUMMY: dimer 0.9914909 taxonomy_domain cleaner0 2023-07-27T12:37:17Z DUMMY: mouse 0.9991703 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.999255 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.99884343 oligomeric_state cleaner0 2023-07-27T10:37:33Z DUMMY: dimer 0.9989561 chemical cleaner0 2023-07-27T12:40:29Z CHEBI: Mg2+ 0.98754936 structure_element cleaner0 2023-07-27T10:36:20Z SO: GTPase domain 0.998869 oligomeric_state cleaner0 2023-07-27T10:37:33Z DUMMY: dimer 0.9723335 structure_element cleaner0 2023-07-27T10:36:20Z SO: GTPase domain 0.99883145 oligomeric_state cleaner0 2023-07-27T10:37:33Z DUMMY: dimer 0.9263503 site cleaner0 2023-07-27T12:39:04Z SO: contact sites 0.9949509 experimental_method cleaner0 2023-07-27T12:44:02Z MESH: Superposition 0.9954356 site cleaner0 2023-07-27T10:35:35Z SO: switch I site SO: cleaner0 2023-07-27T10:35:35Z switch I 0.99632245 site cleaner0 2023-07-27T10:35:35Z SO: Switch I 0.99873453 structure_element cleaner0 2023-07-27T12:34:00Z SO: A 0.999233 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.99642575 protein cleaner0 2023-07-27T10:47:22Z PR: immunity-related GTPase 6 RESULTS paragraph 8117 Like other dynamin superfamily members, the GTPase domain of Irga6 comprises a canonical GTPase domain fold, with a central β-sheet surrounded by helices on both sides (Fig. 1a-c). The helical domain is a bipartite structure composed of helices αA-C at the N-terminus and helix αF-L at the C-terminus of the GTPase domain. Overall, the seven molecules in the asymmetric unit are very similar to each other, with root mean square deviations (rmsd) ranging from 0.32 – 0.45 Å over all Cα atoms. The structures of the seven molecules also agree well with the previously determined structure of native GMPPNP-bound Irga6 (PDB: 1TQ6; rmsd of 1.00-1.13 Å over all Cα atoms). protein_type MESH: cleaner0 2023-07-27T10:36:30Z dynamin 0.99935675 structure_element cleaner0 2023-07-27T10:36:20Z SO: GTPase domain 0.9993537 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 structure_element SO: cleaner0 2023-07-27T10:36:20Z GTPase domain 0.99903613 structure_element cleaner0 2023-07-27T12:34:04Z SO: β-sheet 0.99515283 structure_element cleaner0 2023-07-27T12:34:08Z SO: helices 0.9993558 structure_element cleaner0 2023-07-27T10:41:52Z SO: helical domain 0.9986992 structure_element cleaner0 2023-07-27T12:34:10Z SO: helices 0.999157 structure_element cleaner0 2023-07-27T12:34:13Z SO: αA-C 0.99909174 structure_element cleaner0 2023-07-27T12:34:15Z SO: helix 0.9992301 structure_element cleaner0 2023-07-27T12:34:18Z SO: αF-L 0.99927294 structure_element cleaner0 2023-07-27T10:36:20Z SO: GTPase domain 0.9981309 evidence cleaner0 2023-07-27T10:48:13Z DUMMY: root mean square deviations 0.9978098 evidence cleaner0 2023-07-27T10:48:17Z DUMMY: rmsd 0.998288 evidence cleaner0 2023-07-27T12:42:29Z DUMMY: structures 0.99807537 evidence cleaner0 2023-07-27T12:42:34Z DUMMY: structure 0.99919444 protein_state cleaner0 2023-07-27T10:48:08Z DUMMY: native 0.9989428 protein_state cleaner0 2023-07-27T10:45:26Z DUMMY: GMPPNP-bound 0.999371 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.99834836 evidence cleaner0 2023-07-27T10:48:17Z DUMMY: rmsd RESULTS paragraph 8805 The seven Irga6 molecules in the asymmetric unit form various higher order contacts in the crystals. Within the asymmetric unit, six molecules dimerize via the symmetric backside dimer interface (buried surface area 930 Å2), and the remaining seventh molecule forms the same type of interaction with its symmetry mate of the adjacent asymmetric unit (Additional file 1: Figure S2a, b, Figure S3). This indicates that the introduced mutations in the secondary patch, from which only Lys176 is part of the backside interface, do, in fact, not prevent this interaction. 0.9991252 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.98424673 evidence cleaner0 2023-07-27T12:42:37Z DUMMY: crystals 0.99772984 oligomeric_state cleaner0 2023-07-27T12:30:12Z DUMMY: dimerize 0.96250945 site cleaner0 2023-07-27T10:48:42Z SO: backside dimer interface 0.9550488 experimental_method cleaner0 2023-07-27T12:44:31Z MESH: mutations 0.9822448 site cleaner0 2023-07-27T10:48:50Z SO: secondary patch 0.9995739 residue_name_number cleaner0 2023-07-27T10:48:33Z DUMMY: Lys176 0.9989483 site cleaner0 2023-07-27T10:46:44Z SO: backside interface RESULTS paragraph 9373 Another assembly interface with a buried surface area of 450 Å2, which we call the “tertiary patch”, was formed via two interaction sites in the helical domains (Additional file 1: Figure S2c, d, S3). In this interface, helices αK from two adjacent molecules form a hydrogen bonding network involving residues 373-376. Furthermore, two adjacent helices αA form hydrophobic contacts. It was previously shown that the double mutation L372R/A373R did not prevent GTP-induced assembly, so there is currently no evidence supporting an involvement of this interface in higher-order oligomerization. 0.99892914 site cleaner0 2023-07-27T12:39:10Z SO: assembly interface 0.99890065 site cleaner0 2023-07-27T10:48:59Z SO: tertiary patch 0.99861753 site cleaner0 2023-07-27T12:39:15Z SO: interaction sites 0.9989256 structure_element cleaner0 2023-07-27T12:34:22Z SO: helical domains 0.99846673 site cleaner0 2023-07-27T12:39:18Z SO: interface 0.999212 structure_element cleaner0 2023-07-27T12:34:25Z SO: helices 0.9995142 structure_element cleaner0 2023-07-27T12:34:28Z SO: αK 0.99816227 bond_interaction cleaner0 2023-07-27T10:49:12Z MESH: hydrogen bonding network 0.99330187 residue_range cleaner0 2023-07-27T10:49:40Z DUMMY: 373-376 0.9990397 structure_element cleaner0 2023-07-27T12:34:30Z SO: helices 0.9995098 structure_element cleaner0 2023-07-27T12:34:33Z SO: αA 0.99618495 bond_interaction cleaner0 2023-07-27T10:49:17Z MESH: hydrophobic contacts 0.9097497 protein_state cleaner0 2023-07-27T10:49:30Z DUMMY: double mutation 0.9991792 mutant cleaner0 2023-07-27T10:49:33Z MESH: L372R 0.999008 mutant cleaner0 2023-07-27T10:49:37Z MESH: A373R 0.9986527 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.998389 site cleaner0 2023-07-27T12:39:24Z SO: interface RESULTS paragraph 9974 Strikingly, molecule A of one asymmetric unit assembled with an equivalent molecule of the adjacent asymmetric unit via the G-interface in a symmetric parallel fashion via a 470 Å2 interface. This assembly results in a butterfly-shaped Irga6 dimer in which the helical domains protrude in parallel orientations (Fig. 1b, Additional file 1: Figure S3). In contrast, the other six molecules in the asymmetric unit do not assemble via the G interface. 0.76502633 structure_element cleaner0 2023-07-27T10:49:47Z SO: A 0.9990626 site cleaner0 2023-07-27T12:39:30Z SO: G-interface protein_state DUMMY: cleaner0 2023-07-27T12:09:49Z parallel 0.99863386 protein_state cleaner0 2023-07-27T10:49:48Z DUMMY: butterfly-shaped 0.99940443 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.99864584 oligomeric_state cleaner0 2023-07-27T10:37:33Z DUMMY: dimer 0.9984453 structure_element cleaner0 2023-07-27T12:34:39Z SO: helical domains protein_state DUMMY: cleaner0 2023-07-27T12:09:49Z parallel 0.99906284 site cleaner0 2023-07-27T10:43:00Z SO: G interface RESULTS paragraph 10425 The G interface in molecule A can be subdivided into three distinct contact sites (Fig. 1c, d). Contact site I is formed between R159 and K161 in the trans stabilizing loops, and S132 in the switch II regions of the opposing molecules. Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. In contact site III, G103 of switch I interacts via its main chain nitrogen with the exocyclic 2’-OH and 3’-OH groups of the opposing ribose in trans, whereas the two opposing exocyclic 3’-OH group of the ribose form hydrogen bonds with each other. Via the ribose contact, switch I is pulled towards the opposing nucleotide (Fig. 1e). In turn, E106 of switch I reorients towards the nucleotide and now participates in the coordination of the Mg2+ ion (Fig. 1e, Additional file 1: Figure S4). E106 was previously shown to be essential for catalysis, and the observed interactions in contact site III explain how dimerization via the ribose is directly coupled to the activation of GTP hydrolysis. 0.9990487 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.9987433 site cleaner0 2023-07-27T12:39:36Z SO: contact sites 0.99637073 site cleaner0 2023-07-27T10:51:05Z SO: Contact site I 0.9995734 residue_name_number cleaner0 2023-07-27T10:51:14Z DUMMY: R159 0.9995622 residue_name_number cleaner0 2023-07-27T10:51:19Z DUMMY: K161 structure_element SO: cleaner0 2023-07-27T12:31:43Z trans stabilizing loops 0.999587 residue_name_number cleaner0 2023-07-27T10:51:25Z DUMMY: S132 0.9942776 site cleaner0 2023-07-27T10:35:39Z SO: switch II 0.9976573 site cleaner0 2023-07-27T10:51:10Z SO: Contact site II 0.99633247 bond_interaction cleaner0 2023-07-27T10:51:30Z MESH: polar and hydrophobic interactions 0.998207 site cleaner0 2023-07-27T10:35:35Z SO: switch I 0.9996013 residue_name_number cleaner0 2023-07-27T10:51:34Z DUMMY: V104 0.9996019 residue_name_number cleaner0 2023-07-27T10:51:38Z DUMMY: V107 0.7647327 structure_element cleaner0 2023-07-27T12:34:43Z SO: helix 0.99911284 structure_element cleaner0 2023-07-27T10:50:53Z SO: guanine specificity motif 0.9983238 structure_element cleaner0 2023-07-27T10:50:57Z SO: G4 helix 0.99954104 residue_name_number cleaner0 2023-07-27T10:51:42Z DUMMY: K184 0.99954873 residue_name_number cleaner0 2023-07-27T10:51:47Z DUMMY: S187 structure_element SO: cleaner0 2023-07-27T12:27:32Z trans stabilizing loop 0.9995708 residue_name_number cleaner0 2023-07-27T10:51:53Z DUMMY: T158 0.99916863 structure_element cleaner0 2023-07-27T10:36:20Z SO: GTPase domain 0.99693394 site cleaner0 2023-07-27T10:52:29Z SO: contact site III 0.9996068 residue_name_number cleaner0 2023-07-27T10:52:22Z DUMMY: G103 0.997262 site cleaner0 2023-07-27T10:35:35Z SO: switch I 0.99728584 chemical cleaner0 2023-07-27T12:32:30Z CHEBI: ribose 0.9979128 chemical cleaner0 2023-07-27T12:32:33Z CHEBI: ribose 0.99671197 bond_interaction cleaner0 2023-07-27T10:52:41Z MESH: hydrogen bonds 0.97772825 chemical cleaner0 2023-07-27T12:32:37Z CHEBI: ribose 0.9978828 site cleaner0 2023-07-27T10:35:35Z SO: switch I 0.85826856 chemical cleaner0 2023-07-27T12:32:41Z CHEBI: nucleotide 0.9996145 residue_name_number cleaner0 2023-07-27T10:52:35Z DUMMY: E106 0.997312 site cleaner0 2023-07-27T10:35:35Z SO: switch I 0.88709295 chemical cleaner0 2023-07-27T12:32:44Z CHEBI: nucleotide bond_interaction MESH: cleaner0 2023-07-27T10:52:59Z coordination of 0.9984646 chemical cleaner0 2023-07-27T10:52:25Z CHEBI: Mg2+ 0.999617 residue_name_number cleaner0 2023-07-27T10:52:35Z DUMMY: E106 0.99557066 site cleaner0 2023-07-27T10:52:29Z SO: contact site III 0.99209434 chemical cleaner0 2023-07-27T12:32:53Z CHEBI: ribose 0.9988549 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP RESULTS paragraph 11606 The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis. All of these residues directly participate in contacts (G103, S132, R159, and K161) or are in direct vicinity to the interface (E77, E106, K162, D164, and N191). Residues E77, K162, and D164 appear to orient the trans stabilizing loop which is involved in interface formation in contact site II. In the earlier model of an anti-parallel G interface, it was not possible to position the side chain of R159 to avoid steric conflict. In the present structure, the side-chain of R159 projects laterally along the G interface and, therefore, does not cause a steric conflict. 0.999061 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.9994791 residue_name_number cleaner0 2023-07-27T12:08:18Z DUMMY: E77 0.9994943 residue_name_number cleaner0 2023-07-27T10:52:22Z DUMMY: G103 0.9994764 residue_name_number cleaner0 2023-07-27T10:52:35Z DUMMY: E106 0.99947697 residue_name_number cleaner0 2023-07-27T10:51:25Z DUMMY: S132 0.9993932 residue_name_number cleaner0 2023-07-27T10:51:15Z DUMMY: R159 0.9993832 residue_name_number cleaner0 2023-07-27T10:51:20Z DUMMY: K161 0.9993795 residue_name_number cleaner0 2023-07-27T12:08:37Z DUMMY: K162 0.99940157 residue_name_number cleaner0 2023-07-27T12:08:41Z DUMMY: D164 0.999419 residue_name_number cleaner0 2023-07-27T12:08:45Z DUMMY: N191 0.999411 residue_name_number cleaner0 2023-07-27T12:08:50Z DUMMY: K196 0.9982338 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.9994584 residue_name_number cleaner0 2023-07-27T10:52:22Z DUMMY: G103 0.999456 residue_name_number cleaner0 2023-07-27T10:51:25Z DUMMY: S132 0.9993513 residue_name_number cleaner0 2023-07-27T10:51:15Z DUMMY: R159 0.99934167 residue_name_number cleaner0 2023-07-27T10:51:20Z DUMMY: K161 0.99899274 site cleaner0 2023-07-27T12:39:43Z SO: interface 0.9994425 residue_name_number cleaner0 2023-07-27T12:08:18Z DUMMY: E77 0.99947435 residue_name_number cleaner0 2023-07-27T10:52:35Z DUMMY: E106 0.99937785 residue_name_number cleaner0 2023-07-27T12:08:37Z DUMMY: K162 0.99940526 residue_name_number cleaner0 2023-07-27T12:08:41Z DUMMY: D164 0.99940145 residue_name_number cleaner0 2023-07-27T12:08:46Z DUMMY: N191 0.9994404 residue_name_number cleaner0 2023-07-27T12:08:18Z DUMMY: E77 0.9993716 residue_name_number cleaner0 2023-07-27T12:08:37Z DUMMY: K162 0.9993992 residue_name_number cleaner0 2023-07-27T12:08:41Z DUMMY: D164 structure_element SO: cleaner0 2023-07-27T12:27:32Z trans stabilizing loop 0.9987205 site cleaner0 2023-07-27T12:39:48Z SO: interface 0.99573773 site cleaner0 2023-07-27T10:51:10Z SO: contact site II protein_state DUMMY: cleaner0 2023-07-27T12:08:08Z anti-parallel site SO: cleaner0 2023-07-27T10:43:00Z G interface 0.9994363 residue_name_number cleaner0 2023-07-27T10:51:15Z DUMMY: R159 0.9985574 evidence cleaner0 2023-07-27T12:42:42Z DUMMY: structure 0.9994172 residue_name_number cleaner0 2023-07-27T10:51:15Z DUMMY: R159 0.999063 site cleaner0 2023-07-27T10:43:00Z SO: G interface 12915_2016_236_Fig2_HTML.jpg Fig2 FIG fig_caption 12414 A conserved dimerization mode via the G interface in dynamin and septin GTPases. The overall architecture of the parallel GTPase domain dimer of Irga6 is related to that of other dynamin and septin superfamily proteins. The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. The GTPase domains of the left molecules are shown in orange, helical domains or extensions in blue. Nucleotide, Mg2+ (green) and AlF4 - are shown in sphere representation, the buried interface sizes per molecule are indicated on the right. Irga6 immunity-related GTPase 6, GMPPNP 5'-guanylyl imidodiphosphate, GTP guanosine-triphosphate, BDLP bacterial dynamin like protein, GIMAP2, GTPase of immunity associated protein 2 0.9989195 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.998784 protein_type cleaner0 2023-07-27T10:36:30Z MESH: dynamin 0.99875796 protein_type cleaner0 2023-07-27T12:10:14Z MESH: septin GTPases protein_state DUMMY: cleaner0 2023-07-27T12:09:49Z parallel structure_element SO: cleaner0 2023-07-27T10:36:20Z GTPase domain 0.9963877 oligomeric_state cleaner0 2023-07-27T10:37:33Z DUMMY: dimer 0.99923456 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9982521 protein_type cleaner0 2023-07-27T10:36:30Z MESH: dynamin protein_type MESH: cleaner0 2023-07-27T10:36:52Z septin 0.99592704 evidence cleaner0 2023-07-27T12:42:47Z DUMMY: structures 0.9990865 structure_element cleaner0 2023-07-27T10:34:08Z SO: GTPase domains 0.9987429 protein_state cleaner0 2023-07-27T10:45:26Z DUMMY: GMPPNP-bound 0.99917185 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.99885464 oligomeric_state cleaner0 2023-07-27T10:37:33Z DUMMY: dimer 0.99870783 protein_state cleaner0 2023-07-27T12:33:13Z DUMMY: GDP-AlF4 --bound 0.9958458 protein cleaner0 2023-07-27T12:10:59Z PR: dynamin 1 structure_element SO: cleaner0 2023-07-27T12:36:38Z GTPase-minimal 0.9989162 protein_state cleaner0 2023-07-27T10:44:10Z DUMMY: GDP-bound 0.99906284 protein cleaner0 2023-07-27T12:11:08Z PR: atlastin 1 0.9988581 oligomeric_state cleaner0 2023-07-27T10:37:33Z DUMMY: dimer 0.99890757 protein_state cleaner0 2023-07-27T12:33:17Z DUMMY: GDP-AlF3- bound 0.99915755 protein cleaner0 2023-07-27T12:28:42Z PR: GBP1 0.99882436 structure_element cleaner0 2023-07-27T10:36:20Z SO: GTPase domain 0.9982911 oligomeric_state cleaner0 2023-07-27T10:37:33Z DUMMY: dimer 0.9982279 protein_type cleaner0 2023-07-27T10:44:54Z MESH: BDLP 0.9988255 oligomeric_state cleaner0 2023-07-27T10:37:33Z DUMMY: dimer 0.9990673 protein_state cleaner0 2023-07-27T10:34:29Z DUMMY: bound to 0.9962359 chemical cleaner0 2023-07-27T12:40:34Z CHEBI: GDP 0.99897075 protein_state cleaner0 2023-07-27T12:11:28Z DUMMY: GTP-bound 0.9991867 protein cleaner0 2023-07-27T12:12:18Z PR: GIMAP2 0.998862 oligomeric_state cleaner0 2023-07-27T10:37:33Z DUMMY: dimer 0.9988321 structure_element cleaner0 2023-07-27T10:34:08Z SO: GTPase domains 0.9896811 structure_element cleaner0 2023-07-27T12:36:44Z SO: helical domains 0.9989772 chemical cleaner0 2023-07-27T12:11:32Z CHEBI: Mg2+ 0.99882233 chemical cleaner0 2023-07-27T12:11:37Z CHEBI: AlF4 - 0.99710995 site cleaner0 2023-07-27T12:39:54Z SO: interface 0.99926573 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.998515 protein cleaner0 2023-07-27T10:47:22Z PR: immunity-related GTPase 6 0.99926454 chemical cleaner0 2023-07-27T10:43:26Z CHEBI: GMPPNP 0.99905527 chemical cleaner0 2023-07-27T12:40:39Z CHEBI: 5'-guanylyl imidodiphosphate 0.9871029 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.99913836 chemical cleaner0 2023-07-27T12:40:43Z CHEBI: guanosine-triphosphate 0.99609816 protein_type cleaner0 2023-07-27T10:44:54Z MESH: BDLP taxonomy_domain DUMMY: cleaner0 2023-07-27T10:42:55Z bacterial protein_type MESH: cleaner0 2023-07-27T12:12:13Z dynamin like protein 0.99891984 protein cleaner0 2023-07-27T12:12:18Z PR: GIMAP2 0.99787754 protein cleaner0 2023-07-27T12:12:25Z PR: GTPase of immunity associated protein 2 RESULTS paragraph 13467 The buried surface area per molecule (BSA) of the G interface in Irga6 is relatively small (470 Å2) compared to that of other dynamin superfamily members, such as dynamin (BSA: 1400 Å2), atlastin (BSA: 820 Å2), GBP-1 (BSA: 2060 Å2), BDLP (BSA: 2300 Å2) or the septin-related GTPase of immunity associated protein 2 (GIMAP2) (BSA: 590 Å2) (Fig. 2). However, the relative orientations of the GTPase domains in these dimers are strikingly similar, and the same elements, such as switch I, switch II, the trans activating and G4 loops are involved in the parallel dimerization mode in all of these GTPase families. 0.9989669 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.99922585 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 protein_type MESH: cleaner0 2023-07-27T10:36:30Z dynamin 0.99929154 protein_type cleaner0 2023-07-27T10:36:30Z MESH: dynamin 0.99930716 protein_type cleaner0 2023-07-27T12:13:53Z MESH: atlastin 0.99926883 protein cleaner0 2023-07-27T10:42:47Z PR: GBP-1 0.9990445 protein_type cleaner0 2023-07-27T10:44:54Z MESH: BDLP 0.99843323 protein cleaner0 2023-07-27T12:12:49Z PR: septin-related GTPase of immunity associated protein 2 0.9993419 protein cleaner0 2023-07-27T12:12:18Z PR: GIMAP2 0.99648523 structure_element cleaner0 2023-07-27T10:34:08Z SO: GTPase domains 0.9986093 oligomeric_state cleaner0 2023-07-27T12:30:22Z DUMMY: dimers 0.9986375 site cleaner0 2023-07-27T10:35:35Z SO: switch I 0.9985064 site cleaner0 2023-07-27T10:35:39Z SO: switch II 0.9989953 structure_element cleaner0 2023-07-27T12:36:52Z SO: trans activating and G4 loops 0.89991593 protein_state cleaner0 2023-07-27T12:09:49Z DUMMY: parallel protein_type MESH: cleaner0 2023-07-27T12:28:06Z GTPase DISCUSS title_1 14085 Discussion DISCUSS paragraph 14096 IRG proteins are crucial mediators of the innate immune response in mice against a specific subset of intracellular pathogens, all of which enter the cell to form a membrane-bounded vacuole without engagement of the phagocytic machinery. As members of the dynamin superfamily, IRGs oligomerize at cellular membranes in response to GTP binding. Oligomerization and oligomerization-induced GTP hydrolysis are thought to induce membrane remodeling events ultimately leading to disruption of the PVM. Recent structural and mechanistic analyses have begun to unravel the molecular basis for the membrane-remodeling activity and mechano-chemical function of some members (reviewed in). For example, for dynamin and atlastin, it was shown that GTP binding and/or hydrolysis leads to dimerization of the GTPase domains and to the reorientation of the adjacent helical domains. The resulting domain movement was suggested to act as a “power stroke” during membrane remodeling events. However, for other dynamin superfamily members such as IRGs, the molecular basis for GTP hydrolysis and the exact role of the mechano-chemical function are still unclear. protein_type MESH: cleaner0 2023-07-27T10:37:26Z IRG 0.99626875 taxonomy_domain cleaner0 2023-07-27T10:38:34Z DUMMY: mice protein_type MESH: cleaner0 2023-07-27T10:36:30Z dynamin 0.99929845 protein_type cleaner0 2023-07-27T10:33:43Z MESH: IRGs 0.99870706 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.9958644 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.9985744 experimental_method cleaner0 2023-07-27T12:44:55Z MESH: structural and mechanistic analyses 0.97879887 protein_type cleaner0 2023-07-27T10:36:30Z MESH: dynamin 0.99768 protein_type cleaner0 2023-07-27T12:13:52Z MESH: atlastin 0.9983102 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.9991524 structure_element cleaner0 2023-07-27T10:34:08Z SO: GTPase domains 0.99791276 structure_element cleaner0 2023-07-27T12:36:56Z SO: helical domains protein_type MESH: cleaner0 2023-07-27T10:36:30Z dynamin 0.99784553 protein_type cleaner0 2023-07-27T10:33:43Z MESH: IRGs 0.9967393 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP DISCUSS paragraph 15246 Our structural analysis of an oligomerization- and GTPase-defective Irga6 mutant indicates that Irga6 dimerizes via the G interface in a parallel orientation. Only one of the seven Irga6 molecules in the asymmetric unit formed this contact pointing to a low affinity interaction via the G interface, which is in agreement with its small size. In the crystals, dimerization via the G interface is promoted by the high protein concentrations which may mimic a situation when Irga6 oligomerizes on a membrane surface. Such a low affinity interaction mode may allow reversibility of oligomerization following GTP hydrolysis. Similar low affinity G interface interactions were reported for dynamin and MxA. 0.9986919 experimental_method cleaner0 2023-07-27T12:44:58Z MESH: structural analysis protein_state DUMMY: cleaner0 2023-07-27T12:14:27Z oligomerization- and GTPase-defective 0.99571157 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.99869686 protein_state cleaner0 2023-07-27T10:34:25Z DUMMY: mutant 0.99924856 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.99752396 oligomeric_state cleaner0 2023-07-27T10:33:31Z DUMMY: dimerizes 0.9991144 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.79277337 protein_state cleaner0 2023-07-27T12:09:49Z DUMMY: parallel 0.9992861 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9991021 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.99705374 evidence cleaner0 2023-07-27T12:42:53Z DUMMY: crystals 0.99911535 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.99934715 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9984047 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.9991163 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.9990403 protein_type cleaner0 2023-07-27T10:36:30Z MESH: dynamin 0.9990915 protein cleaner0 2023-07-27T10:42:39Z PR: MxA DISCUSS paragraph 15948 The dimerization mode is strikingly different from the previously proposed anti-parallel model that was based on the crystal structure of the signal recognition particle GTPase, SRP54 and its homologous receptor. However, the G dimer interface is reminiscent of the GTPase domain dimers observed for several other dynamin superfamily members, such as dynamin, GBP1, atlastin, and BDLP. It was recently shown that septin and septin-related GTPases, such as the Tocs GTPases or GTPases of immunity related proteins (GIMAPs), also employ a GTP-dependent parallel dimerization mode. Based on phylogenetic and structural analysis, these observations suggest that dynamin and septin superfamilies are derived from a common ancestral membrane-associated GTPase that featured a GTP-dependent parallel dimerization mode. Importantly, our analysis indicates that IRGs are not outliers, but bona-fide representatives of the dynamin superfamily. 0.90689725 protein_state cleaner0 2023-07-27T12:10:00Z DUMMY: anti-parallel 0.99784887 evidence cleaner0 2023-07-27T12:42:57Z DUMMY: crystal structure protein_type MESH: cleaner0 2023-07-27T12:29:15Z signal recognition particle GTPase 0.99929106 protein cleaner0 2023-07-27T12:29:19Z PR: SRP54 0.9989607 site cleaner0 2023-07-27T12:14:51Z SO: G dimer interface structure_element SO: cleaner0 2023-07-27T12:15:22Z GTPase domain oligomeric_state DUMMY: cleaner0 2023-07-27T12:15:33Z dimers protein_type MESH: cleaner0 2023-07-27T10:36:30Z dynamin 0.9990158 protein_type cleaner0 2023-07-27T10:36:30Z MESH: dynamin 0.9991697 protein cleaner0 2023-07-27T12:29:23Z PR: GBP1 0.9991943 protein_type cleaner0 2023-07-27T12:13:53Z MESH: atlastin 0.9991836 protein_type cleaner0 2023-07-27T10:44:54Z MESH: BDLP 0.9989994 protein_type cleaner0 2023-07-27T10:36:52Z MESH: septin 0.99870986 protein_type cleaner0 2023-07-27T12:15:51Z MESH: septin-related GTPases 0.99816024 protein_type cleaner0 2023-07-27T12:28:22Z MESH: Tocs GTPases 0.9955791 protein_type cleaner0 2023-07-27T12:15:54Z MESH: GTPases of immunity related proteins 0.9981299 protein_type cleaner0 2023-07-27T12:15:58Z MESH: GIMAPs 0.9936714 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.9250834 protein_state cleaner0 2023-07-27T12:09:50Z DUMMY: parallel 0.9989378 experimental_method cleaner0 2023-07-27T12:45:04Z MESH: phylogenetic and structural analysis 0.99865425 protein_type cleaner0 2023-07-27T10:36:30Z MESH: dynamin protein_type MESH: cleaner0 2023-07-27T10:36:52Z septin 0.998357 protein_type cleaner0 2023-07-27T12:16:01Z MESH: membrane-associated GTPase 0.9872011 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.71070504 protein_state cleaner0 2023-07-27T12:09:50Z DUMMY: parallel 0.99881625 protein_type cleaner0 2023-07-27T10:33:43Z MESH: IRGs protein_type MESH: cleaner0 2023-07-27T10:36:30Z dynamin DISCUSS paragraph 16882 Whereas the overall dimerization mode is similar in septin and dynamin GTPases, family-specific differences in the G interface and the oligomerization interfaces exist. For example, the involvement of the 2’ and 3’-OH groups of the ribose in the dimerization interface of Irga6 has not been observed for other dynamin and septin superfamily members. The surface-exposed location of the ribose in the Irga6 structure, with a wide-open nucleotide-binding pocket, facilitates its engagement in the dimerization interface. This contact, in turn, appears to activate GTP hydrolysis by inducing rearrangements in switch I and the positioning of the catalytic E106. During dimerization of GBP1, an arginine finger from the P loop reorients towards the nucleotide in cis to trigger GTP hydrolysis. In dynamin, the corresponding serine residue coordinates a sodium ion that is crucial for GTP hydrolysis. Irga6 bears Gly79 at this position, which in the dimerizing molecule A appears to approach the bridging imido group of GMPPNP via a main chain hydrogen bond. Higher resolution structures of the Irga6 dimer in the presence of a transition state analogue are required to show whether Gly79 directly participates in GTP hydrolysis or whether it may also position a catalytic cation. 0.99877435 protein_type cleaner0 2023-07-27T10:36:52Z MESH: septin 0.9981336 protein_type cleaner0 2023-07-27T12:16:25Z MESH: dynamin GTPases 0.9990227 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.9990978 site cleaner0 2023-07-27T12:39:59Z SO: oligomerization interfaces 0.99857116 chemical cleaner0 2023-07-27T12:40:48Z CHEBI: ribose 0.9990822 site cleaner0 2023-07-27T10:41:24Z SO: dimerization interface 0.99931896 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.99840385 protein_type cleaner0 2023-07-27T10:36:30Z MESH: dynamin protein_type MESH: cleaner0 2023-07-27T10:36:52Z septin 0.9993469 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9976046 evidence cleaner0 2023-07-27T12:43:05Z DUMMY: structure 0.95810074 protein_state cleaner0 2023-07-27T12:33:29Z DUMMY: wide-open 0.9990585 site cleaner0 2023-07-27T12:40:04Z SO: nucleotide-binding pocket 0.9991007 site cleaner0 2023-07-27T10:41:24Z SO: dimerization interface 0.9983584 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.9976095 site cleaner0 2023-07-27T10:35:35Z SO: switch I 0.9610252 protein_state cleaner0 2023-07-27T12:17:07Z DUMMY: catalytic 0.9995932 residue_name_number cleaner0 2023-07-27T10:52:35Z DUMMY: E106 0.9994085 protein cleaner0 2023-07-27T12:29:27Z PR: GBP1 0.9987834 structure_element cleaner0 2023-07-27T12:17:24Z SO: arginine finger 0.99936163 structure_element cleaner0 2023-07-27T12:17:30Z SO: P loop 0.9880989 chemical cleaner0 2023-07-27T12:40:53Z CHEBI: nucleotide 0.9986308 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.99870455 protein_type cleaner0 2023-07-27T10:36:30Z MESH: dynamin 0.99738985 residue_name cleaner0 2023-07-27T12:17:18Z SO: serine 0.97945315 chemical cleaner0 2023-07-27T12:17:15Z CHEBI: sodium 0.99860805 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP 0.99933213 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9995295 residue_name_number cleaner0 2023-07-27T12:16:51Z DUMMY: Gly79 0.9914948 oligomeric_state cleaner0 2023-07-27T12:30:30Z DUMMY: dimerizing 0.6915696 structure_element cleaner0 2023-07-27T12:37:02Z SO: A 0.99933606 chemical cleaner0 2023-07-27T10:43:26Z CHEBI: GMPPNP 0.9966464 bond_interaction cleaner0 2023-07-27T12:16:45Z MESH: hydrogen bond 0.99851006 evidence cleaner0 2023-07-27T12:43:10Z DUMMY: structures 0.99937075 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9987482 oligomeric_state cleaner0 2023-07-27T10:37:33Z DUMMY: dimer 0.8419397 protein_state cleaner0 2023-07-27T10:44:05Z DUMMY: presence of 0.99955744 residue_name_number cleaner0 2023-07-27T12:16:52Z DUMMY: Gly79 0.99833554 chemical cleaner0 2023-07-27T10:35:02Z CHEBI: GTP DISCUSS paragraph 18162 In dynamin, further assembly sites are provided by the helical domains which assemble in a criss-cross fashion to form a helical filament. In dynamin-related Eps15 homology domain containing proteins (EHDs), a second assembly interface is present in the GTPase domain. For Irga6, additional interfaces in the helical domain are presumably involved in oligomerization, such as the secondary patch residues whose mutation prevented oligomerization in the crystallized mutant. Further structural studies, especially electron microscopy analysis of the Irga6 oligomers, are required to clarify the assembly mode via the helical domains and to show how these interfaces cooperate with the G interface to mediate the regulated assembly on a membrane surface. Notably, we did not observe major rearrangements of the helical domain versus the GTPase domain in the Irga6 molecules that dimerized via the G interface. In a manner similar to BDLP, such large-scale conformational changes may be induced by membrane binding. Our structural analysis and the identification of the G-interface paves the way for determining the specific assembly of Irga6 into a membrane-associated scaffold as the prerequisite to understand its action as an anti-parasitic machine. 0.8942208 protein_type cleaner0 2023-07-27T10:36:30Z MESH: dynamin 0.99581313 site cleaner0 2023-07-27T12:17:56Z SO: assembly sites 0.99914 structure_element cleaner0 2023-07-27T12:18:05Z SO: helical domains 0.9960346 structure_element cleaner0 2023-07-27T12:18:07Z SO: helical filament 0.99539596 protein_type cleaner0 2023-07-27T12:17:42Z MESH: dynamin-related Eps15 homology domain containing proteins 0.99789655 protein_type cleaner0 2023-07-27T12:17:45Z MESH: EHDs 0.998976 site cleaner0 2023-07-27T12:17:58Z SO: second assembly interface 0.9990665 structure_element cleaner0 2023-07-27T10:36:20Z SO: GTPase domain 0.99937326 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9970872 site cleaner0 2023-07-27T12:40:07Z SO: interfaces 0.99917567 structure_element cleaner0 2023-07-27T10:41:52Z SO: helical domain 0.99605507 site cleaner0 2023-07-27T10:48:51Z SO: secondary patch 0.9845604 experimental_method cleaner0 2023-07-27T12:45:15Z MESH: mutation 0.60325915 evidence cleaner0 2023-07-27T12:43:14Z DUMMY: crystallized 0.53057414 protein_state cleaner0 2023-07-27T10:34:25Z DUMMY: mutant 0.9892376 experimental_method cleaner0 2023-07-27T12:45:18Z MESH: structural studies experimental_method MESH: cleaner0 2023-07-27T12:45:38Z electron microscopy analysis 0.99935025 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.9985233 oligomeric_state cleaner0 2023-07-27T10:40:32Z DUMMY: oligomers 0.9989483 structure_element cleaner0 2023-07-27T12:37:06Z SO: helical domains 0.99807835 site cleaner0 2023-07-27T12:40:10Z SO: interfaces 0.99912065 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.99904406 structure_element cleaner0 2023-07-27T10:41:52Z SO: helical domain 0.99897075 structure_element cleaner0 2023-07-27T10:36:20Z SO: GTPase domain 0.9993631 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 0.99770314 protein_state cleaner0 2023-07-27T12:30:44Z DUMMY: dimerized 0.9990767 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.6128288 protein_type cleaner0 2023-07-27T10:44:54Z MESH: BDLP 0.995021 experimental_method cleaner0 2023-07-27T12:45:44Z MESH: structural analysis 0.999104 site cleaner0 2023-07-27T12:39:31Z SO: G-interface 0.99935454 protein cleaner0 2023-07-27T10:33:26Z PR: Irga6 METHODS title_1 19413 Methods METHODS title_2 19421 Protein expression and purification METHODS paragraph 19457 Selenomethionine-substituted Mus musculus Irga6R31E, K32E, K176E, K246E was expressed as a GST-fusion from the vector pGEX-4T-2 in BL21 Rosetta2(DE3) cells according to reference. Protein was purified as previously described and the protein stored in small aliquots at a concentration of 118 mg/mL in 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 2 mM DTT. METHODS title_2 19804 Biochemical analyses METHODS paragraph 19825 Oligomerization and GTPase assays for the Irga6R31E, K32E, K176E, K246E mutant were carried out as described in. METHODS title_2 19938 Protein crystallization METHODS paragraph 19962 The protein was gently thawed on ice and diluted to a final concentration of 10 mg/mL with buffer containing 20 mM Tris-HCl, pH 7.5, 8 mM MgCl2, 3 mM DTT. GMPPNP was added to a final concentration of 2 mM. Crystallization was carried out in a 96 well format using the sitting drop vapor diffusion method. The reservoir contained 100 mM HEPES-NaOH pH 7.0, 9 % PEG4000, 6 % isopropanol. The sitting drop was set up using an Art Robbins Gryphon system and consisted of 200 nL protein solution and 200 nL reservoir solution. METHODS paragraph 20483 For cryo-protection, crystals were transferred into a cryo solution containing 33 % PEG4000, 3 % isopropanol, 50 mM HEPES pH 7.0, 4 mM MgCl2, 2 mM DTT, and 2 mM GMPPNP at 4 °C for at least 5 sec. Crystals were screened for diffraction at beamline BL 14.1 at BESSY II, Berlin, Germany. METHODS title_2 20769 Data collection METHODS paragraph 20785 All data were recorded at beamline P11 at PETRA III, DESY Hamburg, Germany using a PILATUS 6 M detector. To achieve spot separation along the long cell axis, three data sets were collected with a φ increment of 0.05/0.1° at a temperature of 100 K using detector distances between 1300 and 598.5 mm (Additional file 1: Table S1). The wavelength was 0.972/0.979 Å. Calculation of an optimal data collection strategy was done with the Mosflm software. The high- and low-resolution datasets were processed and merged using the XDS program suite. METHODS title_2 21330 Structure solution and refinement METHODS paragraph 21364 Structure solution was done by molecular replacement with Phaser employing the structure of Irga6 without nucleotide [PDB: 1TQ2] as search model. Atomic model building was done by Coot. Iterative refinement was done using Phenix at a maximum resolution of 3.2 Å. For the refinement strategy, a seven-fold non-crystallographic symmetry as well as one molecule of Irga6 [PDB: 1TQ4] as high resolution reference structure was chosen. Five percent of the measured X-ray intensities were set aside from the refinement as cross-validation. Methionine sites in the protein were confirmed by the anomalous signal of the selenium atoms. Protein superposition was done with lsqkab and the PyMol Molecular Graphics System, Version 1.3 Schrödinger, LLC. Figures were prepared using the PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC. Evaluation of atom contacts and geometry of the atomic model was done by the Molprobity server. Interface sizes were calculated by the PISA server. METHODS title_2 22354 Accession numbers METHODS paragraph 22372 The Irga6 coordinates were submitted to the Protein Data Bank (pdb) database with accession code 5fph. http://www.rcsb.org/pdb/explore/explore.do?structureId=5fph. CONCL title_1 22536 Conclusions CONCL paragraph 22548 Our study indicates that Irg proteins dimerize via the G interface in a parallel head-to-head fashion thereby facilitating GTPase activation. These findings contribute to a molecular understanding of the anti-parasitic action of the Irg protein family and suggest that Irgs are bona-fide members of the dynamin superfamily. protein_type MESH: cleaner0 2023-07-27T10:37:26Z Irg 0.9966157 oligomeric_state cleaner0 2023-07-27T12:30:12Z DUMMY: dimerize 0.9990368 site cleaner0 2023-07-27T10:43:00Z SO: G interface 0.9982672 protein_state cleaner0 2023-07-27T10:33:36Z DUMMY: parallel head-to-head 0.82288593 protein_type cleaner0 2023-07-27T12:28:26Z MESH: GTPase protein_type MESH: cleaner0 2023-07-27T10:37:26Z Irg 0.999027 protein_type cleaner0 2023-07-27T10:33:43Z MESH: Irgs protein_type MESH: cleaner0 2023-07-27T10:36:30Z dynamin CONCL title_1 22872 Additional file ABBR title 22888 Abbreviations ABBR paragraph 22902 BDLP ABBR paragraph 22907 Bacterial dynamin like protein ABBR paragraph 22938 EHD2 ABBR paragraph 22943 Eps15 homology domain containing protein 2 ABBR paragraph 22986 GBP ABBR paragraph 22990 Guanylate-binding protein ABBR paragraph 23016 GDP ABBR paragraph 23020 Guanosine-diphosphate ABBR paragraph 23042 GIMAP2 ABBR paragraph 23049 GTPase of immunity associated protein 2 ABBR paragraph 23089 GMPPNP ABBR paragraph 23096 5'-guanylyl imidodiphosphate ABBR paragraph 23125 GTP ABBR paragraph 23129 Guanosine-triphosphate ABBR paragraph 23152 IRG ABBR paragraph 23156 Immunity-related GTPase ABBR paragraph 23180 Irga6 ABBR paragraph 23186 Immunity-related GTPase 6 ABBR paragraph 23212 MxA ABBR paragraph 23216 Myxovirus resistance protein A ABBR paragraph 23247 PVM ABBR paragraph 23251 Parasitophorous vacuolar membrane ABBR footnote 23285 Competing interests ABBR footnote 23305 The authors declare that they have no competing interests. ABBR footnote 23364 Authors’ contributions ABBR footnote 23389 All authors planned the experimental design. NP cloned, characterized, and purified the Irga6 construct and found initial crystallization conditions. KS and CF optimized the crystallization condition and found suitable cryo conditions. KS and KF collected data, KF solved, and KS and KF refined the structure. KS, NP, CF, JCH, and OD wrote the manuscript. All authors read and approved the final manuscript. 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