Phosphoinositide binding domains: embracing 3-phosphate.

A variety of signaling proteins, including protein-Tyr kinases, Ser/Thr kinases, and GDP/GTP exchange factors have PH domains that are important for function (3xCorvera, S and Czech, M.P. Trends Cell Biol. 1998; 8: 442–446Abstract | Full Text | Full Text PDF | PubMed | Scopus (174)See all References, 8xLeevers, S.J, Vanhaesebroeck, B, and Waterfield, M.D. Curr. Opin. Cell Biol. 1999; 11: 219–225Crossref | PubMed | Scopus (471)See all References, 13xRameh, L.E and Cantley, L.C. J. Biol. Chem. 1999; 274: 8347–8350Crossref | PubMed | Scopus (766)See all References). More than 100 PH domain–containing proteins are predicted based on sequence data banks. Most have not yet been characterized, but where investigated, phosphoinositides have been shown to bind to most of these domains (Rameh et al. 1997axRameh, L, Tolias, K, Duckworth, B, and Cantley, L. Nature. 1997; 390: 192–196Crossref | PubMed | Scopus (291)See all ReferencesRameh et al. 1997a). Those that have been characterized to date fall into four major groups in regard to their phosphate binding specificity (Figure 2BFigure 2B). The PH domains that bind with high specificity to PtdIns-3,4,5-P3 or PtdIns-3,4-P2 (Groups I and III, respectively) have been of particular interest in recent years because of their role in transmitting growth and survival signals downstream of PI3K.Figure 2Phosphoinositide-Binding Pocket of PH Domains(A) The crystal structure of the BTK PH domain bound to inositol-1,3,4,5-tetrakisphosphate (IP4) is presented on the left, and the PH domain of PLCδ1 bound to inositol-1,4,5-trisphosphate is presented on the right. Basic residues from the β1-β2 loop region that interact with the phosphates numbered are indicated. Adapted by Dr. M. Yaffe based on structure from Baraldi et al. 1999xBaraldi, E, Carugo, K.D, Hyvonen, M, Surdo, P.L, Riley, A.M, Potter, B.V, O’Brien, R, Ladbury, J.E, and Saraste, M. Structure. 1999; 7: 449–460Abstract | Full Text | Full Text PDF | PubMed | Scopus (131)See all ReferencesBaraldi et al. 1999.(B) Alignment of residues from the β1-β2 region of the BTK PH domain with homologous regions of other Group I, Group II, Group III, and Group IV PH domains (adapted from Rameh et al., 1997). The numbers above residues of BTK and PLCδ PH domains indicate side chains that contact phosphates at the 1, 3, 4, or 5 positions of the inositol ring. Basic residues are indicated in blue. Lys-12 and Arg-28 of BTK, Lys-30 and Arg-40 of PLCδ, and analogous residues of the other PH domains are indicated in bold.View Large Image | View Hi-Res Image | Download PowerPoint SlidePrevious structures of PH domains bound to the headgroup of PtdIns-4,5-P2, inositol-1,4,5-trisphosphate (IP3), have provided information about residues involved in headgroup recognition but have not revealed how discrimination among related phosphoinositides is accomplished. The most informative of these structures was the phospholipase C (PLC) δ PH domain/IP3 complex (Figure 2AFigure 2A) (Ferguson et al. 1995xFerguson, K.M, Lemmon, M.A, Schlessinger, J, and Sigler, P.B. Cell. 1995; 83: 1037–1046Abstract | Full Text PDF | PubMed | Scopus (460)See all ReferencesFerguson et al. 1995). This structure revealed that the inositol moiety is sandwiched between the β1-β2 loop and the β3-β4 loop at one end of a twisted β barrel. Most of the contact with the phosphates occurs with basic residues from the β1 and β2 strands, most notably, Lys-30 from β1 and Arg-40 from β2 that interact with the 5- and 4-phosphates of IP3. The 1-phosphate is not deeply buried, explaining how, in the case of PtdIns-4,5-P2 binding, linkage through the 1-phosphate to diacylglycerol is accomodated.The crystal structure of the Bruton’s tyrosine kinase (BTK) PH domain/IP4 complex reveals similarities but crucial differences from the PLCδ structure (Baraldi et al. 1999xBaraldi, E, Carugo, K.D, Hyvonen, M, Surdo, P.L, Riley, A.M, Potter, B.V, O’Brien, R, Ladbury, J.E, and Saraste, M. Structure. 1999; 7: 449–460Abstract | Full Text | Full Text PDF | PubMed | Scopus (131)See all ReferencesBaraldi et al. 1999). The IP4 is located at an analogous position between the β1-β2 loop and the β3-β4 loop of the BTK PH domain, and the 1-phosphate is pointed out of the pocket to allow accomodation of the diacyglycerol moiety of PtdIns-3,4,5-P3. However, the inositol ring is flipped over 180° such that the 3-phosphate occupies a position analogous to the 5-phosphate of IP3 in the PLCδ structure. This places the 3-phosphate of IP4 at the deepest part of the pocket in contact with Arg-28 (analogous to Arg-40 of PLCδ) and Lys-12 (analogous to Lys-30 of PLCδ). This structure explains why mutation of Arg-28 to Cys decreases the affinity of the BTK PH domain for PtdIns-3,4,5-P3 but does not significantly affect the ability to bind to PtdIns-4,5-P2 (Rameh et al. 1997bxRameh, L.E, Arvidsson, A, Carraway, K.L.R, Couvillon, A.D, Rathbun, G, Crompton, A, VanRenterghem, B, Czech, M.P, Ravichandran, K.S, Burakoff, S.J et al. J. Biol. Chem. 1997; 272: 22059–22066Crossref | PubMed | Scopus (386)See all ReferencesRameh et al. 1997b). The Arg-28 to Cys mutation was identified as a natural mutation causing a defect in B cell development and signaling, emphasizing the importance of the PtdIns-3,4,5-P3 binding for BTK signaling in B cells. Mutation of the analogous Arg residue in other PtdIns-3,4,5-P3-binding PH domains also compromises binding to PtdIns-3,4,5-P3, suggesting that these domains also bind this lipid in the orientation found in BTK rather than the orientation found in PLCδ.Alignments of the β1-β2 loop regions of Group I, II, III, and IV PH domains (Figure 2BFigure 2B) allows one to rationalize the lipid binding specificity with the primary structure. For example, the Group I, II, and III PH domains have conserved the two critical basic residues in the β1 and β2 strands (analogous to Lys-12 and Arg-28 of BTK). In contrast, Group IV PH domains, such as dynamin, do not have these residues conserved. This probably explains the relatively low affinity of these domains for phosphoinositides. The Group I PH domains have large loop regions between the β1 and β2 strands and have multiple basic residues in this loop region. The BTK crystal structure reveals that the e amino group of Lys-18 in this loop forms a salt bridge with the 5-phosphate of IP4 and that this phosphate is further stabilized by interactions with the backbone of the loop and by the local basic charge provided by Lys-17 and Lys-19 (Figure 2AFigure 2A).Finally, the crystal structure of the BTK PH domain/IP4 complex suggests that binding to PtdIns-3,4,5-P3 on a membrane surface stabilizes BTK in a dimeric state (Figure 3AFigure 3A). Dimer formation could enhance transphosphorylation and activation of BTK, both of which depend on PI3K activation in vivo (Scharenberg et al. 1998xScharenberg, A.M, El-Hillal, O, Fruman, D.A, Beitz, L.O, Li, Z, Lin, S, Gout, I, Cantley, L.C, Rawlings, D.J, and Kinet, J.P. EMBO J. 1998; 17: 1961–1972Crossref | PubMed | Scopus (349)See all ReferencesScharenberg et al. 1998). Interestingly, the crystal structure of the PH domain of an activating mutant of BTK (Glu-41 to Lys) revealed a second, low-affinity IP4-binding site per monomer on the same face as the high-affinity site found on wild-type BTK (Figure 3BFigure 3B). Lys-41 of the mutant contributes a salt bond to the new IP4 site.Figure 3Dimers in the Crystal Structures of BTK PH Domains Bound to IP4(A) Wild-type BTK PH domain.(B) E41K BTK PH domain, each bound to two molecules of IP4. Note that each of the 1-phosphates points in the same direction, suggesting a mechanism for strengthening the avidity of the interaction with PIP3-containing membranes. Adapted by Dr. M. Yaffe based on structure from Baraldi et al. 1999xBaraldi, E, Carugo, K.D, Hyvonen, M, Surdo, P.L, Riley, A.M, Potter, B.V, O’Brien, R, Ladbury, J.E, and Saraste, M. Structure. 1999; 7: 449–460Abstract | Full Text | Full Text PDF | PubMed | Scopus (131)See all ReferencesBaraldi et al. 1999.View Large Image | View Hi-Res Image | Download PowerPoint SlideIn summary, the structural basis for recognition of specific phosphoinositides by FYVE domains and PH domains is beginning to emerge. It is now clear that multiple types of domains have independently evolved pockets for recognition of specific phosphoinositides as a means of regulated recruitment to membranes. In addition, a surprisingly large number of FYVE domain–containing proteins and PH domain–containing proteins with diverse functions are predicted to bind to PI3K products based on the structures discussed here. Whether all these proteins function in PI3K signaling pathways remains to be determined, yet it is clear that localization of signaling proteins at membrane sites where PI3K becomes activated is critical for many types of signaling networks.*To whom correspondence should be addressed (e-mail: cantley@helix.mgh.harvard.edu).