John T. Evans

The family of Ras-like GTPases consists of over 150 different members, regulated by an even larger number of guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs) that comprise cellular switch networks that govern cell motility, growth, polarity, protein trafficking, and gene expression. Efforts to develop selective small molecule probes and drugs for these proteins have been hampered by the high affinity of guanosine triphosphate (GTP) and lack of allosteric regulatory sites. This paradigm was recently challenged by the discovery of a cryptic allosteric pocket in the switch II region of K-Ras. Here, we ask whether similar pockets are present in GTPases beyond K-Ras. We systematically surveyed members of the Ras, Rho, and Rab family of GTPases and found that many GTPases exhibit targetable switch II pockets. Notable differences in the composition and conservation of key residues offer potential for the development of optimized inhibitors for many members of this previously undruggable family.

Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled receptors (GPCRs) and Ras. Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, we identify molecular differences between p110γ-p84 and p110γ-p101 that explain their differential membrane recruitment and activation by Ras and GPCRs. The p110γ-p84 complex is dynamic compared with p110γ-p101. While p110γ-p101 is robustly recruited by Gβγ subunits, p110γ-p84 is weakly recruited to membranes by Gβγ subunits alone and requires recruitment by Ras to allow for Gβγ activation. We mapped two distinct Gβγ interfaces on p101 and the p110γ helical domain, with differences in the C-terminal domain of p84 and p101 conferring sensitivity of p110γ-p101 to Gβγ activation. Overall, our work provides key insight into the molecular basis for how PI3Kγ complexes are activated.

The formation of complexes between Rab11 and its effectors regulates multiple aspects of membrane trafficking, including recycling and ciliogenesis. WD repeat–containing protein 44 (WDR44) is a structurally uncharacterized Rab11 effector that regulates ciliogenesis by competing with prociliogenesis factors for Rab11 binding. Here, we present a detailed biochemical and biophysical characterization of the WDR44–Rab11 complex and define specific residues mediating binding. Using AlphaFold2 modeling and hydrogen/deuterium exchange mass spectrometry, we generated a molecular model of the Rab11–WDR44 complex. The Rab11-binding domain of WDR44 interacts with switch I, switch II, and the interswitch region of Rab11. Extensive mutagenesis of evolutionarily conserved residues in WDR44 at the interface identified numerous complex-disrupting mutations. Using hydrogen/deuterium exchange mass spectrometry, we found that the dynamics of the WDR44–Rab11 interface are distinct from the Rab11 effector FIP3, with WDR44 forming a more extensive interface with the switch II helix of Rab11 compared with FIP3. The WDR44 interaction was specific to Rab11 over evolutionarily similar Rabs, with mutations defining the molecular basis of Rab11 specificity. Finally, WDR44 can be phosphorylated by Sgk3, with this leading to reorganization of the Rab11-binding surface on WDR44. Overall, our results provide molecular detail on how WDR44 interacts with Rab11 and how Rab11 can form distinct effector complexes that regulate membrane trafficking events. The formation of complexes between Rab11 and its effectors regulates multiple aspects of membrane trafficking, including recycling and ciliogenesis. WD repeat–containing protein 44 (WDR44) is a structurally uncharacterized Rab11 effector that regulates ciliogenesis by competing with prociliogenesis factors for Rab11 binding. Here, we present a detailed biochemical and biophysical characterization of the WDR44–Rab11 complex and define specific residues mediating binding. Using AlphaFold2 modeling and hydrogen/deuterium exchange mass spectrometry, we generated a molecular model of the Rab11–WDR44 complex. The Rab11-binding domain of WDR44 interacts with switch I, switch II, and the interswitch region of Rab11. Extensive mutagenesis of evolutionarily conserved residues in WDR44 at the interface identified numerous complex-disrupting mutations. Using hydrogen/deuterium exchange mass spectrometry, we found that the dynamics of the WDR44–Rab11 interface are distinct from the Rab11 effector FIP3, with WDR44 forming a more extensive interface with the switch II helix of Rab11 compared with FIP3. The WDR44 interaction was specific to Rab11 over evolutionarily similar Rabs, with mutations defining the molecular basis of Rab11 specificity. Finally, WDR44 can be phosphorylated by Sgk3, with this leading to reorganization of the Rab11-binding surface on WDR44. Overall, our results provide molecular detail on how WDR44 interacts with Rab11 and how Rab11 can form distinct effector complexes that regulate membrane trafficking events. Coordinated membrane trafficking of vesicular cargo to the correct intracellular locations is fundamental to complex eukaryotic life. A critical player in membrane trafficking are the Rab GTPases, a subfamily of the Ras superfamily of GTPases that play important roles in membrane identity and trafficking (1Zhen Y. Stenmark H. Cellular functions of Rab GTPases at a glance.J. Cell. Sci. 2015; 128: 3171-3176Crossref PubMed Scopus (292) Google Scholar, 2Müller M.P. Goody R.S. Molecular control of Rab activity by GEFs, GAPs and GDI.Small GTPases. 2017; 13: 1-17Google Scholar, 3Pylypenko O. Hammich H. Yu I.-M. Houdusse A. Rab GTPases and their interacting protein partners: structural insights into Rab functional diversity.Small GTPases. 2017; 113: 1-27Google Scholar, 4Wandinger-Ness A. Zerial M. Rab proteins and the compartmentalization of the endosomal system.Cold Spring Harb. Perspect. Biol. 2014; 6: a022616Crossref PubMed Scopus (324) Google Scholar). Rab GTPases regulate many steps of membrane trafficking by interacting with effector proteins along actin and tubulin networks (5Welz T. Wellbourne-Wood J. Kerkhoff E. Orchestration of cell surface proteins by Rab11.Trends Cell Biol. 2014; 24: 407-415Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Rab GTPases act as molecular switches, alternating between a GTP-bound “on” state and a GDP-bound “off” state, with binding to effectors being dependent on conformational changes of the switch regions driven by nucleotide binding (2Müller M.P. Goody R.S. Molecular control of Rab activity by GEFs, GAPs and GDI.Small GTPases. 2017; 13: 1-17Google Scholar, 3Pylypenko O. Hammich H. Yu I.-M. Houdusse A. Rab GTPases and their interacting protein partners: structural insights into Rab functional diversity.Small GTPases. 2017; 113: 1-27Google Scholar). The Rab GTPase Rab11 is a master regulator of exocytic and recycling processes and critical in guiding proteins to the cell surface (5Welz T. Wellbourne-Wood J. Kerkhoff E. Orchestration of cell surface proteins by Rab11.Trends Cell Biol. 2014; 24: 407-415Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Rab11 is a family of GTPases encompassing three unique genes Rab11a, Rab11b, and Rab11c (Rab25), sharing 89% sequence identity for Rab11a/Rab11b and 61% for Rab11a/Rab11c. Rab11a localizes to early endosomes, recycling endosomes, trans-Golgi network, and post-Golgi vesicles and is important in endosomal recycling and ciliogenesis (5Welz T. Wellbourne-Wood J. Kerkhoff E. Orchestration of cell surface proteins by Rab11.Trends Cell Biol. 2014; 24: 407-415Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 6Knödler A. Feng S. Zhang J. Zhang X. Das A. Peränen J. et al.Coordination of Rab8 and Rab11 in primary ciliogenesis.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 6346-6351Crossref PubMed Scopus (370) Google Scholar). Active GTP-bound Rab11 at intracellular membranes recruits an extensive set of effector proteins, including tethering factors, molecular motors, scaffolding proteins, and kinases to control vesicular trafficking events (3Pylypenko O. Hammich H. Yu I.-M. Houdusse A. Rab GTPases and their interacting protein partners: structural insights into Rab functional diversity.Small GTPases. 2017; 113: 1-27Google Scholar). Understanding the molecular basis of how unique effectors bind to Rab11 is essential in defining its various roles in fundamental membrane trafficking events. Binding of Rab GTPases to their effectors is driven through nucleotide-dependant conformational changes in regions defined as switch I and switch II, with most effectors only binding to GTP-bound Rab (7Eathiraj S. Pan X. Ritacco C. Lambright D.G. Structural basis of family-wide Rab GTPase recognition by rabenosyn-5.Nature. 2005; 436: 415-419Crossref PubMed Scopus (193) Google Scholar, 8Wittinghofer A. Vetter I. Structure-function relationships of the G domain, a canonical switch motif.Annu. Rev. Biochem. 2011; 80: 943-971Crossref PubMed Scopus (338) Google Scholar). The switches are canonically disordered when Rab is bound to GDP, with GTP binding stabilizing both switch I and II, which increases the affinity for effectors (3Pylypenko O. Hammich H. Yu I.-M. Houdusse A. Rab GTPases and their interacting protein partners: structural insights into Rab functional diversity.Small GTPases. 2017; 113: 1-27Google Scholar). Multiple different Rab11 effectors have been identified, including the mammalian family of Rab11-interacting proteins (FIPs) (9Horgan C.P. McCaffrey M.W. The dynamic Rab11-FIPs.Biochem. Soc. Trans. 2009; 37: 1032-1036Crossref PubMed Scopus (124) Google Scholar). FIPs share a homologous C-terminal Rab11-binding domain interaction with Rab11 in complexes S. A. Lambright D.G. Structural basis for of to recycling Biol. PubMed Scopus Google Scholar, T. H. M. et basis for membrane of a family of Rab11-interacting protein Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, McCaffrey M.W. of in complex with family interacting protein Full Text Full Text PDF PubMed Scopus Google Scholar). Rab11 interacts with the C-terminal domain of and the S. interacts with Rab11 a domain and Rab11 in Biol. 2005; PubMed Scopus Google in a through different compared with family to canonical Rab11 is unique in that can bind to effectors through an interface the switch including the domain of the Rab8 nucleotide exchange M. C. E. of binding of and effectors to Biol. 2015; PubMed Scopus Google as as O. et of complexes of Rab11 and its 2014; PubMed Scopus Google Scholar). interface with Rab11 for formation of complexes with The complex increases the affinity of Rab11 for through both Rab11 and binding M. C. E. of binding of and effectors to Biol. 2015; PubMed Scopus Google Scholar). of the complex critical roles in ciliogenesis through of Rab8 at membranes A. Feng S. Zhang J. Zhang X. Das A. Peränen J. et al.Coordination of Rab8 and Rab11 in primary ciliogenesis.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 6346-6351Crossref PubMed Scopus (370) Google Scholar, O. et membrane is by Rab11 and protein II trafficking of to the Natl. Acad. Sci. U. S. A. 2011; PubMed Scopus Google Scholar). of the identified Rab11 effectors was the protein WDR44 repeat–containing protein as its in Rab11 membrane trafficking A. T. I. T. Y. a of G protein in Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. C. M. H. et of a effector protein for that in Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). WDR44 is for its C-terminal WD domain, which a The Rab11-binding was identified to this with a region from residues to identified as the J. M. C. M. H. et of a effector protein for that in Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). WDR44 localizes to endosomes, which to and as a scaffolding protein to the family of membrane proteins with Rab11 S. Y. M. and of and Cell Biol. PubMed Scopus Google Scholar). of WDR44 with this by WDR44 for Rab11 binding with the A. Vetter M. C. S. et regulates a switch for Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The binding affinity of WDR44 for Rab11 can be of WDR44 by R.S. J. T. M. et that the endosomal proteins including and J. PubMed Scopus Google A. Vetter M. C. S. et regulates a switch for Cell. Full Text Full Text PDF PubMed Scopus Google of by of trafficking of with phosphorylated WDR44 to of the to how WDR44 with is the of molecular on its complex with Rab11. the present we the molecular of WDR44 binding to and to only Rab11 over Rab GTPases. and the of this complex a hydrogen/deuterium exchange mass AlphaFold2 and mutagenesis defined a of WDR44 and identified residues for WDR44 binding. The WDR44 interface with Rab11 is distinct from the complex of with with to the dynamics of phosphorylated WDR44 at in with stabilizing the of WDR44. our results molecular Rab11–WDR44 with important for the of Rab11 in ciliogenesis and membrane The WDR44 protein is structurally with a domain to identified through a of WDR44 J. M. C. M. H. et of a effector protein for that in Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). provide into the of we the AlphaFold2 model of WDR44 M. S. M. S. C. et protein the structural of with PubMed Scopus Google which a C-terminal domain, with a region the domain and The to be dynamic to the domain, with between the that a of the similar binding to the was a helix from the C-terminal of the that the domain Using this we and protein for WDR44 of and their binding protein for in this are in the WDR44 with an that the WDR44 bound with the affinity compared with the WDR44 that a of WDR44 that Rab11a was between residues and was to the between Rab11a and WDR44 The was to be was with binding the of WDR44 with Rab11a A. Vetter M. C. S. et regulates a switch for Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Using the AlphaFold2 J. A. T. M. O. et protein with PubMed Scopus Google Scholar, M. Y. S. M. et protein to PubMed Scopus Google we generated three different WDR44 of different and with Rab11. in complexes that for the and of a for the interface The with the was the WDR44 complex and is in the in the of we compared the to of Rab11 bound to different The more with the of Rab11 bound to the of Rab11 bound to which is with WDR44 binding S. J. J. et structural of Rab11 a interface in the dynamics of recycling Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). are structural in the switch I region between and GDP-bound Rab11a, with the GDP-bound with WDR44 The of the WDR44 of a and that a The interface of WDR44 is of residues in helix the and helix with the interface of Rab11a of residues at the residues in switch I and II, the interswitch and of the C-terminal The interface is of a surface with extensive between both switch I and The residues of Rab11a residues important for complex formation E. S. Lambright D.G. et of an in the switch regions of Rab GTPases is a of effector Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the we of WDR44 and Rab11a to our model and the interface between WDR44 and the of with the exchange being dependent on S. dynamics by Sci. PubMed Scopus Google Scholar, of exchange mass in 2017; PubMed Scopus Google Scholar). is a to conformational changes and define WDR44 with Rab11a with of with of WDR44 and of Rab11a was sequence of the region of with of the disordered in exchange between defined as at the three and with a three and complex. in exchange WDR44 Rab11a binding and in Rab11a WDR44 binding in WDR44 to the with changes in the disordered of the with in WDR44 from to which to the of the helix and into the of WDR44 which extensive with Rab11a in the AlphaFold2 in Rab11a switch I, and switch II regions which is with effectors binding to Rab GTPases (2Müller M.P. Goody R.S. Molecular control of Rab activity by GEFs, GAPs and GDI.Small GTPases. 2017; 13: 1-17Google Scholar, 3Pylypenko O. Hammich H. Yu I.-M. Houdusse A. Rab GTPases and their interacting protein partners: structural insights into Rab functional diversity.Small GTPases. 2017; 113: 1-27Google Scholar). The Rab11a regions with the WDR44 binding switch I and II changes with the AlphaFold2 model and into the of the complex. the of the complex and of the WDR44 we extensive mutagenesis The WDR44 region with the in exchange Rab11a binding to this along with the AlphaFold2 as as evolutionarily conserved residues in we found at the interface with residues in WDR44 in helix which switch II, the which switch I, and helix which switch II, and a between of WDR44 and of switch I. the residues and and residues at the Rab11a interface with in a in complex formation by with the this of the AlphaFold2 The of Rab11a bound to WDR44 a distinct interface compared with the complex S. A. Lambright D.G. Structural basis for of to recycling Biol. PubMed Scopus Google Scholar, T. H. M. et basis for membrane of a family of Rab11-interacting protein Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the dynamic between the complex and effector we to the and which to changes complex formation in Rab11a and in Rab11a between the was in Rab11a WDR44 binding in regions switch I and II and the helix that switch II in exchange binding similar regions of Rab11a, with dynamics when we the between the and Rab11a was more in switch II and the helix that switch II in the WDR44 complex compared with FIP3. switch I of Rab11a the for both WDR44 and with a more extensive interface in the switch II region for which is with the Overall, this that both and WDR44 similar with switch I, with WDR44 more extensive at switch II compared with FIP3. provide into the of the we for WDR44 binding to a set of conserved Rab GTPases and of binding to WDR44 the evolutionarily similar a in Rab11a effector binding is the of the switch regions compared with Rab GTPases (3Pylypenko O. Hammich H. Yu I.-M. Houdusse A. Rab GTPases and their interacting protein partners: structural insights into Rab functional diversity.Small GTPases. 2017; 113: 1-27Google Scholar, E. S. Lambright D.G. et of an in the switch regions of Rab GTPases is a of effector Biol. Full Text Full Text PDF PubMed Scopus Google we to specific residues in Rab11a that play a in the of this the of switch II for a of Rab GTPases and a conserved in and that in a of the WDR44 interface that this to a as in Rab GTPases binding to WDR44. Rab11 binding to through between switch II and WDR44 been that WDR44 can be phosphorylated at by with this ciliogenesis by the formation of the complex A. Vetter M. C. S. et regulates a switch for Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). was that WDR44 is phosphorylated by at only as a at the with WDR44 a at the R.S. J. T. M. et that the endosomal proteins including and J. PubMed Scopus Google Scholar). define conformational changes of we WDR44 with WDR44 was phosphorylated by as by with a the of phosphorylated in was only in of the of leading to of in was in the in the the being we the of a which on phosphorylated WDR44 as by compared with WDR44 in the and of compared different phosphorylated and phosphorylated complex. phosphorylated WDR44 exchange for phosphorylated WDR44 in the of the is that of in between and phosphorylated WDR44 be by the exchange of we a between and phosphorylated on a that the that this exchange is driven by conformational changes driven by in exchange for phosphorylated WDR44 binding Rab11a compared with WDR44 binding Rab11a the region by in WDR44 is in the Rab11a that the interface in WDR44. The formation of complexes critical roles membrane trafficking including the recycling endosomal (5Welz T. Wellbourne-Wood J. Kerkhoff E. Orchestration of cell surface proteins by Rab11.Trends Cell Biol. 2014; 24: 407-415Abstract Full Text Full Text PDF PubMed Scopus (220) Google and in ciliogenesis A. Vetter M. C. S. et regulates a switch for Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The complex ciliogenesis through of the complex. the molecular how Rab11a interacts with WDR44 is compared with Rab11 structural and biochemical of the complex identified residues and dynamic changes that both Rab11 binding and of WDR44. Overall, this fundamental into how Rab11 its effectors as as how can this WDR44 was of the identified Rab11 effectors A. T. I. T. Y. a of G protein in Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. C. M. H. et of a effector protein for that in Natl. Acad. Sci. U. S. A. PubMed Scopus Google its roles in processes have only to be The AlphaFold2 model J. A. T. M. O. et protein with PubMed Scopus Google of the WDR44 complex that the of WDR44 to be dynamic to the C-terminal is only a dynamic between a helix that the domain, and the of the which the of of Rab11 binding in the with structural to this Using a and AlphaFold2 modeling to a model of the complex. to many Rab11 the primary interface was of switch I II and the interswitch is a of structural of the switch regions of Rab11 with its different effector proteins, in switch II, with this region unique in bound to FIP3, and S. A. Lambright D.G. Structural basis for of to recycling Biol. PubMed Scopus Google Scholar, O. C. M. et basis of Rab cargo Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, O. et of complexes of Rab11 and its 2014; PubMed Scopus Google Scholar). is unique from Rab GTPases, which the switch binding effectors (7Eathiraj S. Pan X. Ritacco C. Lambright D.G. Structural basis of family-wide Rab GTPase recognition by rabenosyn-5.Nature. 2005; 436: 415-419Crossref PubMed Scopus (193) Google Scholar, C. Structural basis of effector of the G protein with the effector domain of Full Text Full Text PDF PubMed Scopus Google Scholar). The interface of WDR44 with Rab11a from the model a unique of switch II compared with Rab11 of the of nucleotide in the AlphaFold2 structural be to this specific conformational The model a more extensive interface with switch II compared with the S. A. Lambright D.G. Structural basis for of to recycling Biol. PubMed Scopus Google which is by our in switch II for WDR44 compared with FIP3. The Rab11 interface in WDR44 is conserved through with of both and leading to of the Rab11–WDR44 complex. residues in three of WDR44 in with with three being critical in complex The of complex is essential in defining the of WDR44 in ciliogenesis. is to the that the can ciliogenesis by multiple of WDR44 by of can the binding affinity for Rab11 A. Vetter M. C. S. et regulates a switch for Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, R.S. J. T. M. et that the endosomal proteins including and J. PubMed Scopus Google of and M. C. E. of binding of and effectors to Biol. 2015; PubMed Scopus Google and Rab8 of can nucleotide exchange leading to Rab8 trafficking A. Vetter M. C. S. et regulates a switch for Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). is the of between and as the in can be by of of the of in of WDR44 S. et interacts with to and 2014; PubMed Scopus Google Scholar). act as important that can be in defining the of the specific WDR44–Rab11 complex in ciliogenesis. found that WDR44 was phosphorylated by an in with results R.S. J. T. M. et that the endosomal proteins including and J. PubMed Scopus Google Scholar). to phosphorylated WDR44 for by the of the including regions that with Rab11. is that of Rab GTPases can their roles in membrane trafficking of Rab GTPases in the of membrane PubMed Scopus Google with the a of GTPases through M. M. Vetter M. et that regulates a of Rab Google Scholar, M. R.S. O. et of Rab GTPase a to 2017; PubMed Scopus Google Scholar). of the roles of is through ciliogenesis by the interaction of phosphorylated Rab8 with its effectors M. R.S. O. et of Rab GTPase a to 2017; PubMed Scopus Google Scholar, E. E. Structural basis for of of switch Full Text Full Text PDF PubMed Scopus Google Scholar). of phosphorylated WDR44 for structural and biochemical to define the of WDR44 in Rab11 and binding. Rab GTPases play roles in steps of membrane trafficking by specific effectors on their The formation of the Rab11–WDR44 complex an important in its been by of on its molecular the molecular basis for complex how been conserved over and its dynamics from WDR44 in this can be in in to the of WDR44 and Rab11 in membrane trafficking and ciliogenesis. and proteins in WDR44 WDR44 WDR44 WDR44 WDR44 WDR44 WDR44 and in a The Rab genes as U. A. et of the nucleotide exchange Biol. PubMed Scopus Google Scholar, U. et into activity of the mammalian Biol. Scopus Google Scholar). The WDR44 was from the WDR44 and into a with Rab an affinity A in this is in Multiple and the by X. E. and sequence and from of PubMed Scopus Google to conserved The for the in and are and is an by that protein from a primary sequence J. 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