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    • Main Text There are over small GTPases that play critical

    2021-10-25

    Main Text There are over 200 small GTPases that play critical roles in diverse fundamental cellular processes such as signal transduction, A 83-01 dynamics, and intracellular trafficking. Most of the GTPases act as switch-like molecules by cycling between the guanosine triphosphate (GTP)-bound “ON” and guanosine diphosphate (GDP)-bound “OFF” states. This ON/OFF switch is controlled by guanine nucleotide exchange factors (GEFs), which load GTP to GTPases, and GTPase-activating proteins (GAPs), which catalyze the hydrolysis of GTP to GDP. GTP induces the active conformation of GTPases, allowing them to bind their effector proteins and thereby initiate downstream events. The mechanisms of GTP loading, hydrolysis, GTP-induced conformational changes, and effector binding for many GTPases have been extensively studied and are well understood. However, one question that remains incompletely addressed is how GTPases, many of which are highly similar in sequence and structure, achieve specific interactions with their respective effector proteins. This point is of particular importance for the Rab GTPases, a group with over 70 members that constitutes the largest subfamily in the small GTPase family (Pylypenko et al., 2018). The large number of Rab GTPases allow them to interact with diverse effector proteins to fulfill their functions in the regulation of intracellular trafficking, an extremely complex process that is essential for defining the identity of sub-cellular membrane compartments and the exchange of proteins and lipids among these compartments. The study by Lin et al. (2019) in this issue of Structure sheds light on this question by revealing the structural basis of how Rab35, but not other closely related Rab GTPases, binds two effector proteins, ACAP2 and RUSC2. GTP controls the conformation of two segments in small GTPases, named switches I and II, that are essential for effector binding (Figure 1). The γ-phosphate group in GTP and the bound magnesium ion (Mg2+) make many interactions with switch I, confining it in a relatively rigid conformation, with one side intimately “glued” to the body of the protein while the other side’s surface is exposed for binding effectors. Switch I and GTP/Mg2+ together stabilize the conformation of switch II, which forms a characteristic helical structure at its C-terminal part. In contrast, switches I and II in GDP-bound GTPases are usually flexible and less ordered. For some Rabs, such as Rab6 and Rab11, conformational plasticity of switches I and II plays a role in specific binding to different effectors (Pylypenko et al., 2018). However, in most cases the conformations of switches I and II in GTP-bound Rabs are rather similar, suggesting that the specificity to effectors is not usually a result of gross structural differences in the effector binding region. Some non-conserved residues surrounding the effector binding region are known to influence the precise arrangement of effector-interacting residues, thereby indirectly regulating the binding specificity of effectors (Pylypenko et al., 2018). A more direct mechanism for achieving effector specificity is sequence variations in the effector binding region. While these mechanisms of specificity determination are known in general, pinpointing residues in individual Rab proteins responsible for the specificity to particular effectors requires high resolution structures of the Rab/effector complexes. The crystal structures presented in the paper by Lin et al. (2019) does exactly that. This paper (Lin et al., 2019) first confirmed the remarkable mutual specificity between Rab35 and the two effectors ACAP2 and RUSC2 reported by previous studies (Etoh and Fukuda, 2015, Fukuda et al., 2011). They then determined the crystal structures of GTP-bound Rab35 in complex with the ankyrin repeat (ANK) domain of ACAP2 and the RPIP8/UNC-14/NESCA (RUN) domain of RUSC2, respectively, representing the first set of structures of Rab35/effector complexes. As expected, Rab35 uses primarily switch I (residues 31–42) and switch II (residues 65–82) to interact with both ACAP2 and RUSC2. The conformations of switches I and II of Rab35 in the two crystal structures are nearly identical and highly similar to the active conformations seen for most other Rabs. Notably, the conformation of switch II in Rab35 and the binding mode between Rab35 and RUSC2-RUN are quite different from those shown in the complex structure between Rab6 and the RUN domain of Rab6-interacting protein 1 reported previously (Recacha et al., 2009). On the effector side, two helices in either ACAP2 or RUSC2 make major contributions to the recognition of the switch regions in Rab35, which is also a common feature in many Rab/effector complexes. However, the orientation of the two helices in ACAP2 and RUSC2 are completely different. These differences under a common theme suggest that the binding mode and details of the Rab/effector interactions cannot be readily inferred from available structures of the complexes. More structures are therefore required for better understanding of the plasticity and specificity in Rab/effector interactions.