Structural Plasticity of an Invariant Hydrophobic Triad in the Switch Regions of Rab GTPases Is a Determinant of Effector Recognition*

Eric Merithew, Scott Hatherly, John J. DumasDagger, Deirdre C. Lawe, Robin Heller-Harrison, and David G. Lambright§

From the Program in Molecular Medicine and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

Received for publication, October 26, 2000, and in revised form, January 11, 2001




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Rab GTPases function as regulatory components of an evolutionarily conserved machinery that mediates docking, priming, and fusion of vesicles with intracellular membranes. We have previously shown that the active conformation of Rab3A is stabilized by a substantial hydrophobic interface between the putative conformational switch regions (Dumas, J. J., Zhu, Z., Connolly, J. L., and Lambright, D. G. (1999) Structure 7, 413-423). A triad of invariant hydrophobic residues at this switch interface (Phe-59, Trp-76, and Tyr-91) represents a major interaction determinant between the switch regions of Rab3A and the Rab3A-specific effector Rabphilin3A (Ostermeier, C., and Brunger, A. T. (1999) Cell 96, 363-374). Here, we report the crystal structure of the active form of Rab5C, a prototypical endocytic Rab GTPase. As is true for Rab3A, the active conformation of Rab5C is stabilized by a hydrophobic interface between the switch regions. However, the conformation of the invariant hydrophobic triad (residues Phe-58, Trp-75, and Tyr-90 in Rab5C) is dramatically altered such that the resulting surface is noncomplementary to the switch interaction epitope of Rabphilin3A. This structural rearrangement reflects a set of nonconservative substitutions in the hydrophobic core between the central beta  sheet and the alpha 2 helix. These observations demonstrate that structural plasticity involving an invariant hydrophobic triad at the switch interface contributes to the mechanism by which effectors recognize distinct Rab subfamilies. Thus, the active conformation of the switch regions conveys information about the identity of a particular Rab GTPase as well as the state of the bound nucleotide.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

As general regulators of intracellular vesicle transport between donor and acceptor membranes, Rab proteins comprise the largest GTPase family with 11 distinct homologues in yeast and more than 50 known Rab GTPases in mammals (3-7). As is true of other GTPases of the Ras superfamily, Rabs cycle between active (GTP-bound) and inactive (GDP-bound) conformations (3, 8, 9). A key question concerns the molecular and structural mechanisms by which Rab GTPases generate specificity for a diverse spectrum of effectors and regulatory factors. Biochemical and genetic studies of chimeric and mutant Rab proteins have identified several hypervariable regions, including the N and C termini and the alpha 3/beta 5 loop, that play an important role in determining functional specificity (10, 11). However, interactions involving hypervariable regions cannot explain the ability of Rab GDI1 (GDP dissociation inhibitor) to recognize most or all Rab GTPases yet still discriminate against other GTPase families or the ability of certain regulatory factors to recognize particular Rab subfamilies (12-15). These observations imply the existence of specificity determinants that are common to all Rab GTPases but not other GTPase families, as well as determinants that are conserved only within particular Rab subfamilies.

Crystallographic studies of Rab GTPases have identified structural motifs and modes of effector interaction that are distinct from those of other GTPase families. The active conformation is stabilized by additional hydrogen bonding interactions with the gamma  phosphate of GTP, mediated by serine residues in the P-loop and switch I region, as well as an extensive hydrophobic interface between the switch I and II regions (1, 16). The structure of the complex between a constitutively active mutant of Rab3A and a putative effector, Rabphilin3A, revealed an interaction epitope that extends from the relatively conserved switch interface to an adjacent pocket formed by three hypervariable "complementary determining regions" (CDRs) corresponding to the N and C termini of the GTPase domain and the alpha 3/beta 5 loop (2). A triad of invariant hydrophobic residues at the switch I/switch II interface of Rab3A mediates a central interaction with Rabphilin3A. In a recent analysis of the primary structure of all known Rab GTPases, five distinctive motifs (known as the Rab family motifs or RabFs) were identified that distinguish Rab proteins from other GTPase families (17). The RabF motifs are located either in the putative switch I and II regions or in the adjacent beta 3 and beta 4 strands. It was also noted that the Rab family could be further subdivided based on the presence of four subfamily motifs, which include all three Rab CDRs as well as a region corresponding to the alpha 1 helix and most of the alpha 1/beta 2 loop.

Rab5 is an essential regulator of early endosome fusion (18, 19). Several putative Rab5 effectors have been identified including EEA1 (early endosomal auto antigen), a large protein consisting of an N-terminal Zn2+ finger, four long heptad repeats having weak homology with myosins, and a compact C-terminal region containing an IQ motif (putative calmodulin binding site), a Rab5 interaction site, and a FYVE domain that specifically binds phosphatidylinositol 3-phosphate (20-26). Interactions with both phosphatidylinositol 3-phosphate and Rab5 are essential for the fusion of endocytic vesicles with early endosomes in an in vitro reconstitution assay (27, 28). Although lacking the corresponding C-terminal regions, the FYVE domain of EEA1 is structurally similar to the ring finger domain of Rabphilin3A (29). Moreover, a predicted helical region N-terminal to the FYVE domain of EEA1 exhibits weak homology with the N-terminal helix of Rabphilin3A and has been implicated in the interaction with Rab5 (25, 30). However, critical hydrophobic residues in Rabphilin3A are not conserved in EEA1. Thus, the extent to which the Rab3A-Rabphilin3A complex can be regarded as a paradigm for other Rab-effector complexes remains uncertain (31).

The crystallographic and biochemical studies cited above are consistent with the hypothesis that the relatively conserved switch regions convey information regarding the state of the bound nucleotide, whereas independent Rab CDRs determine the specificity of Rab-effector interactions. Here, we report the crystal structure of Rab5 bound to GppNHp. Despite strong homology, the characteristic RabF motifs in the switch regions of Rab5 and Rab3A do not encode the same active conformation. In particular, the invariant triad of partially exposed hydrophobic residues located at the switch I/switch II interface adopts a dramatically different conformation in Rab5 such that the resulting hydrophobic surface is noncomplementary to Rabphilin3A. These changes reflect an alternative packing arrangement in response to a concerted set of nonconservative substitutions in the hydrophobic core. Moreover, the packing constraints on the conformation of the invariant hydrophobic triad appear to be engaged only in the active form. Thus, structural plasticity within the relatively conserved switch regions is evolutionarily coupled to the conformational switching mechanism and represents a potentially general determinant of Rab-effector recognition.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression and Purification-- The GTPase domain of Rab5C (residues 16-186) was expressed in Escherichia coli using a modified pET15b vector containing an N-terminal His10 peptide (MGHHHHHHHHHHGS). BL21(DE3) cells harboring the modified pET15b plasmid containing the Rab5 insert were grown at 37 °C in 2× YT medium (16 g of Bacto tryptone, 10 g of Bacto yeast extract, and 5 g of sodium chloride liter of water) induced at an A600 of ~0.6 by the addition of 1 mM isopropyl-1-thio-beta -D-galactopyranoside, and harvested after 3 h at 37 °C. The cell pellet was resuspended in 50 mM Tris, pH 8.5, and 0.1% 2-mercaptoethanol, lysed by sonication, and was centrifuged at 35,000 × g for 1 h. The supernatant was loaded onto a nickel-nitrilotriacetic acid-agarose column (Qiagen). After washing with 11 column volumes of 50 mM Tris, pH 8.5, 500 mM NaCl, 10 mM imidazole, and 0.1% 2-mercaptoethanol, the fusion protein was eluted with a gradient of 10-500 mM imidazole. The protein was further purified by ion exchange chromatography on Resource Q and Resource S (Amersham Pharmacia Biotech) followed by gel filtration on Superdex-75 (Amersham Pharmacia Biotech). Roughly 30 mg of >99% pure protein were obtained from a 6-liter culture.

Crystallization and Data Collection-- The GppNHp-bound form of Rab5C was prepared as described for Rab3A (1). Crystals of the Rab5-GppNHp complex were grown at 4 °C by vapor diffusion in hanging drops containing 10% polyethylene glycol-6000, 50 mM MES, pH 6.0, 0.2 M NaCl, 0.5 mM MgCl2, and 0.1% 2-mercaptoethanol. Single crystals appeared overnight and grew to maximum dimensions of 0.3 × 0.3 × 0.2 after several days. The crystals are in the primitive orthorhombic space group P212121 with cell constants a = 35.9 Å, b = 64.0 Å, and c = 65.9 Å. The volume of the unit cell is consistent with one molecule in the asymmetric unit and a solvent content of 35%. Crystals were soaked for 5 min at 4 °C in a cryoprotectant-stabilizer solution (30% polyethylene glycol-6000 and 20% glycerol) prior to flash freezing in a nitrogen cryostream. A native data set complete to 1.8 Å was collected on a Rigaku RUH3R/Mar 30-cm image plate detector equipped with focusing mirrors (Charles Supper). The crystal was maintained at 100 K using a nitrogen cryostream (Oxford Cryosystems). All data were processed with Denzo and scaled with Scalepack (32).

Structure Determination and Refinement-- The structure of GppNHp-bound Rab5C was solved by molecular replacement using a polyalanine search model derived from the coordinates of the Rab3A structure (1). A translation search that included the top 50 rotation function solutions yielded a unique solution with an R value of 50.5% after rigid body refinement against data from 8 to 3 Å. Initial difference maps calculated with Sigma A weights to reduce model bias revealed poor density for the putative switch I and II regions and several loop regions. Multiple rounds of simulated annealing and manual fitting were interleaved with gradual extension of the resolution to 1.8 Å. Manual fitting of the omitted regions and placement of the side chains were followed by additional rounds of simulated annealing, positional, and B factor refinement. The final refined model includes residues 19-182, one molecule of GppNHp, a Mg2+ ion, and 153 ordered water molecules and has a crystallographic R value of 0.198 and a free R value of 0.246 based on a 5% subset of reflections randomly omitted prior to refinement. Molecular replacement was conducted with AMORE as implemented in CCP4, refinement with X-PLOR, and interactive model building with O (33-36). Structural images were generated with MOLSCRIPT or GRASP, combined with GL  RENDER (provided by Dr. L. Esser), and rendered with RASTER 3D (37-40).

Other Crystallographic Models-- For comparison with Rab5C, models for Rab3A, Ypt51, Rab6, and the Rab3A-Rabphilin3A complex were rendered from the coordinates of the corresponding crystal structures (PDB ID codes 3RAB, 1EK0, 1D5C, and 1ZBD, respectively).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Overall Structure and Comparison with Other Rab GTPases-- The GTPase domain of Rab5C bound to GppNHp was crystallized, and the structure was determined by molecular replacement (Table I). The final refined model, which includes residues 19-182, one molecule of GppNHp, a Mg2+ ion, and 153 ordered water molecules, has a working R value of 0.198 and a free R value of 0.246. The stereochemistry is excellent, and there are no backbone torsion angles outside the allowed regions of the Ramachandran plot. Residues 16-18 at the N terminus, residues 65-69 in the beta 2/beta 3 loop, and residues 183-186 at the C terminus are poorly ordered.


                              
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Table I
Structure determination and refinement

Like other members of the GTPase superfamily, Rab5C possesses a characteristic nucleotide binding fold consisting of a six-stranded beta  sheet surrounded by five alpha  helices (Fig. 1). In the absence of a structure for the GDP-bound form of Rab5C, a precise definition of the conformational switch regions is not possible. However, a comparison of the GppNHp-bound structure of Rab3A with that of the GDP-bound form of Rab6 from the malaria parasite Plasmodium falciparum suggests that the conformational changes accompanying nucleotide exchange will be localized to regions analogous to those in p21ras (41). As a working hypothesis, we will assume that the conformational changes in Rab5 are localized to the corresponding regions, although the precise nature and extent of the conformational changes will not alter the observations described below. To facilitate discussion, we will also refer to the Rab CDRs as defined for Rab3A based on the complex with Rabphilin3A (2). Whether these and/or other variable regions mediate effector interactions with Rab5C remains to be determined.



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Fig. 1.   Overall structure of Rab5C. Ribbon representation of the Rab5C structure with the putative switch regions highlighted in purple, the regions corresponding to the CDRs of Rab3A highlighted in orange, and the nucleotide highlighted in green.

Although the overall fold of Rab5C resembles that of Rab3A, significant structural differences are evident throughout the GTPase domain (Fig. 2). The structures are most similar in the regions in contact with the nucleotide, whereas the largest differences occur in or adjacent to regions implicated in the interaction with effectors and/or regulatory factors. The GTPase domains of Rab5C and Rab3A share 37% sequence identity overall; however, the homology within the switch regions is considerably higher. For example, the switch II regions of Rab5C and Rab3A are 63% identical, and substitutions either are conservative or occur at exposed positions. Moreover, the majority of intramolecular interactions with the switch II region involves residues from the highly conserved RabF motifs. Interestingly, the magnitude of the displacement of Calpha atoms does not strictly correlate with sequence variability. Indeed, the displacements in the relatively conserved switch II region are considerably larger than the overall root-mean-square deviation and comparable with the displacements in the poorly conserved alpha 4 helix. Moreover, the largest main chain rearrangement within the switch II region is centered on an invariant Tyr residue in one of the highly conserved RabF motifs. The origin and functional consequences of these changes with respect to effector recognition are considered below.



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Fig. 2.   Comparison of the active conformations of endocytic and exocytic Rab GTPases. A, superposition of the Rab5C structure (blue) with the structures of Rab3A alone (purple), the yeast Rab5 homologue Ypt51 (semitransparent blue), and Rab3A from the complex with Rabphilin3A (semitransparent purple). The superposition is based on Calpha atoms. Models for Rab3A alone (1), Ypt51 (16), and Rab3A from the Rab3A-Rabphilin3A complex (2) were derived from the coordinates of the corresponding crystal structures. B, distance between pairs of Calpha atoms in the Rab5C and Rab3A structures following the superposition. Also shown is the secondary structure with the putative switch regions highlighted in magenta and the CDRs of Rab3A highlighted in orange.

Structural Variability in the Rab CDRs-- Although the large structural differences in the variable Rab CDRs are due in part to the inherent disorder in the absence of interactions with effectors, critical changes can be attributed to specific sequence determinants. For example, proline residues from a PXXXP motif in Rab5 result in an alpha 5 helix that is truncated by two turns relative to Rab3A. The additional turns of the alpha 5 helix in Rab3A comprise an essential element of the binding pocket for a C-terminal hydrophobic motif in Rabphilin3A (2). The differences in backbone conformation are further augmented by nonconservative substitutions in which hydrophobic residues in Rab3A are substituted for charged or polar residues in Rab5. Thus, the specificity of effector interactions with the Rab CDRs is determined by backbone conformational constraints as well as the stereochemical and electrostatic complementarity of the relevant molecular surfaces. These specificity determinants are strongly correlated with a high degree of variability in the sequences comprising the Rab CDRs.

Structural Rearrangement of an Invariant Hydrophobic Triad at the Switch Region Interface-- As shown in Fig. 3, A and B, a triad of invariant hydrophobic residues encoded by three of the characteristic RabF motifs undergoes a dramatic structural rearrangement. In Rab5, the aromatic ring of Tyr-90 in the switch II region is buried in the hydrophobic core formed between the switch regions and the central beta  sheet, whereas the aromatic ring of Phe-58 in the beta 2 strand is partially buried, leaving the indole ring of Trp-75 in the beta 3 strand significantly exposed. In this packing arrangement, the aromatic rings of Trp-75 and Tyr-90 lie in a van der Waals contact with the methyl group of Ala-56 in the switch I region. Consequently, the conformation adopted by the invariant hydrophobic triad in Rab5 requires a residue with a small side chain at position 56. In Rab3A, a nonconservative alanine to isoleucine substitution at the corresponding position displaces the aromatic side chains of Tyr-91 and Trp-76. The displacement of Trp-76 in turn forces the side chain of Phe-59 to adopt an alternative rotomer conformation. Thus, the aromatic side chains of Tyr-91 and Phe-59 in Rab3A protrude from either side of the indole ring of Trp-76, which is considerably more buried than its counterpart in Rab5. Despite the dramatic conformational rearrangement of the invariant hydrophobic triad, the alternative packing arrangement in Rab5 preserves the hydrophobic interface between the switch regions.



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Fig. 3.   Structural rearrangement of an invariant hydrophobic triad at the switch region interface. A, semitransparent molecular surface of Rab5C in the vicinity of the invariant hydrophobic triad (Phe-58, Trp-75, and Tyr-90). B, semitransparent molecular surface of Rab3A (1) in the vicinity of the invariant hydrophobic triad (Phe-59, Trp-76, and Tyr-91). C, overlay following the superposition of Calpha atoms in the independently determined active structures of Rab5C (blue and orange), Ypt51 (semitransparent blue and orange), Rab3A alone (gray and purple), and Rab3A from the complex with Rabphilin3A (semitransparent gray and purple). Models for Rab3A alone (1), Ypt51 (16), and Rab3A from the Rab3A-Rabphilin3A complex (2) were derived from the coordinates of the corresponding crystal structures. D, sigma A-weighted 2Fo - Fc map countered at 1.2 sigma  showing residues in the vicinity of the invariant hydrophobic triad.

Because of crystal contacts involving the hydrophobic surfaces at the switch interface, it is reasonable to question whether the observed active conformations might be influenced by crystal packing or by their engagement with effectors. Several lines of evidence indicate that the structural plasticity of the invariant hydrophobic triad is not a crystallographic artifact but rather an inherent property of Rab GTPases. First, the hydrophobic triad in the independently determined structure of the yeast Rab5 homologue Ypt51 (16) adopts a nearly identical conformation despite crystallizing in a different space group (Fig. 3C). This observation eliminates crystal packing as a likely explanation. Second, the conformation of the invariant hydrophobic triad in Rab3A is not altered by the interaction with Rabphilin3A (2), indicating that these residues are preoriented (Fig. 3C). Consistent with this observation, the side chains of the invariant triad are well ordered as a result of intramolecular packing constraints (Fig. 3D). Finally, as described below, a concerted set of nonconservative substitutions in the hydrophobic core between the switch regions and the central beta  sheet provides a simple and plausible explanation for the observed structural rearrangement.

Sequence Determinants of the Active Conformation-- As illustrated in Fig. 4, exocytic Rab GTPases conserve either valine or isoleucine at the equivalent of position 57 in Rab3A, whereas a subset of endocytic Rab GTPases (Rab5, Rab7, Rab20, Rab21, Rab22, and Rab24) substitutes residues with small side chains (alanine or glycine). In yeast, the distinction is absolute; all exocytic Rab homologues conserve an isoleucine or valine, and all endocytic Rab homologues conserve an alanine. Although the alanine to isoleucine substitution in the switch I region represents an obvious proximal determinant of the observed differences, other nonconservative substitutions could contribute to the stabilization of the alternative conformation.



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Fig. 4.   Structure-based sequence alignment of representative endocytic and exocytic Rab GTPases. Invariant hydrophobic residues with significantly altered conformations are shown in purple, and the corresponding conformation determining residues are shown in green. Rab3A residues in the interface with Rabphilin3A are shown in blue (2). Residues that are highly conserved in the Rab GTPase family are indicated in boldface type. Residue numbering and secondary structure correspond to Rab5C. The complementary determining regions of Rab3A are highlighted in orange. The conformational switch regions defined by the comparison of GppNHp-bound Rab3A and GDP-bound Rab6 are highlighted in purple. The horizontal lines below the sequences denote the RabF and Rab subfamily (RabSF) regions (17).

To determine whether the conformation of the invariant hydrophobic triad is likely to vary from one Rab to another or simply reflects two general arrangements, we have systematically analyzed each position in or adjacent to the switch regions. Residues were segregated into three groups based on the sequences of all known Rab GTPases and the available structural data for the active conformations of Rab3A and Rab5. The first group includes invariant residues as well as residues that are conservatively substituted. Although these residues are not determinants of the active conformation, it is nevertheless clear that structural rearrangements involving conserved residues (e.g. the invariant hydrophobic triad) can mediate and amplify the effects of adjacent nonconservative substitutions. The second group consists of nonconservative substitutions of residues that occupy solvent-exposed positions. Such substitutions could well contribute to the conventional mechanism of specificity determination; however, the effects are not expected to propagate beyond the immediate vicinity. The third group corresponds to nonconservative substitutions of residues that are substantially buried in either the Rab3A or Rab5 structures. The effects of such substitutions will necessarily propagate to surrounding regions and thus have the potential to be key conformational determinants of specificity. A surprising number of nonconservative substitutions occur at interior positions. As shown in Fig. 5, the majority of such substitutions involve a cluster of hydrophobic residues situated in the hydrophobic core formed between the switch regions, the beta 1-beta 4 strands and the alpha 3 helix. Substitutions at these positions are concerted and, to a first approximation, they can be correlated with the overall similarity in the GTPase domains of the previously identified Rab subfamilies (17).



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Fig. 5.   Location of nonconservative substitutions predicted to influence the active conformation of invariant residues in the switch regions. A, Rab5. B, Rab3A from the GppNHp-bound structure (1). Variable residues are highlighted in green, and invariant residues are highlighted in purple. The view is from the interior of the protein.

Structural Plasticity in the Switch Regions Is a Determinant of Effector Recognition-- As shown in Fig. 6, the invariant hydrophobic triad represents a major site of interaction between Rabphilin3A and the switch regions of Rab3A (2). The protruding aromatic side chains of Phe-59 and Tyr-91 engage complementary hydrophobic surfaces in Rabphilin3A. Because of the structural rearrangement described above, the corresponding surface in Rab5 is noncomplementary to the switch interaction epitope of Rabphilin3A. These observations clearly demonstrate that structural plasticity involving a partially exposed invariant hydrophobic triad contributes to the specificity of Rab-effector interactions. Thus, in addition to signaling the state of the bound nucleotide, the active conformation of the switch regions conveys information about the subfamily of a particular Rab GTPase.



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Fig. 6.   Complementarity of the invariant hydrophobic triad in Rab3A and Rab5C with the switch region interaction epitope of Rabphilin3A. A, molecular surface of Rabphilin3A in contact with the switch regions of Rab3A (semitransparent gray and purple). B, hypothetical Rab5C-Rabphilin3A interface following the superposition of Rab5C (semitransparent blue and orange) with Rab3A. Note that the alternative conformation of the invariant hydrophobic triad in Rab5C lacks complementarity with Rabphilin3A. The Rabphilin3A and Rab3A models in this figure were generated from the coordinates of the Rab3A-Raphilin3A structure (2).

A General Model for Activation of Rab GTPases-- The recent crystal structure of a GDP-bound Rab6 homologue provides the opportunity to assess whether the structural changes accompanying activation are likely to influence the conformation of the invariant hydrophobic triad. Interestingly, the invariant hydrophobic triad in GDP-bound Rab6 adopts a conformation distinct from that of the active forms of either Rab3A or Rab5 (Fig. 7). In particular, the conformation determining isoleucine in the switch I region and invariant tyrosine in the switch II region are displaced relative to their counterparts in Rab3A and Rab5. Although the main chain atoms of the invariant tryptophan and phenylalanine are not substantially displaced, the aromatic side chains of these residues adopt different rotomer conformations. Moreover, crystallographic and NMR studies of a large number of GTP-binding proteins support the general conclusion that the switch regions are highly flexible in the GDP-bound form but adopt specific ordered conformations in the GTP-bound form because of the additional hydrogen bonding interactions with the gamma  phosphate (9). These observations suggest a simple structural model for the activation of Rab GTPases, leading to distinct active conformations for each Rab subfamily. Upon GTP binding, an extensive hydrophobic interface forms between the switch I and II regions. The formation of this interface engages the aromatic side chains of the invariant hydrophobic triad in a Rab subfamily-specific conformation determined by van der Waals interactions with the critical conformation determining residues in the hydrophobic core. Finally, the presence of a substantial exposed hydrophobic patch adjacent to the Rab CDRs suggests that the nucleotide-dependent engagement of the invariant hydrophobic triad could play a general role in Rab activation and effector recognition.



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Fig. 7.   Distinct conformations for the invariant hydrophobic triad in the active and inactive forms of Rab GTPases. Overlay following the superposition of Calpha atoms in GppNHp-bound Rab3A (blue and purple) and GDP-bound Rab6 (gray and orange). The Rab3A and Rab6 models in this figure were generated from the coordinates of the GppNHp-bound Rab3A (1) and GDP-bound Rab6 structures (16).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Given the large number of Rab GTPases, it is plausible that the structural plasticity of the invariant hydrophobic triad evolved primarily as a mechanism to augment other specificity-determining interactions with the Rab CDRs. In addition to the obvious implications with respect to effector interactions, differences in the active conformation of the invariant hydrophobic triad could potentially contribute to the specificity of interactions with Rab GAPs (42-47). On the other hand, a comparison of the GppNHp-bound Rab5C and Rab3A structures with the GDP-bound Rab6 structure suggests that the constraints on the conformation of the invariant hydrophobic triad are engaged only in the active form (1, 16). Consequently, the conformation of the invariant hydrophobic triad is unlikely to affect the specificity of interactions with GDI, guanine nucleotide exchange factors, GDI displacement factors, or other factors that interact with the GDP-bound form (12, 48-57). Indeed, one or more residues from the invariant hydrophobic triad could well contribute to the interaction with Rab GDI, which recognizes the GDP-bound conformation of all Rab GTPases (12).

The use of invariant hydrophobic residues in the switch regions as nucleotide-dependent recognition determinants appears to be unique to the Rab family. For example, invariant hydrophobic residues in the switch II region of heterotrimeric G protein alpha  subunits engage hydrophobic residues in the alpha 3 helix with little or no difference in conformation in the crystal structures of the active forms of Gtalpha , Gialpha , and Gsalpha (58-60). Interestingly, however, the specificity of the Gsalpha interaction with adenylyl cyclase is primarily attributable to a conformational rearrangement in the alpha 3/beta 5 loop (compared with Gialpha , which does not interact with the same site on adenylyl cyclase) rather than differences in the composition of residues in the effector interaction epitope (60). Thus, in both Rab GTPases and Galpha subunits, the conformational plasticity of invariant residues provides a structural mechanism that propagates and amplifies the effects of key nonconservative amino acid substitutions. It is likely that similar structural mechanisms of specificity determination have evolved in other large protein or domain families.

When compared with other Rab family GTPases, the Rab5 structure provides compelling evidence for an evolutionarily conserved mechanism of effector recognition in which the structural plasticity of an invariant hydrophobic triad at the switch region interface is coupled to nonconservative substitutions in the hydrophobic core. The conformation of the invariant hydrophobic triad reflects the state of the bound nucleotide as well as the Rab subfamily identity and thus appears to be an integral component of the switching mechanism. Although the extent to which Rab effectors other than Rabphilin3A will interact with the invariant hydrophobic triad remains to be established experimentally, the presence of a substantial nucleotide-dependent hydrophobic surface adjacent to the Rab CDRs suggests the possibility of a general role with respect to activation and effector recognition.


    ACKNOWLEDGEMENTS

We thank Silvia Corvera, Michael Czech, and Jennifer O'Neil for comments on the manuscript.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM56324 (to D. G. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1HUQ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Dagger National Institutes of Health postdoctoral fellow.

§ A Leukemia and Lymphoma Society Scholar. To whom correspondence should be addressed: Program in Molecular Medicine, Two Biotech, 373 Plantation St., Worcester, MA 01605. Tel.: 508-856-6876; Fax: 508-856-4289; E-mail: David.Lambright@umassmed.edu.

Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M009771200


    ABBREVIATIONS

The abbreviations used are: GDI, GDP dissociation inhibitor; CDR, complementary determining region; RabF, Rab family; MES, 4-morpholineethanesulfonic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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