Structural basis of family-wide Rab GTPase recognition by rabenosyn-5

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Abstract

Rab GTPases regulate all stages of membrane trafficking, including vesicle budding, cargo sorting, transport, tethering and fusion1,2. In the inactive (GDP-bound) conformation, accessory factors facilitate the targeting of Rab GTPases to intracellular compartments3,4,5,6,7,8. After nucleotide exchange to the active (GTP-bound) conformation, Rab GTPases interact with functionally diverse effectors including lipid kinases, motor proteins and tethering complexes. How effectors distinguish between homologous Rab GTPases represents an unresolved problem with respect to the specificity of vesicular trafficking. Using a structural proteomic approach, we have determined the specificity and structural basis underlying the interaction of the multivalent effector rabenosyn-5 with the Rab family. The results demonstrate that even the structurally similar effector domains in rabenosyn-5 can achieve highly selective recognition of distinct subsets of Rab GTPases exclusively through interactions with the switch and interswitch regions. The observed specificity is determined at a family-wide level by structural diversity in the active conformation, which governs the spatial disposition of critical conserved recognition determinants, and by a small number of both positive and negative sequence determinants that allow further discrimination between Rab GTPases with similar switch conformations.

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Figure 1: Mapping of the rabenosyn-5 Rab GTPase binding domains.
Figure 2: Quantitative family-wide analysis of Rab GTPase–effector specificity.
Figure 3: Structural basis of Rab recognition by rabenosyn-5.
Figure 4: Structure-based mutational analysis of Rab and rabenosyn-5 interaction specificity.

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Acknowledgements

We are grateful to M. Zerial for a full-length clone of rabenosyn-5; E. Kittler and M. Zapp for assistance with surface plasmon resonance experiments; and A. Delprato for Rab5 mutants. Surface plasmon resonance data were collected in the UMASS Center for AIDS Research Molecular Biology Core. This work was supported by an NIH grant.

Author information

Correspondence to David G. Lambright.

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Competing interests

Coordinates and structure factors have been deposited in the Protein Data Bank under the codes 1YZM (Rbsn(458–503)), 1Z0J (Rab22–Rbsn(728–784)), 1Z0K (Rab4–Rbsn(440–503)) and as listed in Supplementary Table 3 (Rab GTPases). Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure S1

Electron density for functionally relevant regions (PDF 589 kb)

Supplementary Figure S2

Influence of crystal packing on the active and inactive structures of Rab GTPases (PDF 498 kb)

Supplementary Figure S3

Survey of structural similarity, variability and plasticity in the Rab family (PDF 502 kb)

Supplementary Figure S4

Mapping and structure of the Rabenosyn-5 Rab binding domains (PDF 426 kb)

Supplementary Figure S5

Quantitative family-wide analysis of Rab-effector specificity. (PDF 240 kb)

Supplementary Figure S6

Schematic diagram of intermolecular interactions in the Rab-Rabenosyn-5 complexes. (PDF 319 kb)

Supplementary Figure S7

Reversal of specificity mutations in the Rabenosyn-5 Rab binding domains. (PDF 324 kb)

Supplementary Table S1

Rab constructs and constructs used for crystallization. (PDF 42 kb)

Supplementary Table S2

Crystallization, data collection and refinement statistics. (PDF 84 kb)

Supplementary Table S3

Summary of Rab GTPase structures analyzed with references to original work (PDF 51 kb)

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Eathiraj, S., Pan, X., Ritacco, C. et al. Structural basis of family-wide Rab GTPase recognition by rabenosyn-5. Nature 436, 415–419 (2005) doi:10.1038/nature03798

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