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

Abstract

Rab GTPases regulate all stages of membrane trafficking, including vesicle budding, cargo sorting, transport, tethering and fusion. In the inactive (GDP-bound) conformation, accessory factors facilitate the targeting of Rab GTPases to intracellular compartments. 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.

Figure 1

Mapping of the Rabenosyn-5 Rab GTPase binding domains. Mean K_d values and standard deviations were obtained from 2–4 SPR experiments using purified Rab GTPases and Rabenosyn-5 constructs. The oligomeric state (n = 1 for monomer; n = 2 for dimer) was determined from sedimentation equilibrium experiments over the concentration range from 5 – 50 mM. Values represent the mean and standard deviation for 3 measurements.

Figure 2

Quantitative family-wide analysis of Rab GTPase-effector specificity. a, Initial screen for the interaction of 6xHis (or GST) fusions of Rab GTPases with GST (or 6xHis) fusions of Rbsn_440–503 and Rbsn_728–784. For each potential interaction, the equilibrium SPR signal (R_eq) was measured at four concentrations of the 6xHis Rab GTPase or 6xHis Rbsn construct. b, Concentration dependence of the equilibrium SPR signal (R_eq) for the binding of 6xHis Rab GTPases to GST fusions of Rbsn_440–503 and Rbsn_728–784. Mean K_d values and standard deviations for 2–4 independent experiments are tabulated on the right.

Figure 3

Structural basis of Rab recognition by Rabeonsoyn-5. a, Ribbon rendering of GTP-bound Rab4 Q67L and Rab22 Q64L in complex with the minimal Rab binding domains of Rabenosyn-5. b, Conservation and variability in the Rab22-Rbsn_728–784 interface. Spheres covered by a semitransparent surface represent Rab22 (left panel) or Rbsn_728–784 (right panel). Hydrogen bonding interactions are depicted in the right panel. c, Conservation and variability in the Rab4-Rbsn_440–503 interface. Spheres covered by a semitransparent surface represent Rab4 (left panel) or Rbsn_440–503 (right panel). Hydrogen bonding interactions are depicted in the right panel.

Figure 4

Structure-based mutational analysis of Rab and Rabeonsoyn-5 interaction specificity. a, Local alignment of representative Rab GTPases and Rabenosyn-5 homologs. Residues within the binding interfaces for Rab22-Rbsn_728–784, Rab4-Rbsn_440–503, Rab5-Rabaptin-5, and Rab3-Rabphilin are highlighted according to conservation. b, Effect of site specific substitutions on the interaction between Rab5 and Rbsn_728–784 or Rab4 and Rbsn_440–503. Mean values and standard deviations were calculated from 2 experiments. c, Views of the Rab22-Rbsn_728–784 and Rab4-Rbsn_440–503 interfaces relevant to the mutations discussed in the text. Also shown (middle panel) is the structure of the Rab5 G55Q mutant following superposition with Rab22.