Structure of the DENND1B-S/Rab35 complex. (A) Sequence of DENND1B-S in crystallization construct. Secondary structure as in the crystal structure is indicated. Helices are orange bars and strands blue. Disordered regions are indicated by dotted lines. Sixteen residues at the C terminus and a 20-residue loop in wild-type protein were deleted; their locations are indicated by green and red arrowheads. (B) The DENND1B-S construct used for crystallization is equally efficient in catalyzing guanine nucleotide exchange for Rab35 as wild-type protein. MantGDP dissociation from Rab35 was continuously monitored by FRET (λ_ex = 280 nm, λ_em = 435 nm) with increasing concentrations of either the crystallization construct or wild-type DENN domain. The DENND1C DENN domain is approximately 10-fold less efficient, as is a DENND1B mutant, where residues at the Rab35 interface were changed to corresponding residues in DENND1C. See Fig. S5A for representative time course data. (C) Ribbons diagram of DENND1B-S, colored with blue at the N terminus and red at the C terminus, complexed with Rab35 (blue). (D) DENND1B-S alone with secondary structure elements labeled. (E) Rab35 is rendered as a ribbon, DENND1B-S as a surface representation.

Interactions between DENND1B-S and Rab35. (A) Residues in DENND1B-S within 5 Å of Rab35 are labeled (left), as are residues in Rab35 within 5 Å of the DENN-domain (right). In Rab35, interacting residues in switches I or II are orange and residues outside the switch region blue. A 180° rotation about a vertical axis would position the Rab to interact with DENND1B-S. (B) DENND1B-S oriented as in A with residues within 4.5 Å of Rab35 in orange and labeled. (C) DENND1B-S as in B, with Rab35 switch regions I and II bound. Rab35 residues are labeled. (D) Sequence conservation in 18 human DENN domains mapped onto the surface of DENND1B-S. Strictly, highly, and weakly conserved residues (identical in 16–18, 11–15, and 8–10 sequences, respectively) are purple, magenta, and lilac, respectively. (E) The Rab35 interaction surface is similar in DENND1B and −1A. The Rab35 interaction surface on DENND1B is as in B, with residues in DENND1B within 10 Å of Rab35 that are different in DENND1A indicated (blue). Residues further than 5 Å from Rab35 are in parentheses. (F) As in E but comparing DENND1B and −1C.

Rearrangement of Rab35 and switches I and II in the Rab/GEF complex. (A) Sequences in the switch regions of Rab35 and Rab1 are highly conserved. See also Fig. S2F for a comparison of Rab1 and Rab35 surfaces. (B) Comparison of Rab35 (purple) in the complex with GDP-bound Rab1 (lilac; PDB ID code 2FOL ). Switches I and II are orange or yellow, respectively, and GDP and Mg²⁺ are yellow. F33 (Y33 in Rab1) moves out of the nucleotide-binding pocket as switch I alters its conformation. F33 is not well ordered and that the position of its side chain is uncertain. Arrows indicate conformational changes. (C) As in B but comparing Rab35 in the complex with GTP-bound Rab1 (PDB ID code 1YZN). (D) Switch I in Rab35 must rearrange as Rab35 binds DENND1B because it would otherwise clash sterically with H7 in the DENN domain (brown). Rab35 in the Rab/GEF complex is purple/orange; GDP-bound Rab is lilac/yellow. Arrows indicate clashes. (E) DENND1B residues predicted to affect either switch I or II binding were mutated, as indicated (blue and lilac for I and II, respectively). Compare this panel to Fig. 2 B–C. (F) The catalytic efficiency of the mutants is reduced compared to wild type as assessed by monitoring mantGDP release. A representative time course with DENN domains at 4 μM. (G) [³H]GDP dissociates approximately 20-fold faster from the Rab35 F33A mutant than from Rab35. A time course of [³H]GDP dissociating from Rab35 or Rab35-F33A (both at 200 nM concentration) is shown. The solid line is the best fit of a single exponential function, yielding a GDP dissociation rate constant (k_-GDP) of 1.35 ± 0.07 × 10^-4 s^-1 for Rab35 and 25.6 ± 2.8 × 10^-4 s^-1 for Rab35-F33A. (H) mantGDP association rates are similar for Rab35 and the F33A mutant. MantGDP binding to nucleotide-free Rab was monitored by FRET (Fig. S5B). The concentration dependence of the observed GDP association rate constant (k_+GDP) on [mantGDP] is shown. The rate constants from the slope of the best linear fit are 2.09 ± 0.03 μM^-1 s^-1 for Rab35 and 2.59 ± 0.09 μM^-1 s^-1 for Rab35 (F33A). Data were generated considering only the fast k_obs from the double-exponential fit. The true concentration dependence may be hyperbolic, but we were not able to work at sufficiently high mantGDP concentration to see saturation.