1. Rab35/ACAP2 and Rab35/RUSC2 Complex Structures Reveal Molecular Basis for Effector Recognition by Rab35 GTPase 
Lin Lin, Yingdong Shi, Mengli Wang, Chao Wang, Jinwei Zhu, Rongguang Zhang 
Structure 
DOI: /j.str
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2. Structure DOI: (10.1016/j.str.2019.02.008)
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3. Figure 1 Specific Binding of Rab35 to ACAP2 and RUSC2
(A) Schematic diagrams showing the domain organizations of Rab35 and its selective effectors. The switch-I and switch-II of Rab35 are colored in red and blue, respectively. The Rab-binding domains of OCRL (ASH), MICAL1 (CC), ACAP2 (ANK), and RUSC2 (RUN) used in this study are colored in blue, orange, green, and pink, respectively.
(B and C) GST pull-down assays showing that ACAP2-ANK (B) and RUSC2-RUN (C) specifically binds to Rab35, but not Rab1, Rab8, or Rab13.
(D and E) Quantitative measurement of the binding affinities of Rab35/ACAP2-ANK (D) and Rab35/RUSC2-RUN (E) interactions using the ITC-based assay.
Structure DOI: ( /j.str )
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4. Figure 2
Crystal Structures of the Rab35/ACAP2-ANK and Rab35/RUSC2-RUN Complexes
(A and B) Ribbon diagram of the structures of Rab35/ACAP2-ANK complex (A) and Rab35/RUSC2-RUN complex (B). Rab35, ACAP2-ANK, and RUSC2-RUN are colored in gray, green, and pink, respectively. In this drawing, the switch-I and switch-II of Rab35 are colored in red and blue, respectively. Mg2+ ions are shown as balls and the GTP as sticks.
(C and D) Combined surface and ribbon diagram of the Rab35-contacting interfaces of ACAP2-ANK (C) and RUSC2-RUN (D).
(E and F) The detailed interface of Rab35/ACAP2 complex showing the hydrophobic contact sites (E) and the polar contact sites (F) respectively.
(G and H) The detailed interface of Rab35/RUSC2-RUN complex showing the interactions between RUSC2-RUN-α1 and Rab35 (G), RUSC2-RUN-α7/α8 and Rab35 (H), respectively. Residues involved in bindings are shown with the stick model. Salt bridges and hydrogen bonds are indicated with dashed lines. Residues mutated in the patients with cancers are highlighted with orange box.
See also Figures S1 and S4.
Structure DOI: ( /j.str )
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5. Figure 3 The Pseudo-arginine Finger of RUSC2
(A) Ribbon diagram of the Rab35/RUSC2-RUN structure showing that R1015RUSC2 forms specific hydrogen bonds with GTP via two water molecules. The omit maps of the GTP, H2O, and R1015RUSC2 are also shown here.
(B) GAP activity assays showing that RUSC2 is not a GAP for Rab35.
(C) Superposition of the structures of Rab35/RUSC2 and Rab33/Gyp1 complexes. Rabs, RUSC2, and Gyp1 are colored in gray, pink, and yellow, respectively. The orientation of R343Gyp1 and R1015RUSC2 (with stick mode) toward Rabs is distinct.
(D and E) Interactions in the active site of the Rab33/Gyp1 (D), and Rab35/RUSC2 (E) complexes.
Structure DOI: ( /j.str )
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6. Figure 4
Effects of Mutations on Rab35/ACAP2 and Rab35/RUSC2 Interactions
(A) The bindings of ACAP2-ANK or its various mutants to Rab35 by the GST pull-down assay.
(B) The bindings of Rab35 or its various mutants to wild-type ACAP2-ANK by the GST pull-down assay.
(C) Summary of ITC-based measurements of binding affinities between wild-type or mutants of Rab35 and ACAP2-ANK.
(D) The bindings of RUSC2-RUN or its various mutants to Rab35 by the GST pull-down assay.
(E) The bindings of Rab35 or its various mutants to wild-type RUSC2-RUN by the GST pull-down assay.
(F) Summary of ITC-based measurements of binding affinities between wild-type or mutants of Rab35 and RUSC2-RUN. n.d., not detectable.
(G) The cellular colocalization analyses of Rab35, ACAP2 and their mutations in transfected HEK293T cells. Noted that wild-type Rab35 (GFP) and ACAP2 (RFP) colocalized at the membrane protrusions of cells, whereas the binding-deficient mutants cannot. Scale bars, 5 μm.
(H) The cellular colocalization analyses of Rab35, RUSC2 and their mutations in transfected HEK293T cells. Noted that wild-type Rab35 (GFP) and RUSC2 (RFP) colocalized at the membrane protrusions of cells, whereas the binding-deficient mutants cannot. Scale bars, 5 μm.
Structure DOI: ( /j.str )
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7. Figure 5 Comparison of Rab35-Effector Complex Structures
(A) Amino acid sequence alignment of effector-contacting regions of Rab35, Rab1, Rab8, Rab10, and Rab13. The totally conserved residues are highlighted with red box, and conserved residues are colored in red. The residues involved in binding to ACAP2, RUSC2, OCRL, and MICAL1 are annotated below as green, red, blue, and orange dots at the bottom.
(B) Surface representation showing the overall architecture of Rab35. Residues in the switch-I, -II, and interswitch are color in red, blue, and yellow, respectively.
(C) Projection of the key determinant residues for recognition of ACAP2 (c1), RUSC2 (c2), OCRL (c3), and MICAL1 (c4) on the surface of active Rab35.
(D) GST pull-down assay showing that T72A mutant of Rab35 specifically eliminated its binding to ACAP2, but not other effectors.
(E) GST pull-down assay showing that D4A mutant of Rab35 specifically disrupted its binding to RUSC2, but not other effectors.
See also Figure S2.
Structure DOI: ( /j.str )
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8. Figure 6
The Effects of Disease-Associated Mutations on the Formations of the Rab35/ACAP2 and Rab35/RUSC2 Complexes
(A) Selective disease-associated mutations occurred in Rab35 and its effectors (ACAP2-ANK and RUSC2-RUN). Noted that T72 of Rab35 is the phosphorylation site for LRRK2.
(B and C) Effects of the cancer-associated mutations on the formations of the Rab35/ACAP2-ANK (B) and Rab35/RUSC2-RUN (C) complexes based on GST pull-down assays.
(D) GST pull-down assays showing that both the phosphor-deficient mutant (T72ARab35) and the phosphor-mimetic mutant (T72DRab35) eliminated their bindings to ACAP2, but not RUSC2.
See also Figure S3.
Structure DOI: ( /j.str )
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