Allosteric binding sites in Rab11 for potential drug candidates

<div><p>Rab11 is an important protein subfamily in the RabGTPase family. These proteins physiologically function as key regulators of intracellular membrane trafficking processes. Pathologically, Rab11 proteins are implicated in many diseases including cancers, neurodegenerative diseases and type 2 diabetes. Although they are medically important, no previous study has found Rab11 allosteric binding sites where potential drug candidates can bind to. In this study, by employing multiple clustering approaches integrating principal component analysis, independent component analysis and locally linear embedding, we performed structural analyses of Rab11 and identified eight representative structures. Using these representatives to perform binding site mapping and virtual screening, we identified two novel binding sites in Rab11 and small molecules that can <i>preferentially</i> bind to different conformations of these sites with high affinities. After identifying the binding sites and the residue interaction networks in the representatives, we computationally showed that these binding sites may <i>allosterically</i> regulate Rab11, as these sites communicate with switch 2 region that binds to GTP/GDP. These two allosteric binding sites in Rab11 are also similar to two allosteric pockets in Ras that we discovered previously.</p></div

Ligands that are scored best only in GNP-bound conformations of Rab11a.

<p>Ligands that are scored best only in GNP-bound conformations of Rab11a.</p

Allosteric binding sites in Rab11 for potential drug candidates - Fig 11

<p><b>Sequence map showing identities of (A) site 1 and (B) site 2 between Rab1 and Rab11 proteins.</b> Proteins are represented as circles and the identities between them are indicated on the edges. Site 1 is conserved in Rab1 and Rab11 proteins having more than 50% identity. Site 2 is less conserved with less than 40% identity.</p

Top scoring ligands ZINC18057104 and ZINC04783229 and their interactions with Rab11.

<p>The figure shows the interactions of the ligands (labeled) with the residues of the Rab11 structures in which they scored the best. The ligand and Rab11 side chains are shown in ball-and-stick representation. Black circles denote carbon atoms, red circles denote oxygen atoms, and blue circles denote nitrogen atoms. The ligand bonds are colored in purple. Residues in Rab11 interacting with the ligand are labeled. Hydrogen bonds are shown as green dotted lines. The Rab11 residues making nonbonded contacts with the ligand are shown as spoked arcs. Figures are generated using LigPlot+ [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198632#pone.0198632.ref066" target="_blank">66</a>][<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198632#pone.0198632.ref067" target="_blank">67</a>].</p

Site 1 and Site 2 in Rab11.

<p>The figure shows site 1 (colored in green) and site 2 (colored in blue) in Rab11 representative structure 1OIV_A. Guanosine-5'-Diphosphate (GDP) in the active site near site 1 is shown as red sticks. The Rab11 structure is rotated by 180⁰ along the Y axis to show the location of site 2. The figures are generated using UCSF Chimera [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198632#pone.0198632.ref065" target="_blank">65</a>].</p

Allosteric binding sites in Rab11 for potential drug candidates - Fig 10

<p><b>Communications between switch 2 and site 1 in (A) 1YZK_A, (B) 4C4P_A, (C) 4LX0_C, (D) 5JCZ_D, (E) 4OJK_A and (F) 1OIV_A.</b> Switch 1 and switch 2 regions are shown in cyan and magenta, respectively. Site 1 and site 2 residues are colored in green and blue, respectively. The ligands at the active site (i.e. GTP analogs or GDP) are shown as red sticks. Berryliumflouride (BEF) in (C) and (D) are shown as yellow sticks. In (D) ethylene glycol (EDO) molecules are shown as orange sticks. For each structure, the magnesium ion at the active site is represented as a green sphere, water molecules are shown as red spheres and the residues that undergo conformational changes are labeled. Hydrogen bonds are shown as thin blue lines. In (A) GLY69 of switch 2 has hydrogen bond interaction with the ligand. Switch 2 is perturbed and GLU71 (switch 2) makes contacts with ARG104 (site 1) and ALA75 (switch 2), through hydrogen bonds. In (B) GLY69 and SER20 have hydrogen bond interactions with the ligand. The interaction with Rab11a-effector causes additional conformational changes in switch 2 region comprising ARG72, and there are associated conformational changes in residue ARG104 of site 1. In (C), in relation to switch 2 conformational changes, ARG72 and SER20 have hydrogen bonds with GLN70, and ARG74 and SER78 have hydrogen bonds with GLU108. In (D), there are two EDO molecules shown on protein structure. ARG72 and GLU108 have hydrogen bond interactions with EDO. ARG72 has hydrogen bond interaction with GLY69. In (E), there are conformational changes in GLN70, GLU71, ARG74, THR77 and GLU108 associated with perturbation of switch 2. In (F), the GDP-bound representative structure of Rab11a is shown, the switch 2 region is structurally different from the other representative structures and it is in no position to have hydrogen bond interactions with site 1. Figures are generated using UCSF Chimera [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198632#pone.0198632.ref065" target="_blank">65</a>].</p

Structural dynamics of Rab11 protein as quantified by residual flexibility.

<p>Switches 1 and 2 are colored in cyan and pink, respectively. P-loop is highlighted in green. RabF and RabSF regions are colored in magenta and orange, respectively.</p

Top scoring ligands ZINC01694053 and ZINC01707130 and their interactions with Rab11.

<p>The figure shows the interactions of the ligands (labeled) with the residues of the Rab11 structures in which they scored the best. The ligand and Rab11 side chains are shown in ball-and-stick representation. Black circles denote carbon atoms, red circles denote oxygen atoms, and blue circles denote nitrogen atoms. The ligand bonds are colored in purple. Residues in Rab11 interacting with the ligand are labeled. Hydrogen bonds are shown as green dotted lines. The Rab11 residues making nonbonded contacts with the ligand are shown as spoked arcs. Figures are generated using LigPlot+ [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198632#pone.0198632.ref066" target="_blank">66</a>][<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198632#pone.0198632.ref067" target="_blank">67</a>].</p

Top scoring ligands docked at different Rab11 representative structures.

<p>(A) ZINC18141294 docked at site 1 of PDB entry 1OIV_A, (B) ZINC29590259 docked at site 1 of PDB entry 4LX0_C, (C) ZINC04773602 docked at site 2 of PDB entry 1YZK_A, (D) ZINC01572309 docked at site 2 of PDB entry 4C4P_A, (E) ZINC13152284 docked at site 2 of PDB entry 4UJ5_B and (F) ZINC29590263 docked at site 2 of PDB entry 4OJK_A. Ligands are shown in brown sticks. Site 1 and Site 2 are colored in green and blue, respectively. Ligands at the active sites of Rab11 in (A) and (B) are shown as red spheres.</p