Insights into Oncogenic Mutations of Plexin-B1 Based on the Solution Structure of the Rho GTPase Binding Domain  Yufeng Tong, Prasanta K. Hota, Mehdi Bagheri Hamaneh, Matthias Buck  Structure  Volume 16, Issue 2, Pages 246-258 (February 2008) DOI: 10.1016/j.str.2007.12.012 Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 1 Structure of the Plexin-B1 RBD (A) Backbone trace of 20 structures of plexin-B1 RBD W1830F with the lowest energy. (B) Ribbon representation of the average RBD structure with elements of secondary structure labeled. (C) Superposition of the Cα trace of the average structure of the plexin-B1 RBD W1830F (PBD: 2JPH, red) and of human ubiquitin (PDB code: 1UBQ, green). Structure 2008 16, 246-258DOI: (10.1016/j.str.2007.12.012) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 2 Topology and Hydrogen Bond Pattern in the Plexin-B1 RBD Structure (A) Cartoon representation of the topology of plexin-B1 RBD structure and hydrogen bonding pattern. Side chains of residues N1834 and Q1837, and amide or carbonyl groups of A1756, D1773, I1777 and S1818, are shown in ball-sticks. (B) Cartoon representation of the topology of ubiquitin structure and hydrogen bonding pattern. α Helices are colored in orange, β strands in blue, and 3,10 helices in pink. Protons are shown in gray, carbons in black, nitrogens in blue, and oxygens in red. Hydrogen bonds are labeled with orange dashed arrows. Direction of arrow head indicates donor to acceptor connection, thus a double-headed arrow implies hydrogen bonding from both the amide proton of residue A to the carbonyl oxygen of residue B and the amide proton of residue B to the carbonyl oxygen of residue A. Structure 2008 16, 246-258DOI: (10.1016/j.str.2007.12.012) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 3 Residue Conservation in the Plexin Family for the RBD Region Conserved residues by ConSurf (Armon et al., 2001) indicated on the RBD main chain (A) and protein surface (B). Proteins on the right are back views of the left images by rotating 180° around an x axis, horizontal on the page. Structure 2008 16, 246-258DOI: (10.1016/j.str.2007.12.012) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 4 Surface Charge of the Plexin-B1 RBD Surface charge and nonpolar regions mapped onto the structure of the RBD (A and B) and ubiquitin (C and D). View of (B) and (D) is the same as in Figure 3. Structure 2008 16, 246-258DOI: (10.1016/j.str.2007.12.012) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 5 Location of the Oncogenic Mutations and the Rho GTPase Binding Region Mutation sites are shown with side chains as ball-and-stick relative to the Rho GTPase binding interface as determined by NMR (Tong et al., 2007). Two plexin Rho GTPase association motifs (PRAMs), residues 1805–1817 and 1834–1841, colored green, are brought together by the tertiary fold. Structure 2008 16, 246-258DOI: (10.1016/j.str.2007.12.012) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 6 Binding between Rho GTPases and the Plexin-B1 RBD Detected by Isothermal Titration Calorimetry Typical data for the Plexin-B1: Rac1 interaction monitored by isothermal titration calorimetry in phosphate buffer, pH 7.0, containing 4 mM MgCl2 and 1 mM TCEP at 25°C. (Top) raw data; (bottom) peak integrated enthalpy change; (A) for plexin W1830F and (B) L1815P/W1830F. Structure 2008 16, 246-258DOI: (10.1016/j.str.2007.12.012) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 7 Chemical Shift Perturbation Due to the Mutations Mapped onto the Structure Magenta, δΔ > 0.25 ppm; dark blue, δΔ > 0.35 ppm. Structure 2008 16, 246-258DOI: (10.1016/j.str.2007.12.012) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 8 Molecular Dynamics Analysis of the Plexin-B1 RBD MD analyses of mutants T1795A (green), T1802A (magenta), L1815P (blue), L1815F (red) compared to the wild-type protein (black). (A) Main chain rmsd of the protein secondary structure from the starting conformation as a function of simulation time. (B and C) Same rmsd, but on a residue basis for (B) loop L2 and (C) helix α2. (D) Main chain rms fluctuation over the last 2 ns for residues in helix α2. (E and F) Distances between main chain nitrogen/oxygen atoms of residues 1815 and 1807. A window averaging of 100 ps was used. (G) χ1 angle of Y1839. (H) Angle between ring planes Y1839…W1807. Structure 2008 16, 246-258DOI: (10.1016/j.str.2007.12.012) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 9 Conformational Changes in L1815F and L1815P Mutants (A) Two regions of interest are shown, with the positions of certain side-chains superimposed on the main chain conformation. (B) Binding site for Rho GTPases viewed from a different angle (as seen from the back of the structure in [A]). Snap-shots of the structure of models after 5 ns of MD at 300K. Region around residue 1815: (C) wild type; (D) L1815F; (E) L1815P. Structure 2008 16, 246-258DOI: (10.1016/j.str.2007.12.012) Copyright © 2008 Elsevier Ltd Terms and Conditions