Structural Basis of Rnd1 Binding to Plexin Rho GTPase Binding Domains (RBDs)

Hui Wang,Prasanta K Hota,Yufeng Tong,Buren Li,Limin Shen,Lyudmila Nedyalkova,Susmita Borthakur,Soonjeung Kim,Wolfram Tempel,Matthias Buck,Hee-Won Park

doi:10.1074/jbc.m110.197053

Abstract

Plexin receptors regulate cell adhesion, migration, and guidance. The Rho GTPase binding domain (RBD) of plexin-A1 and -B1 can bind GTPases, including Rnd1. By contrast, plexin-C1 and -D1 reportedly bind Rnd2 but associate with Rnd1 only weakly. The structural basis of this differential Rnd1 GTPase binding to plexin RBDs remains unclear. Here, we solved the structure of the plexin-A2 RBD in complex with Rnd1 and the structures of the plexin-C1 and plexin-D1 RBDs alone, also compared with the previously determined plexin-B1 RBD.Rnd1 complex structure. The plexin-A2 RBD·Rnd1 complex is a heterodimer, whereas plexin-B1 and -A2 RBDs homodimerize at high concentration in solution, consistent with a proposed model for plexin activation. Plexin-C1 and -D1 RBDs are monomeric, consistent with major residue changes in the homodimerization loop. In plexin-A2 and -B1, the RBD β3-β4 loop adjusts its conformation to allow Rnd1 binding, whereas minimal structural changes occur in Rnd1. The plexin-C1 and -D1 RBDs lack several key non-polar residues at the corresponding GTPase binding surface and do not significantly interact with Rnd1. Isothermal titration calorimetry measurements on plexin-C1 and -D1 mutants reveal that the introduction of non-polar residues in this loop generates affinity for Rnd1. Structure and sequence comparisons suggest a similar mode of Rnd1 binding to the RBDs, whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail. Our results confirm, from a structural perspective, that Rnd1 does not play a role in the activation of plexin-C1 and -D1. Plexin functions appear to be regulated by subfamily-specific mechanisms, some of which involve different Rho family GTPases.

Highlights

JULY 22, 2011 VOLUME 286 NUMBER 29 and sequence comparisons suggest a similar mode of Rnd1 binding to the Rho GTPase binding domain (RBD), whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail
A similar homodimerization interface has been observed in the structure of the Rnd1-free form of plexin-B1 RBD [21]
Rnd1 binding to the plexin-B1 RBD appears to promote ordering of a flexible region, the loop ␤3-␤4, in the plexin-B1 RBD-Rnd1 interface in the crystal (Fig. 1, B and C)

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The genes encoding the RBDs of human plexin-A2 (residues 1490 –1600), plexin-B1 (residues 1746 –1852), plexin-C1 (residues 1198 –1305), plexin-D1 (residues 1553–1678), and Rnd GTPase (residues 5–200) and Rnd (residues 1–227) were amplified by PCR and subcloned into the expression vector pET28-MHL, which encodes an N-terminal His tag followed by the tobacco etch virus protease cleavage site. Plexin-A2 RBD and Rnd were mixed in a 1:1 ratio in the buffer used for protein purification. Plexin-C1 RBD was crystallized using an in situ proteolysis method [27]. A 0.5-␮l protein sample containing chymotrypsin (1:100, w/w) was mixed with 0.5 ␮l of well solution containing 0.1 M HEPES (pH 7.5), 2.0 M (NH4)2SO4, and 0.2 M NaCl. The plexin-D1 RBD was overexpressed in E. coli using a prepacked M9 selenomethionine growth medium kit (Medicilon) and the N-terminal His tag was removed by tobacco etch virus protease cleavage before crystallization. The crystal of the plexin-D1 RBD was grown in 0.1 M sodium cacodylate buffer (pH 5.2) containing 1.39 M sodium citrate and 15 mM tris(2-carboxyethyl)phosphine, using the in situ proteolysis method with chymotrypsin (1:100, w/w). The method and data are shown in the supplemental material

RESULTS

PDB code Diffraction data

DISCUSSION