The Application of Hybrid Methods to Study Complex, Multi-Domain Protein - RhoA-Specific Guanidine Nucleotide Exchange Factor - PDZ-RhoGEF

Abstract

As the focus of structural biology shifts towards studies of increasingly complex, multi-domain proteins and their complexes, the use of hybrid methodologies is becoming a necessity. Here, we present our findings regarding the mechanism of regulation of a multi-domain, RhoA-specific guanine nucleotide exchange factor, PDZ-RhoGEF (PRG). PRG belongs to a small family of RGS GEFs and is an important component of the ‘RhoA cycle’.Activity of RhoA is tightly regulated by a sophisticated interplay between three proteins - guanine nucleotide exchange factor (RhoGEF), GTPase-activating protein (RhoGAP), and guanine nucleotide dissociation inhibitor (RhoGDI). GEFs catalyze the exchange of a nucleotide on RhoA, and consequently cause RhoA activation. Active RhoA transmits signal to its effectors (e.g. Rho-dependent kinase (ROCK)), and consequently triggers a number of physiological responses, including reorganization of actin cytoskeleton, neurite retraction and neuronal morphogenesis, and Ca2+ - sensitization of smooth muscle.The classical paradigm for regulation of multidomain GEFs states that these proteins are autoinhibited by the supramodular arrangement of their domains, which may interfere with the binding of upstream and downstream binding partners, and that a combination of inter-protein interactions initiates a conformational change, leading to the relief of autoinhibition. Using a broad array of biophysical and biochemical techniques, including SAXS, EPR, DLS, CD and others, we are investigating the structure of several multidomain constructs of PRG and we present current data suggesting a functional role for interdomain linkers.