The Role of Mg2+ Cofactor in the Guanine Nucleotide Exchange and GTP Hydrolysis Reactions of Rho Family GTP-binding Proteins

TLDR

Rho family GTPases regulate cellular activities through their guanine nucleotide binding states, and unlike Ras and Rab proteins, they bind GDP and GTP with similar submicromolar affinity whether Mg²⁺ is present or absent. The study investigates how Mg²⁺ influences nucleotide binding and hydrolysis in Rho family GTPases Cdc42, Rac1, and RhoA. They assessed Mg²⁺ impact by measuring nucleotide dissociation rates, GEF‑mediated exchange, and GAP‑catalyzed hydrolysis in these GTPases. Mg²⁺ markedly slows nucleotide dissociation, is displaced by GEFs (which reduce Mg²⁺ affinity), and enhances GTP hydrolysis and GAP activity, with GEF exchange involving both Mg²⁺‑dependent and independent steps and loss of GAP specificity for Cdc42 in the absence of Mg²⁺.

Abstract

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg²⁺ cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which require Mg²⁺ for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg²⁺ similarly, with dissociation constants in the submicromolar concentration. The presence of Mg²⁺, however, resulted in a marked decrease in the intrinsic dissociation rates of the nucleotides. The catalytic activity of the guanine nucleotide exchange factors (GEFs) appeared to be negatively regulated by free Mg²⁺, and GEF binding to Rho GTPase resulted in a 10-fold decrease in affinity for Mg²⁺, suggesting that one role of GEF is to displace bound Mg²⁺ from the Rho proteins. The GDP dissociation rates of the GTPases could be further stimulated by GEF upon removal of bound Mg²⁺, indicating that the GEF-catalyzed nucleotide exchange involves a Mg²⁺-independent as well as a Mg²⁺-dependent mechanism. Although Mg²⁺ is not absolutely required for GTP hydrolysis by the Rho GTPases, the divalent ion apparently participates in the GTPase reaction, since the intrinsic GTP hydrolysis rates were enhanced 4–10-fold upon binding to Mg²⁺, and<i>k</i> _cat values of the Rho GTPase-activating protein (RhoGAP)-catalyzed reactions were significantly increased when Mg²⁺ was present. Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg²⁺ cofactor. These studies directly demonstrate a role of Mg²⁺ in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases. The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg²⁺, whereas RhoGAP utilizes the Mg²⁺ cofactor to achieve high catalytic efficiency and specificity.