Control of the Ability of Profilin to Bind and Facilitate Nucleotide Exchange from G-actin

Kuo-Kuang Wen,Melissa Mckane,Jon C.D Houtman,Peter A Rubenstein

doi:10.1074/jbc.m709806200

Abstract

A major factor in profilin regulation of actin cytoskeletal dynamics is its facilitation of G-actin nucleotide exchange. However, the mechanism of this facilitation is unknown. We studied the interaction of yeast (YPF) and human profilin 1 (HPF1) with yeast and mammalian skeletal muscle actins. Homologous pairs (YPF and yeast actin, HPF1 and muscle actin) bound more tightly to one another than heterologous pairs. However, with saturating profilin, HPF1 caused a faster etheno-ATP exchange with both yeast and muscle actins than did YPF. Based on the -fold change in ATP exchange rate/K(d), however, the homologous pairs are more efficient than the heterologous pairs. Thus, strength of binding of profilin to actin and nucleotide exchange rate are not tightly coupled. Actin/HPF interactions were entropically driven, whereas YPF interactions were enthalpically driven. Hybrid yeast actins containing subdomain 1 (sub1) or subdomain 1 and 2 (sub12) muscle actin residues bound more weakly to YPF than did yeast actin (K(d) = 2 microm versus 0.6 microm). These hybrids bound even more weakly to HPF than did yeast actin (K(d) = 5 microm versus 3.2 microm). sub1/YPF interactions were entropically driven, whereas the sub12/YPF binding was enthalpically driven. Compared with WT yeast actin, YPF binding to sub1 occurred with a 5 times faster k(off) and a 2 times faster k(on). sub12 bound with a 3 times faster k(off) and a 1.5 times slower k(on). Profilin controls the energetics of its interaction with nonhybrid actin, but interactions between actin subdomains 1 and 2 affect the topography of the profilin binding site.

Highlights

The ability of profilin to preferentially sequester ATP G-actin and to facilitate adenine nucleotide exchange from the actin is important, considering the role that an actin-dependent ATP hydrolysis cycle plays in actin dynamics
The small enhancement of nucleotide exchange previously observed with yeast actin and yeast profilin does not result from some maximal rate at which a yeast actin can exchange nucleotide due to its inherently more open conformation [17, 26]
Catalysis of Nucleotide Exchange—We first addressed the ability of profilin to facilitate actin nucleotide exchange under saturating profilin concentrations

Summary

Introduction

The ability of profilin to preferentially sequester ATP G-actin and to facilitate adenine nucleotide exchange from the actin is important, considering the role that an actin-dependent ATP hydrolysis cycle plays in actin dynamics. Based on incomplete data obtained so far, change in enthalpy seems to control the interaction of yeast profilin with muscle actin, whereas change in entropy seems to drive the interaction of human profilin with muscle actin [17]. Whether this difference applies only to these two actin-profilin pairs or is more general is not known. To gain more insight into the mechanism governing the profilin-dependent acceleration of the release of actin-bound nucleotide, we have carried out a detailed study of both the binding and exchange reactions involving the interaction of both yeast and human profilins with both muscle and yeast actins. We present work with a hybrid actin we have constructed in which subdomains 1 and 2 are from muscle actin and subdomains 3 and 4 are from yeast actin to better understand the relative importance of subdomains 1 and 3 in its interaction with profilin

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