Control of the ability of profilin to bind and facilitate nucleotide exchange from G-actin.
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.
Wen
Kuo-Kuang
KK
Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA.
McKane
Melissa
M
Houtman
Jon C D
JC
Rubenstein
Peter A
PA
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D006801:Humans; D007700:Kinetics; D051304:Profilins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D000199:Actins; C495738:PFN1 protein, human; D051304:Profilins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate