Switch II mutants reveal coupling between the nucleotide- and actin-binding regions in myosin V.
Conserved active-site elements in myosins and other P-loop NTPases play critical roles in nucleotide binding and hydrolysis; however, the mechanisms of allosteric communication among these mechanoenzymes remain unresolved. In this work we introduced the E442A mutation, which abrogates a salt-bridge between switch I and switch II, and the G440A mutation, which abolishes a main-chain hydrogen bond associated with the interaction of switch II with the γ phosphate of ATP, into myosin V. We used fluorescence resonance energy transfer between mant-labeled nucleotides or IAEDANS-labeled actin and FlAsH-labeled myosin V to examine the conformation of the nucleotide- and actin-binding regions, respectively. We demonstrate that in the absence of actin, both the G440A and E442A mutants bind ATP with similar affinity and result in only minor alterations in the conformation of the nucleotide-binding pocket (NBP). In the presence of ADP and actin, both switch II mutants disrupt the formation of a closed NBP actomyosin.ADP state. The G440A mutant also prevents ATP-induced opening of the actin-binding cleft. Our results indicate that the switch II region is critical for stabilizing the closed NBP conformation in the presence of actin, and is essential for communication between the active site and actin-binding region.
Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania, USA.
Darshan V
DV
Trivedi
Charles
C
David
Donald J
DJ
Jacobs
Christopher M
CM
Yengo
D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D018360:Crystallography, X-Ray; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism*; D024701:Myosin Type V / Q000737:chemistry* / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013696:Temperature
D000199:Actins; D050505:Mutant Proteins; D009711:Nucleotides; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D024701:Myosin Type V