Switch II mutants reveal coupling between the nucleotide- and actin-binding regions in myosin V.

Abstract:

: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.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Trivedi DV,David C,Jacobs DJ,Yengo CM

doi

10.1016/j.bpj.2012.04.025

subject

Has Abstract

pub_date

2012-06-06 00:00:00

pages

2545-55

issue

11

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(12)00470-5

journal_volume

102

pub_type

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