Electrostatic interactions in the SH1-SH2 helix of human cardiac myosin modulate the time of strong actomyosin binding
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ORIGINAL PAPER
Electrostatic interactions in the SH1-SH2 helix of human cardiac myosin modulate the time of strong actomyosin binding Akhil Gargey1,2 · Shiril Bhardwaj Iragavarapu1 · Alexander V. Grdzelishvili1 · Yuri E. Nesmelov1 Received: 21 April 2020 / Accepted: 2 September 2020 © Springer Nature Switzerland AG 2020
Abstract Two single mutations, R694N and E45Q, were introduced in the beta isoform of human cardiac myosin to remove permanent salt bridges E45:R694 and E98:R694 in the SH1-SH2 helix of the myosin head. Beta isoform-specific bridges E45:R694 and E98:R694 were discovered in the molecular dynamics simulations of the alpha and beta myosin isoforms. Alpha and beta isoforms exhibit different kinetics, ADP dissociates slower from actomyosin containing beta myosin isoform, therefore, beta myosin stays strongly bound to actin longer. We hypothesize that the electrostatic interactions in the SH1-SH2 helix modulate the affinity of ADP to actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutants of the myosin head construct (1–843 amino acid residues) were expressed in differentiated C 2C12 cells, and the duration of the strongly bound state of actomyosin was characterized using transient kinetics spectrophotometry. All myosin constructs exhibited a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. The mutant R694N showed a faster rate of ADP release from actomyosin, compared to the wild type and the E45Q mutant, thus indicating that electrostatic interactions within the SH1-SH2 helix region of human cardiac myosin modulate ADP release and thus, the duration of the strongly bound state of actomyosin. Keywords Electrostatics · Salt bridge · Transient kinetics · Myosin · Actin · ATP · ADP
Introduction Myosin II is a nanoscale motor that transduces the chemical energy of ATP to generate force during muscle contraction and perform transport functions in non-muscle cells. ATP binds actomyosin and initiates its dissociation, and ATP hydrolysis drives myosin conformational change, or the recovery stroke, that primes myosin for subsequent strong binding to actin to produce the power stroke. The rate of ATP binding to actomyosin and the rate of ADP release determine the duration of the strong actomyosin binding, Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10974-020-09588-1) contains supplementary material, which is available to authorized users. * Yuri E. Nesmelov [email protected] 1
Department of Physics and Optical Science, University of North Carolina Charlotte, 9201 University City Blvd, Charlotte, NC 28223, USA
Department of Biological Science, University of North Carolina Charlotte, Charlotte, NC 28223, USA
2
which is directly related to the average force produced by muscle (Harris and Warshaw 1993). General organization of the motor domain is conserved among myosins (Houdusse and Sweeney 200
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