Abstract
Myosin motor proteins convert chemical energy into force and movement through their interactions with nucleotide and filamentous actin (F-actin). The evolutionarily conserved lysine-265 (K265) of the myosin-2 motor from Dictyostelium discoideum (Dd) is proposed to be a key residue in an allosteric communication pathway that mediates actin-nucleotide coupling. To better understand the role of K265, point mutations were introduced within the Dd myosin-2 M765-2R framework, replacing this lysine with alanine (K265A), glutamic acid (K265E) or glutamine (K265Q), and the functional and kinetic properties of the resulting myosin motors were assessed. The alanine and glutamic acid substitutions reduced actin-activated ATPase activity, slowed the in vitro sliding velocity and attenuated the inhibitory potential of the allosteric myosin inhibitor pentabromopseudilin (PBP). However, glutamine substitution did not substantially change these parameters. Structural modelling suggests that K265 interacts with D590 and Q633 to establish a pivotal allosteric branching point. Based on our results, we propose: (1) that the K265-D590 interaction functions to reduce myosins basal ATPase activity in the absence of F-actin, and (2) that the dynamic formation of the K265-Q633 salt bridge upon actin cleft closure regulates the activation of product release by actin filaments.
Highlights
This study sought to characterize the kinetic and structural basis for the K265-mediated modulation of myosin motor function
Based on previously published crystal structures of the myosin motor domain[23,24,25,26], we suggest that K265 is located at a branching point in the allosteric pathway, where its interaction with Q633 at the base of loop-2 supports strong actin-binding and the actin-mediated activation of product release, and thereby myosin ATPase activity
We show that the evolutionary conserved amino acid residue lysine-265 in Dd myosin-2 is implicated in the kinetic and mechanical coupling of nucleotide- and actin-binding sites in the motor domain
Summary
This study sought to characterize the kinetic and structural basis for the K265-mediated modulation of myosin motor function. To define the molecular role of this conserved amino acid in the proposed allosteric pathway, we generated mutant constructs where the lysine at position 265 is substituted for alanine (K265A), glutamic acid (K265E) or glutamine (K265Q). These substitutions, which effectively remove a side chain (K265A), reverse the charge of the amino acid (K265E) or remove the charge and replace the amino acid with one of a similar size (K265Q), give insights regarding the immediate interactions of K265. The interaction of K265 with D590 in the strut loop appears to attenuate the basal ATPase in the absence of actin and to modulate nucleotide-binding properties of the myosin motor domain
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