The Conserved Lysine-265 Allosterically Modulates Nucleotide- and Actin-binding Site Coupling in Myosin-2

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.

Undefeated—Changing the phenamacril scaffold is not enough to beat resistant Fusarium

“…Currently, no phenamacril-FgMyo1 structure derived from X-ray diffraction crystallography is available to aid the interpretation of our findings. From one perspective, residues such as K216 in FgMyo1 (one of the amino acids associated with resistance) is known to constitute part of an allosteric communication pathway which transmits information between the nucleotide-and the actin-binding site [19,20,29,39]. These mechano-chemical events involve larger structural changes such as cleft-opening or closure and rotation of the lever arm.…”

“…This could potentially affect more distal binding-sites. From another perspective, the actin-binding cleft is also known to harbor allosteric binding sites [18][19][20][21]24] and the halogenated pseudilins mediate their inhibitory effect through direct interactions with a conserved lysine residue (K216 in F. graminearum) [19,20,29,39]. This therefore speaks for a binding of phenamacril in the vicinity of the residues associated with resistance.…”

“…Since the establishment of a baseline sensitivity [1,6], several UV-induced phenamacrylicresistant laboratory and more recently resistant field strains of Fusarium and the underlying individual amino acid mutations associated with the resistant phenotypes have been characterized [2,5,10,26,27]. In F. graminearum, the mutations confer either low (A135T, V151M, P204S, I434M, A577T, R580G/H and I581F), moderate (S418R, I424R and A577G) or high (K216R/E, S217P/L and E420K/G/D) levels of resistance [28] and mostly involve amino acid residues that are clustered deep within the actin-binding cleft, in immediate vicinity of an allosteric communication pathway responsible for the coupling of the actin and nucleotide binding sites [19,20,29]. While the qualitative resistance mechanism and the emergence of fieldresistant strains [5] may render the use of phenamacril short-lived, novel cyanoacrylate derivatives based on the phenamacril-scaffold promise to provide a useful strategy and rational approach to overcome resistant genotypes and preserve the usefulness of this new class of fungicides and inhibitors of myosin ATPase activity.…”

Phenamacril is a reversible and noncompetitive inhibitor of Fusarium class I myosin

“…Resistance development was observed to correlate with amino acid mutations, which are primarily located within the actin-binding cleft of the myosin motor domain (4,5,19). This region is known to play a pivotal role in mediating allosteric communication between the nucleotide-and actin-binding sites (21)(22)(23). It is a well-characterized and conserved allosteric site targeted by other classspecific myosin inhibitors such as blebbistatin (24) and the halogenated pseudilins (21,23,25).…”

Structural and Computational Insights into a Blebbistatin-Bound Myosin•ADP Complex with Characteristics of an ADP-Release Conformation along the Two-Step Myosin Power Stoke

“…Pivotal to this allosteric communication is the central seven-stranded β-sheet (the transducer) (twisted in the rigor state, but untwisted in the pre-power stroke state) in the myosin motor domain, which couples the large actin-binding cleft (closed while strongly attached to F-actin, and open in actin-detached states) with the active site phosphate-sensing elements—the P-loop, switch-1 and switch-2—(closed in the hydrolysis-competent pre-power stroke state, but partially or fully opened in all other states) as well as through the relay helix (bent in the pre-power stroke state, while straight in the rigor state) and SH1/SH2-region with the mechanical converter and lever arm (down position in the rigor state, and up position in the pre-power stroke state). We and others have shown that mutations in these key regions disturb the motor function by interfering with chemomechanical coupling pathways [ 4 , 5 , 6 , 7 , 8 , 9 ].…”