Experimental investigation of the seesaw mechanism of the relay region that moves the myosin lever arm.
A seesaw-like movement of the relay region upon the recovery step of myosin was recently simulated in silico. In this model the relay helix tilts around its pivoting point formed by a phenylalanine cluster (Phe(481), Phe(482), and Phe(652)), which moves the lever arm of myosin. To study the effect of the elimination of the proposed pivoting point, these phenylalanines were mutated to alanines in two Dictyostelium myosin II motor domain constructs (M(F481A, F482A) and M(F652A)). The relay movement was followed by the fluorescence change of Trp(501) located in the relay region. The steady-state and transient kinetic fluorescence experiments showed that the lack of the phenylalanine fulcrum perturbs the formation of the "up" lever arm state, and only moderate effects were found in the nucleotide binding, the formation of the "down" lever arm position, and the ATP hydrolysis steps. We conclude that the lack of the fulcrum decouples the distal part of the relay from the nucleotide binding site upon the recovery step. Our molecular dynamics simulations also showed that the conformation of the motor is not perturbed by the mutation in the down lever arm state, however, the lack of the pivoting point rearranges the dynamic pattern of the kink region of the relay helix.
Kintses
Bálint
B
Department of Biochemistry, Eötvös Loránd University, Budapest H-1117, Hungary.
Yang
Zhenhui
Z
Málnási-Csizmadia
András
A
D000199:Actins / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D004023:Dictyostelium; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D024462:Myosin Type II / Q000737:chemistry / Q000502:physiology*; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D000199:Actins; D009711:Nucleotides; D014364:Tryptophan; D000255:Adenosine Triphosphate; D024462:Myosin Type II