Functional adaptation of the switch-2 nucleotide sensor enables rapid processive translocation by myosin-5.
Active site loops that are conserved across superfamilies of myosins, kinesins, and G proteins play key roles in allosteric coupling of NTP hydrolysis to interaction with track filaments or effector proteins. In this study, we investigated how the class-specific natural variation in the switch-2 active site loop contributes to the motor function of the intracellular transporter myosin-5. We used single-molecule, rapid kinetic and spectroscopic experiments and semiempirical quantum chemical simulations to show that the class-specific switch-2 structure including a tyrosine (Y439) in myosin-5 enables rapid processive translocation along actin filaments by facilitating Mg(2+)-dependent ADP release. Using wild-type control and Y439 point mutant myosin-5 proteins, we demonstrate that the translocation speed precisely correlates with the kinetics of nucleotide exchange. Switch-2 variants can thus be used to fine-tune translocation speed while maintaining high processivity. The class-specific variation of switch-2 in various NTPase superfamilies indicates its general role in the kinetic tuning of Mg(2+)-dependent nucleotide exchange.
Nagy
Nikolett T
NT
Department of Biochemistry, Eötvös University, Budapest, Hungary.
Sakamoto
Takeshi
T
Takács
Balázs
B
Gyimesi
Máté
M
Hazai
Eszter
E
Bikádi
Zsolt
Z
Sellers
James R
JR
Kovács
Mihály
M
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D003198:Computer Simulation; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D024701:Myosin Type V / Q000235:genetics* / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics
D000199:Actins; D009711:Nucleotides; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D024701:Myosin Type V; D008274:Magnesium