Motor domain mutation traps kinesin as a microtubule rigor complex.
Conventional kinesin is a highly processive, microtubule-based motor protein that drives the movement of membranous organelles in neurons. Using in vivo genetics in Drosophila melanogaster, Glu164 was identified as an amino acid critical for kinesin function [Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506-31514]. Glu164 is located at the beta-strand 5a/loop 8b junction of the catalytic core and projects toward the microtubule binding face in close proximity to key residues on beta-tubulin helix alpha12. Substitution of Glu(164) with alanine (E164A) results in a dimeric kinesin with a dramatic reduction in the microtubule-activated steady-state ATPase (5 s(-1) per site versus 22 s(-1) per site for wild-type). Our analysis shows that E164A binds ATP and microtubules with a higher affinity than wild-type kinesin. The rapid quench and stopped-flow results provide evidence that ATP hydrolysis is significantly faster and the precise coordination between the motor domains is disrupted. The data reveal an E164A intermediate that is stalled on the microtubule and cannot bind and hydrolyze ATP at the second head.
Klumpp
Lisa M
LM
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Brendza
Katherine M
KM
Rosenberg
John M
JM
Hoenger
Andreas
A
Gilbert
Susan P
SP
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D019281:Dimerization; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008870:Microtubules / Q000201:enzymology* / Q000235:genetics; D020409:Molecular Motor Proteins / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D055672:Static Electricity; D062367:ortho-Aminobenzoates / Q000378:metabolism
D029721:Drosophila Proteins; D020409:Molecular Motor Proteins; D062367:ortho-Aminobenzoates; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D016547:Kinesins; D000409:Alanine