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.

Mapping the microtubule binding regions of calponin.

The smooth muscle basic calponin interacts with F-actin and inhibits the actomyosin ATPase in a calmodulin or phosphorylation modulated manner. It also binds in vitro to microtubules and its acidic isoform, present in nonmuscle cells, and co-localizes with microfilaments and microtubules in cultured neurons. To assess the physiological significance and the molecular basis of the calponin-microtubule interaction, we have first studied the solution binding of recombinant acidic calponin to microtubules using quantitative cosedimentation analyses. We have also characterized, for the first time, the ability of both calponin isoforms to induce the inhibition of the microtubule-stimulated ATPase activity of the cytoskeletal, kinesin-related nonclaret dysjunctional motor protein (ncd) and the abolition of this effect by calcium calmodulin. This property makes calponin a potent inhibitor of all filament-activated motor ATPases and, therefore, a potential regulatory factor of many motor-based biological events. By combining the enzymatic measurements of the ncd-microtubules system with various in vitro binding assays employing proteolytic, recombinant and synthetic fragments of basic calponin, we further unambiguously identified the interaction of microtubules at two distinct calponin sites. One is inhibitory and resides in the segment 145-182, which also binds F-actin and calmodulin. The other one is noninhibitory, specific for microtubules, and is located on the COOH-terminal repeat-containing region 183-292. Finally, quantitative fluorescence studies of the binding of basic calponin to the skeletal pyrenyl F-actin in the presence of microtubules did not reveal a noticeable competition between the two sets of filaments for calponin. This result implies that calponin undergoes a concomitant binding to both F-actin and microtubules by interaction at the former site with actin and at the second site with microtubules. Thus, in the living cells, calponin could potentially behave as a cross-linking protein between the two major cytoskeletal filaments.