Functional elements within the dynein microtubule-binding domain

Dynein interacts with microtubules through an ATP-sensitive linkage mapped to a structurally complex region of the heavy chain following the fourth P- loop motif. Virtually nothing is known regarding how binding affinity is ac hieved and modulated during ATP hydrolysis. We have performed a detailed di ssection of the microtubule contact site, using fragment expression, alanin e substitution, and peptide competition. Our work identifies three clusters of amino acids important for the physical contact with microtubules; two o f these fall within a region sharing sequence homology with MAP1B, the thir d in a region just downstream. Amino acid substitutions within any one of t hese regions can eliminate or weaken microtubule binding (KK3379,80, E3385, K3387, K3397, KK3410,11, W3414, RKK3418-20, F3426, R3464, S3466, and K3467 ), suggesting that their activities are highly coordinated. A peptide that actively displaces MAP1B from microtubules perturbs dynein binding, support ing previous evidence for similar sites of interaction. We have also identi fied four amino acids whose substitutions affect release of the motor from the microtubule (E3413, R3444, E3460, and C3469). These suggest that nucleo tide-sensitive affinity may be locally controlled at the site of contact. O ur work is the first detailed description of dynein-tubulin interactions an d provides a framework for understanding how affinity is achieved and modul ated.