Functional elements within the dynein microtubule-binding domain

• Published in: Molecular biology of the cell 2000-02, Vol.11 (2), p.523-529
• Main Authors: Koonce, M P, Tikhonenko, I
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Amino Acid Substitution; Animals; Binding, Competitive; Dictyostelium - cytology; Dyneins - antagonists & inhibitors; Dyneins - chemistry; Dyneins - genetics; Dyneins - metabolism; Microtubule-Associated Proteins - antagonists & inhibitors; Microtubule-Associated Proteins - metabolism; Microtubules - metabolism; Molecular Motor Proteins - antagonists & inhibitors; Molecular Motor Proteins - chemistry; Molecular Motor Proteins - genetics; Molecular Motor Proteins - metabolism; Molecular Sequence Data; Molecular Weight; Peptide Fragments - antagonists & inhibitors; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Binding; Protein Structure, Tertiary; Sequence Alignment; Sequence Deletion - genetics; Structure-Activity Relationship; Tubulin - metabolism; Tubulin Modulators
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.11.2.523

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 achieved and modulated during ATP hydrolysis. We have performed a detailed dissection of the microtubule contact site, using fragment expression, alanine substitution, and peptide competition. Our work identifies three clusters of amino acids important for the physical contact with microtubules; two of these fall within a region sharing sequence homology with MAP1B, the third in a region just downstream. Amino acid substitutions within any one of these 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, supporting previous evidence for similar sites of interaction. We have also identified four amino acids whose substitutions affect release of the motor from the microtubule (E3413, R3444, E3460, and C3469). These suggest that nucleotide-sensitive affinity may be locally controlled at the site of contact. Our work is the first detailed description of dynein-tubulin interactions and provides a framework for understanding how affinity is achieved and modulated.