The Third P-loop Domain in Cytoplasmic Dynein Heavy Chain Is Essential for Dynein Motor Function and ATP-sensitive Microtubule Binding

Abstract

Sequence comparisons and structural analyses show that the dynein heavy chain motor subunit is related to the AAA family of chaperone-like ATPases. The core structure of the dynein motor unit derives from the assembly of six AAA domains into a hexameric ring. In dynein, the first four AAA domains contain consensus nucleotide triphosphate-binding motifs, or P-loops. The recent structural models of dynein heavy chain have fostered the hypothesis that the energy derived from hydrolysis at P-loop 1 acts through adjacent P-loop domains to effect changes in the attachment state of the microtubule-binding domain. However, to date, the functional significance of the P-loop domains adjacent to the ATP hydrolytic site has not been demonstrated. Our results provide a mutational analysis of P-loop function within the first and third AAA domains of the Drosophila cytoplasmic dynein heavy chain. Here we report the first evidence that P-loop-3 function is essential for dynein function. Significantly, our results further show that P-loop-3 function is required for the ATP-induced release of the dynein complex from microtubules. Mutation of P-loop-3 blocks ATP-mediated release of dynein from microtubules, but does not appear to block ATP binding and hydrolysis at P-loop 1. Combined with the recent recognition that dynein belongs to the family of AAA ATPases, the observations support current models in which the multiple AAA domains of the dynein heavy chain interact to support the translocation of the dynein motor down the microtubule lattice.

Monte Carlo modeling of single-molecule cytoplasmic dynein

“…The ATP hydrolysis primarily responsible for the power stroke occurs at site 1. There is evidence that hydrolysis at site 1, and subsequent molecular function, could be altered by the presence or absence of nucleotides at other secondary binding sites, which are labeled as sites 2, 3, and 4 (20)(21)(22). In our Monte Carlo algorithm, we assume that ATP binding to site 1 has the highest priority, followed by binding at the secondary sites.…”

“…Although ATP hydrolysis predominantly happens at site 1, in principle it could occur at other sites as well. There is evidence that hydrolysis at site 1, and subsequent molecular function, can depend on the presence or absence of nucleotides at other secondary binding sites (20)(21)(22).Monte Carlo is an approach to computer simulations in which an event A occurs with a certain probability P A where 0 Յ P A Յ 1. In practice, during each time step, a random number x is generated with uniform probability between 0 and 1.…”

“…Although ATP hydrolysis predominantly happens at site 1, in principle it could occur at other sites as well. There is evidence that hydrolysis at site 1, and subsequent molecular function, can depend on the presence or absence of nucleotides at other secondary binding sites (20)(21)(22).…”

Molecular motors: not quite like clockwork

“…Nucleotide-dependent conformational shifts in the coiled-coil have been predicted to change the affinity of the globular domain for tubulin [40]. There has been significant progress in defining the complex enzymatic behaviour of P1-P4 [41][42][43][44][45]. The P1 domain hydrolyses ATP and controls the coordination [38,39].…”

“…ATP hydrolysis is also occurs in P3, while nucleotide binding to P2 and P4 may play a regulatory function [38][39][40][41][42][43][44][45]. At low ATP levels, binding to P1 occurs preferentially, then to P3 and finally to P2 and P4 [39,[43][44][45].…”