Structural basis for microtubule binding and release by dynein - PubMed (original) (raw)
Structural basis for microtubule binding and release by dynein
W B Redwine et al. Science. 2012.
Abstract
Cytoplasmic dynein is a microtubule-based motor required for intracellular transport and cell division. Its movement involves coupling cycles of track binding and release with cycles of force-generating nucleotide hydrolysis. How this is accomplished given the ~25 nanometers separating dynein's track- and nucleotide-binding sites is not understood. Here, we present a subnanometer-resolution structure of dynein's microtubule-binding domain bound to microtubules by cryo-electron microscopy that was used to generate a pseudo-atomic model of the complex with molecular dynamics. We identified large rearrangements triggered by track binding and specific interactions, confirmed by mutagenesis and single-molecule motility assays, which tune dynein's affinity for microtubules. Our results provide a molecular model for how dynein's binding to microtubules is communicated to the rest of the motor.
Figures
Fig. 1
Cryo-EM reconstruction of the cytoplasmic dynein high-affinity MTBD bound to a MT. (A) Schematic of dimeric cytoplasmic dynein. Major features relevant to this study are indicated. The MTBD is depicted in its low- (light blue) and high- (dark blue) affinity states during a step along the MT. (B) Schematic of the fusion constructs between the MTBD and seryl-tRNA synthetase (SRS) that fix the heptad registry of the stalk. The low-affinity construct has an additional 4 amino acids (yellow) inserted in CC1 (black) relative to the high-affinity construct. (C) Cryo-EM image of MTs highly decorated with the high-affinity SRS-MTBD construct (top) and a class average generated from segments of decorated 14-protofilament MTs (bottom). Scale bars: 25 nm. (D) Three-dimensional reconstruction of the MTBD-MT complex, filtered to the calculated resolution of 9.7 Å (Fig. S1). The black solid line represents a slice through the volume, which is shown on the right viewed from the minus end of the MT. The MT polarity is indicated and the dashed line shows the location of the MT seam. The SRS has been omitted due to its lower resolution (Fig. S1).
Fig. 2
The high-affinity, MT-bound state of the dynein MTBD is characterized by repositioning of helices H1 and CC1. (A) Rigid-body docking of the low-affinity MTBD structure into our cryo-EM density. (B) Pseudo-atomic model of the high-affinity MTBD bound to MTs generated by Molecular Dynamics Flexible Fitting (MDFF) and Targeted Molecular Dynamics (TMD) (see text for details). (C) Close-up of the structure shown in (A), with its orientation indicated in panel (A). (D) Close-up of the structure shown in (B), with its orientation indicated in panel (B). The cryo-EM map is shown as a transparent grey mesh. The MTBD is colored following the scheme shown at the bottom of the figure and structural elements are indicated in the different views. H1 (orange/red) is the element with the largest movement in the transition to the high-affinity conformation; H3 and H6 (dark blue) are major contact points with the MT (green). α- and β-tubulin are indicated (green). MT polarity is indicated in panels A and B. H1 protrudes from the cryo-EM map and clashes with the MT in the rigid-body docked low-affinity state (A and C).
Fig. 3
Behavior of dynamic salt bridges in the MTBD as determined by MD. (A) K3298 in H1 of the MTBD alternates between an intermolecular salt bridge with E420 on β-tubulin (MTBD-MT) and an intramolecular salt bridge with E3289 on CC1 of the MTBD (intra-MTBD). (B) R3382 in H6 of the MTBD alternates between an intermolecular salt bridge with E414 and E420 on α-tubulin (MTBD-MT) and an intramolecular salt bridge with E3378 in H6 (intra-MTBD). Single letter amino acid code and number are indicated for Bos taurus tubulin and Mus musculus cytoplasmic dynein. Time stamps for frames from MD simulations are indicated. Intermediate refers to a position midway between MTBD-MT and intra-MTBD salt bridges.
Fig. 4
Dynamic salt briges reduce dynein motility. Bar graphs of (A) mean velocities and (B) characteristic run lengths of fluorescently labeled Saccharomyces cerevisiae dynein bearing the equivalent of the indicated Mus musculus mutations moving on MTs. Error bars: standard deviation, SD (A) and standard error of the mean, SE (B). Velocity and run length differences between WT and Q mutants, as well as between Q and K mutations at the same position, are statistically significant (t-test, P < 0.01 for velocity, and two-tailed KS-test, P < 0.01 for run length). The data for the double mutant (E->K at both CC1 and H6) was omitted because run lengths could only be determined under more stringent motility conditions (Fig. S10). (C-E) Molecular model for the coordination of nucleotide state and MT binding by dynein (see text for details). (C) Unbound dynein in the low affinity conformation, H1 is colored orange. (D) Initial interaction with a new binding site. (E) Repositioning of H1 (now in red) leads to the formation of new ionic interactions with β-tubulin (green cylinder) that stabilize the high-affinity state of the MTBD. The repositioning of H1 is accompanied by a movement in CC1; both movements are indicated by solid black arrows. The conformational change in the MTBD biases the registry of the coiled-coil towards the high-affinity α state, a change that can propagate to the motor domain (white/grey arrow). Ionic interactions are indicated with dashed lines, The identities of the helices in the MTBD are indicated by the key.
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