M6 tail domains and experimental constructs. The tail domains of M6 are indicated in the context of the full-length protein, with the position of the first residue of each domain in the human sequence annotated. The calmodulin binding domains are the heretofore known elements of the lever arm (LA); the end of the IQ helix is residue 835. Sequences from the MT from four species are presented to show the repeating-charge pattern, which switches approximately every four residues. The E. coli – expressed tail fragments are shown along with the construct name. The control M6 dimer, the MT locked mutant and the PT mutant constructs were modified by insertion of a GCN4 segment (black regions) to ensure dimerization at the low concentrations used for single-molecule analyses and by replacing the cargo binding domain (CBD) with YFP to provide a specific surface-attachment point via a YFP monoclonal antibody. The location of the randomized PT is gray.

CD spectra for the PT and MT-DT domains. (a) A typical CD spectrum of the PT showing the characteristic double minima of an α-helical protein. Inset, the thermal melt of the PT showing the cooperative melt typical of a folded protein. (b) CD spectra of the MT-DT as a monomer (red) or as an artificial dimer held together by a C-terminal disulfide bridge (blue). Inset, thermal melt curves showing that both constructs have a broad thermal unfolding transition as expected for a single α-helix. The similarity of the spectra and melt curves of the monomer and dimer indicates that no structural changes occur when the MT-DT is placed in conditions mimicking high concentrations. This indicates an inability of the MT-DT to dimerize.

SAXS envelope reconstructions of tail domains. (a) A model of the MT-DT structure with the MT structure (green) derived from the single α-helix prediction and the DT structure (orange) from a Rosetta prediction28. The model was constructed by aligning the peptide backbone manually to the consensus best GASBOR reconstruction and then docking the model into the filtered GASBOR reconstruction envelope using the Situs software package51. (b) A model of the PT-DT structure was constructed by adding a Rosetta prediction for the PT structure (blue) to the N terminus of the MT-DT model in a. This model was docked as above into the filtered GASBOR reconstruction. Note that even though the number of residues has increased from 129 to 201 (56%), the envelope is only ~3 nm longer, indicating a compact PT. Inset, the structure of a segment of the highly charged MT (residues 935–955), with the side chain atoms color coded by charge, revealing bands of charge circling the helix and providing stabilizing i to i+4 charge-charge interactions.

Motility assays for the MT locked mutant compared to control M6 dimer. (a) Plots of log landing rate versus log motor density with lines depicting the processive model and the nonprocessive model superimposed on the data. The control M6 dimer data match the processive model (number of motors for attachment = 1.0), and the MT locked data more closely match the nonprocessive model (number of motors for attachment = 2.0). (b) Plots of the probability of a filament moving further than its length versus log motor density. As described for a, models for processive and nonprocessive movement were superimposed over the data, with the control M6 dimer being in better agreement with the one-motor model (r² processive model = 0.98, r² nonprocessive model = 0.95) and the MT locked mutant being in better agreement with the two-motor model (r² processive model = 0.93, r² nonprocessive model = 0.97). (c) Histogram of measured nonprocessive displacements for the MT locked mutant in a dual-beam optical trap assay. The mean displacement is indicated and represents the length of the power stroke of the motor. All error bars are standard errors.

SAXS envelope and models for full-length myosin VI. (a) A model of an extended full-length M6 containing the published post-stroke crystal structure of the catalytic domain5 with the calmodulin-bound unique insert and IQ regions (purple), the PT-DT model from Figure 3b and the Rosetta prediction of the CBD (magenta) docked into the corresponding SAXS envelope. With the head aligned to one end of the reconstruction and the PT fused to the IQ such that it extends the lever arm in the same conformation as in Figure 6, the rest of the tail lies well outside the calculated scattering envelope. (b) A model of an alternate compact state for monomeric M6, with the CBD folded back onto the lever arm calmodulins, docked into the same SAXS envelope as in a.

A scale model of a M6 dimer moving along an actin filament. The F-actin–docked myosin model is based on that proposed by Holmes et al.54 Monomers corresponding to the two protofilaments are colored in light and dark gray, respectively, to emphasize the pseudorepeat at 36 nm. Using a structural alignment in PyMOL (http://pymol.sourceforge.net), the post-stroke structure of the M6 head (PDB 2BKI5) was docked onto the filament. The prestroke structure (PDB 2V2619) was also docked onto the actin filament 13 monomers removed from the post-stroke head. These structures along with the associated light chains are shown in purple. The tail model presented in Figure 3b, with the same color scheme, has been fused to the end of the IQ domains such that the PT projects along the same vector as the IQ helix (cyan) and then rotates around the Gly839 to remove steric clashes. This represents an orientation where the PT maximally contributes to the M6 stroke, which is one of many potential angles at which it meets the IQ domain. The cargo binding domains are shown in close association, as their dimerization suggests. The SAXS envelopes were then superimposed on the model to place the data in context of a working motor using Chimera55. This model shows that the proposed roles for the tail domains are clearly compatible with a 36-nm processive step for a M6 dimer.