Structural insights into the globular tails of the human type v myosins Myo5a, Myo5b, And Myo5c

Abstract

Vertebrate type V myosins (MyoV) Myo5a, Myo5b, and Myo5c mediate transport of several different cargoes. All MyoV paralogs bind to cargo complexes mainly by their C-terminal globular domains. In absence of cargo, the globular domain of Myo5a inhibits its motor domain. Here, we report low-resolution SAXS models for the globular domains from human Myo5a, Myo5b, and Myo5c, which suggest very similar overall shapes of all three paralogs. We determined the crystal structures of globular domains from Myo5a and Myo5b, and provide a homology model for human Myo5c. When we docked the Myo5a crystal structure into a previously published electron microscopy density of the autoinhibited full-length Myo5a, only one domain orientation resulted in a good fit. This structural arrangement suggests the participation of additional region of the globular domain in autoinhibition. Quantification of the interaction of the Myo5a globular domain with its motor complex revealed a tight binding with dissociation half-life in the order of minutes, suggesting a rather slow transition between the active and inactive states.

Figure 1. X-ray structures and Small-Angle X-ray Scattering (SAXS) models of globular domains from human type V myosins.

(A–C) Surface envelopes calculated from SAXS data revealed that Myo5a GD (A), Myo5b GD (B), and Myo5c GD (C) have very similar shapes in solution. See Figure S1 in File S1 for details of SAXS measurements. (D) Ribbon representation of the Myo5a GD crystal structure at 2.2 Å, with the small C-terminal beta-sheet shown in orange. The missing loop I (1640 to 1658) is depicted as a blue dashed line, loop II is highlighted in green. (E) Same structure as in (D), but rotated 180° around the vertical axis. Rainbow color-coding follows the peptide chain from its N-terminus in blue to its C-terminus in red. (F) Ribbon representation of the Myo5b GD crystal structure at 3.1 Å resolution. First and last residue of the missing loops I and II are depicted in blue or green, respectively. (G) Ribbon representation of the modeled structure of the GD from Myo5c, calculated with the program Modeller and the structures of Myo5a (D–E) and Myo5b (F) as templates. For a Ramachandran plot of the computed model, see Figure S4 in File S1. (H) Close-up of (D), slightly rotated to better show the position of the small beta-sheet (orange) that connects the very N-terminus with the C-terminus. Figures were generated with the program Pymol.

Figure 2. Analyses of surface properties of the globular domains from Myo5a, Myo5b, and Myo5c.

Orientation is as shown in Figure 1D (left) and rotated by 180° around the vertical axis (right). (A) Amino acid conservation between human Myo5a, Myo5b, and Myo5c GDs based on alignment shown in Figure S3 in File S1 and plotted on the structure of Myo5a GD. Green indicates high sequence conservation, yellow partial conservation, and white a lack of conservation. (B–D) Unique surface residues in Myo5a (B), Myo5b (C), or Myo5c (D), when compared to their respective paralogs, are shown in red. (E–G) Representation of surface potentials of Myo5a (E), Myo5b (F), and Myo5c (G). Red and blue indicate surface areas with negative and positive surface charges, respectively. White regions indicate hydrophobic regions. The surface region encircled by a red dotted line is an area with high similarity of overall surface charges amongst the three type V myosins (see also Figure S6 in File S1). This similarity might hint at a common function in all three paralogs. (H) Amino acids required for Rab11a binding are highlighted in magenta, residues in red are essential for motor autoinhibition.

Figure 3. C-terminal globular domains of all three human type V myosins potentially mediate autoinhibition.

(A) Surface representation of Myo5a GD with residues K1708 and K1781 highlighted in red. Their mutation resulted in a loss of autoinhibition . (B) Comparison of electrostatic surface potentials of K1708 and K1781 (red circles) with identical positions in Myo5b and Myo5c and the conservation of these residues in all three paralogs (Figure S3 in File S1) suggests that autoinhibition might be a general feature of human type V myosins. Figures were generated with the program Pymol.

Figure 4. Autoinhibition of type V myosins by their globular domains involves multiple interactions sites.

(A) Docking of our high resolution Myo5a GD structure into a previously published 24 Å electron density map of the inhibited Myo5a motor (PDB-ID of published model lacking the globular domain: 2DFS). Shown are three modeled myosin dimers in the EM density (meshed surface rendering) that are arranged in a flower-like fashion. The color scheme is as follows: blue, Myo5a GD; green, motor domain, lever arm, light chains, coiled coil domain; turquoise, neighboring dimers. (B) Close-up of (A), depicting the Myo5a head complex (green) and the fitted GD (blue). Residues previously reported to be required for autoinhibition are shown as colored spheres. (C) Close-up of the upper rectangle in (B). (D) Close-up of the lower rectangle in (B). Loop I is disordered in the structural data. Depicted are three computed models for the flexible loop I, highlighted in yellow, orange, and magenta. (E) Atomic model of Myo5a. Missing amino acids of the flexible loop I GD are depicted as a blue dashed line (disordered loop I: 1640–1658), loop II in green (1787–1797), and amino acids important for interaction with the motor domain are highlighted in red (K1708, K1781). Residues important for Rab11a binding (Y1721 and Q1755) are shown in magenta and the beta-sheet is depicted in orange. Computed models for the flexible loop I were depicted in yellow, orange, and magenta (see also D). Figures were generated with the program Pymol.

Figure 5. Surface plasmon resonance (SPR) measurements with surface-coupled Myo5a globular domain (GD) and the motor complex.

(A) Diagram shows a representative steady-state binding experiment with surface-coupled Myo5a GD and its motor complex (Myo5a HMM) using a multi-injection protocol. The K_d = 30±20 nM was derived from two independent measurements, as recommended by the manufacturer. (B, C) Kinetic binding experiments of Myo5a GD interaction with its motor complex (Myo5a HMM). (B) shows sensograms with representative kinetic measurements (thick lines) at 77 nM, 38.6 nM, and 9.7 nM Myo5a (HMM) and the corresponding curve fittings (thin lines). Off-rates were determined from this concentration range using bivalent curve fitting. Curve fittings with chi₂<0.2 (n = 5) were used to determine average K_off values (C).