Identification of the Isoform-specific Interactions between the Tail and the Head of Class V Myosin

Abstract

Vertebrates have three isoforms of class V myosin (Myo5), Myo5a, Myo5b, and Myo5c, which are involved in transport of multiple cargoes. It is well established that the motor functions of Myo5a and Myo5b are regulated by a tail inhibition mechanism. Here we found that the motor function of Myo5c was also inhibited by its globular tail domain (GTD), and this inhibition was abolished by high Ca(2+), indicating that the tail inhibition mechanism is conserved in vertebrate Myo5. Interestingly, we found that Myo5a-GTD and Myo5c-GTD were not interchangeable in terms of inhibition of motor function, indicating isoform-specific interactions between the GTD and the head of Myo5. To identify the isoform-specific interactions, we produced a number of Myo5 chimeras by swapping the corresponding regions of Myo5a and Myo5c. We found that Myo5a-GTD, with its H11-H12 loop being substituted with that of Myo5c, was able to inhibit the ATPase activity of Myo5c and that Myo5a-GTD was able to inhibit the ATPase activity of Myo5c-S1 and Myo5c-HMM only when their IQ1 motif was substituted with that of Myo5a. Those results indicate that the H11-H12 loop in the GTD and the IQ1 motif in the head dictate the isoform-specific interactions between the GTD and head of Myo5. Because the IQ1 motif is wrapped by calmodulin, whose conformation is influenced by the sequence of the IQ1 motif, we proposed that the calmodulin bound to the IQ1 motif interacts with the H11-H12 loop of the GTD in the inhibited state of Myo5.

Keywords: ATPase; allosteric regulation; intracellular trafficking; molecular motor; myosin; unconventional myosin.

FIGURE 1.

Sequence alignment of mouse Myo5a and human Myo5c. Top panel, annotated sequence alignment of the converter and the IQ1 of Myo5a and Myo5c. Non-identical residues are colored red. Green and cyan shades indicate the converter and IQ1, respectively. In this study, the residues between two solid triangles were swapped to create Myo5c(5aConv)-S1 and -HMM, and those between two open triangles were swapped to create Myo5c(5aIQ1)-S1 and -HMM. Center panel, schematic of Myo5a and Myo5c. Bottom panel, annotated sequence alignment of Myo5a-GTD and Myo5c-GTD. Yellow shades indicate the α helix. The underlined residues belong to the subdomain II of GTD. Non-identical residues are colored red. Asterisks indicate the two non-conserved residues in the H11-H12 loop, residue 327 (Asn¹⁷⁸⁸ in Myo5a and Asp¹⁶⁷⁷ in Myo5c) and residue 331 (Glu¹⁷⁹² in Myo5a and Lys¹⁶⁸¹ in Myo5c), critical for the isoform-specific interaction between the GTD and the head of Myo5. The two conserved basic residues 245 and 318 (open triangles, corresponding to Lys¹⁷⁰⁶ and Lys¹⁷⁷⁹ in Myo5a, respectively) and the two conserved acidic residues 328 and 330 (solid triangles, corresponding to Glu¹⁷⁸⁹ and Glu¹⁷⁹¹ in Myo5a, respectively) are critical for the interaction between the GTD and the head of Myo5a (7, 22).

FIGURE 2.

The ATPase activity of Myo5c-HMM is inhibited by Myo5c-GTD in a Ca²⁺-dependent manner. A, the ATPase activity of Myo5a-HMM in the presence of Myo5a-GTD. B, the ATPase activity of Myo5c-HMM in the presence of Myo5c-GTD. C, the ATPase activity of Myo5c-S1 in the presence of Myo5c-GTD. The ATPase activity was performed in the presence of 50 mm NaCl (for S1) or 100 mm NaCl (for HMM) under EGTA or pCa4 conditions. The data obtained under EGTA conditions were fit to a hyperbola, defining the affinity of the GTD to Myo5-HMM or Myo5-S1, which was 57 ± 2.3 nm of Myo5a-GTD to Myo5a-HMM, 102 ± 17 nm of Myo5c-GTD to Myo5c-HMM, or 700 ± 16 nm of Myo5c-GTD to Myo5c-S1. The data are mean ± S.D. from three independent assays.

FIGURE 3.

Myo5c-GTD inhibits Myo5a-HMM ATPase activity, but Myo5a-GTD does not inhibit Myo5c-HMM ATPase activity. A, the ATPase activity of Myo5a-HMM in the presence of Myo5c-GTD under EGTA or pCa4 conditions. The K_d of Myo5c-GTD to Myo5a-HMM under EGTA conditions was 1.74 ± 0.23 μm, obtained by a hyperbolic fit. B, the ATPase activity of Myo5c-HMM in the presence of Myo5a-GTD under EGTA conditions. The ATPase activity was performed in the presence of 100 mm NaCl. The data are mean ± S.D. from three independent assays. C, FLAG pulldown of FLAG-tagged Myo5-HMM with GST-tagged Myo5-GTD. The FLAG pulldown assay was performed using 0.5 μm FLAG-tagged Myo5-HMM and 0.5 or 4 μm GST-tagged Myo5-GTD. The samples that were pulled down were separated by SDS-PAGE and visualized by Coomassie Blue staining.

FIGURE 4.

Identification of the critical region in the GTD for the isoform-specific interactions between the GTD and head of Myo5. The GTD chimeras were created by substituting the corresponding region in Myo5a-GTD with that of Myo5c-GTD. A–I, the ATPase activity of Myo5c-HMM in the presence of the GTD chimera. The data were fit to a hyperbola, defining the K_d of the GTD chimera to Myo5c-HMM, which is indicated in the corresponding panel. The ATPase assays were performed in the presence of 100 mm NaCl under EGTA conditions. The data are mean ± S.D. from three independent assays.

FIGURE 5.

Identification of the critical residues in the H11-H12 loop for the isoform-specific interactions between the GTD and the head of Myo5. The ATPase activity of Myo5c-HMM in the presence of Myo5a-GTD mutants N327D (A), E328D (B), E331K (C), or N327D/E331K (D). The ATPase activity was measured in the presence of 100 mm NaCl under EGTA conditions. The K_d of Myo5a-GTD N327D/E331K to Myo5c-HMM was 287 ± 63 nm. The data are mean ± S.D. from three independent assays.

FIGURE 6.

Effects of mutations in the H11-H12 loop on the inhibition of Myo5c-HMM ATPase activity by Myo5c-GTD. The ATPase activity of Myo5c-HMM in the presence of Myo5c-GTD mutants, D327N (A), D328E (B), K331E (C), or D327N/K331E (D). The K_d values are 2.38 ± 1.93 μm for D327N, 110 ± 28 nm for D328E, and 680 ± 159 nm for K331E. The ATPase activity was measured in the presence of 100 mm NaCl under EGTA conditions. The data are mean ± S.D. of three independent assays.

FIGURE 7.

Identification of the critical region in the head of Myo5a for the isoform-specific interactions between the GTD and head of Myo5a. A, relative ATPase activity of Myo5a-S1 and Myo5c-S1 in the presence of Myo5a-GTD. The ATPase activity of Myo5a-S1 and Myo5c-S1 in the absence of Myo5a-GTD was 9.87 ± 0.24 s⁻¹ head⁻¹ and 2.68 ± 0.27 s⁻¹ head⁻¹, respectively. B, the ATPase activity of Myo5c(5aConv)-S1 in the presence of Myo5a-GTD. C, the ATPase activity of Myo5c(5aIQ1)-S1 in the presence of Myo5a-GTD. D, the ATPase activity of Myo5c(5aConv)-HMM and Myo5c(5aIQ1)-HMM in the presence of Myo5a-GTD. The ATPase activities were measured in the presence of 50 mm NaCl (for S1) or 100 mm NaCl (for HMM) under EGTA conditions. The data are mean ± S.D. from three independent assays.

FIGURE 8.

Model for the interaction between the GTD and head of Myo5a. A, proposed model of the Myo5a head-GTD complex. The GTD-binding pocket is composed of loop 65–77 (Pro⁶⁵-Leu⁷⁷), the loop of Asp¹³⁶ (Gly¹²⁰-Asp¹³⁶), the converter (Gly⁶⁹⁶-Asp⁷⁶⁴), and the C-lobe of CaM bound to IQ1. The distances between the oxygen atom (Oδ) of Asp¹³⁶ in the motor domain and the nitrogen atom (Nζ) of Lys³¹⁸ in the GTD (corresponding to Lys¹⁷⁷⁹ in Myo5a) is ∼4 Å. The H11-H12 loop of the GTD (red) is in close proximity to the C-lobe of CaM (green). The H11-H12 loop in this model corresponds to loop II in the model proposed by Velvarska and Niessing (17). The residue numbers refer to mouse Myo5a. B, ribbon drawing of CaM (N-lobe, orange; C-lobe, green) in complex with IQ1 of Myo5a (PDB code 2IX7). The non-identical residues of IQ1 between mouse Myo5a and human Myo5c are shown as sticks.