Structure of Myo7b/USH1C complex suggests a general PDZ domain binding mode by MyTH4-FERM myosins

Abstract

Unconventional myosin 7a (Myo7a), myosin 7b (Myo7b), and myosin 15a (Myo15a) all contain MyTH4-FERM domains (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in their cargo binding tails and are essential for the growth and function of microvilli and stereocilia. Numerous mutations have been identified in the MyTH4-FERM tandems of these myosins in patients suffering visual and hearing impairment. Although a number of MF domain binding partners have been identified, the molecular basis of interactions with the C-terminal MF domain (CMF) of these myosins remains poorly understood. Here we report the high-resolution crystal structure of Myo7b CMF in complex with the extended PDZ3 domain of USH1C (a.k.a., Harmonin), revealing a previously uncharacterized interaction mode both for MyTH4-FERM tandems and for PDZ domains. We predicted, based on the structure of the Myo7b CMF/USH1C PDZ3 complex, and verified that Myo7a CMF also binds to USH1C PDZ3 using a similar mode. The structure of the Myo7b CMF/USH1C PDZ complex provides mechanistic explanations for >20 deafness-causing mutations in Myo7a CMF. Taken together, these findings suggest that binding to PDZ domains, such as those from USH1C, PDZD7, and Whirlin, is a common property of CMFs of Myo7a, Myo7b, and Myo15a.

Keywords: Harmonin; Usher syndrome; microvilli; myo7a; stereocilia.

Fig. 1.

Structure of Myo7b CMF/USH1C PDZ3 complex. (A) Schematic diagrams showing the domain architectures of Myo7b and USH1C. The interaction is mediated by Myo7b CMF and USH1C PDZ3. The color coding of the domains is kept throughout the report. (B) Ribbon representation (Left) and schematic cartoon diagram (Right) of the Myo7b CMF/USH1C PDZ3 complex structure. The disordered loops are drawn as dashed lines in the ribbon representation. (C) Surface representation of the Myo7b CMF structure showing the central pocket. The USH1C PDZ3 domain is shown as ribbon, and its C-terminal tail is shown as stick and transparent spheres. The amino acid sequence of the USH1C C-terminal tail is shown below the surface diagram. (D) Ribbon representation (Left) and schematic cartoon diagram (Right) of the Myo7a NMF/USH1G CEN and Myo7b NMF/ANKS4B CEN complexes. (E) Ribbon representation (Left) and schematic cartoon diagrams (Right) of the Myo10 MF/DCC complex.

Fig. S1.

Structural comparison of all of the MyTH4-FERM structures showing the relative rotations of the three lobes. (A–G) Ribbon diagrams showing all of the MyTH4-FERM structures solved up to date as well as the founding member Moesin FERM domain. (A) Myo7b CMF, this study; (B) Myo7b NMF, PDB ID code: 5F3Y; (C) Myo7a NMF, PDB ID code: 3PVL; (D) Dictyostelium Myo7 CMF, PDB ID code: 5EJR; (E) Dictyostelium Myo7 NMF, PDB ID code: 5EJY; (F) Myo10 MF, PDB ID code: 3PZD; (G) Moesin FERM domain, PDB ID code: 1EF1. All of the structures are shown in the same orientation by aligning the F1 lobes together. The α7 in the MyTH4 domain, α1 in the F1 lobe, α1/α4 in the F2 lobe, and α1 in the F3 lobe are highlighted in color to show the relative orientations of each domains. (H) Superposition of the five helices highlighted in A–F showing the orientation differences of the MyTH4-FERM tandems using the F1 lobe α1 (α1^F1) helix as the reference.

Fig. 2.

Detailed interaction between Myo7b CMF and USH1C PDZ3. (A) Ribbon representation of the Myo7b CMF/USH1C PDZ3 complex structure with the two major interfaces highlighted with the black boxes. (B) Detailed interaction between USH1C C-terminal tail and Myo7b CMF central pocket. Residues involved in binding are highlighted with stick models. Salt bridges and hydrogen bonds are indicated with dashed lines. (C) Surface electrostatic potential of the Myo7b CMF showing the positively charged central pocket. (D) Detailed interaction between USH1C PDZ3 βB/βC loop and Myo7b CMF F3 lobe. (E) Summary of dissociation constants showing that mutations of the critical residues in the interface either weakened or even abolished the binding. (F) Schematic diagrams showing domains organizations of different classes of USH1C spliced isoforms.

Fig. S2.

Sequence alignment of PDZ3 in USH1C isoforms a and b. The secondary structure elements are shown according to the USH1C structure reported in this study. Identical and highly similar residues are colored (yellow for hydrophobic residues, blue for positively charged residues, red for negatively charged residues, and green for other polar residues). The C-terminal-TFF critical for binding to Myo7b CMF are highlighted with a magenta box.

Fig. 3.

Myo7b variants deficient in USH1C binding exhibit decreased colocalization with USH1C and impaired microvillar clustering. (A) Confocal images of 14 DPC Myo7b KD CACO-2_BBE cells expressing the EGFP-tagged Myo7b rescue constructs indicated (green) and stained for USH1C (magenta) and F-actin (gray). (Scale bars, 10 μm.) (B) Colocalization analysis of Myo7b with USH1C using Pearson’s correlation coefficients. n = 5 fields, 111 cells for FL; 4 fields, 30 cells for ΔCMF; 5 fields, 66 cells for K1918E; 5 fields, 84 cells for R1921E. *P < 0.05, **P < 0.01, t test. (C) Colocalization analysis of Myo7b constructs with CDHR2 using Pearson’s correlation coefficients. n = 4 fields, 59 cells for FL; 4 fields, 37 cells for ΔCMF; 4 fields, 46 cells for K1918E; 5 fields, 61 cells for R1921E. *P < 0.01, t test. The representative images are shown in Fig. S3. (D) Quantification of cells with clustering microvilli expressed as a total percentage of cells. For KD rescue cell lines, only EGFP⁺ (i.e., rescue construct-expressing) cells were scored. Bars indicate mean ± SD. n = 606 cells for scramble, 573 cells of Myo7b KD13 experiment, 507 for Myo7b KD15, 271 for FL, 89 for ΔCMF, 261 for K1918E, and 264 for R1921E. *P < 0.002, t test. KD13 and KD15 represent two distinct short hairpin RNA KD lines as described previously (37).

Fig. S3.

Myo7b variants deficient in USH1C binding exhibit decreased colocalization with CDHR2. Confocal images of 14 d post confluency Myo7b KD CACO-2BBE cells expressing the EGFP-tagged Myo7b rescue constructs (A: EGFP-Myo7b-FL; B: EGFP-Myo7b-ΔCMF; C: EGFP-Myo7b-K1918E; D: EGFP-Myo7b-R1921E) indicated (green) and stained for CDHR2 (magenta) and F-actin (gray). (Scale bars, 10 μm.) The colocalization analysis of Myo7b constructs with CDHR2 using Pearson’s correlation coefficients is shown in Fig. 3C.

Fig. 4.

Interaction between Myo7a CMF and USH1C PDZ3. (A) Domain organizations of Myo7a and Myo7b. Sequence identity of their CMF is indicated. (B) The amino acid sequence conservation map of Myo7a/b CMF showing that the residues forming the USH1C PDZ3 binding surface are nearly identical. The conservation map is calculated based on amino acid sequences of mammalian Myo7a and Myo7b. (C) SDS/PAGE analysis showing that coexpression of USH1C PDZ3 can increase the solubility of Myo7a CMF. “W” means whole-cell extract; “S” denotes supernatant after centrifugation of the cell lysate; “E” stands for the fraction after elution from the Ni²⁺-NTA column. (D) The elution profiles of Myo7a CMF/USH1C PDZ3 complex in the buffer containing 100 mM NaCl (black) or 300 mM NaCl (gray). The elution positions of molecular size markers are indicated at the top (D1). The corresponding SDS/PAGE analysis of the eluted peaks (D2: in buffer containing 100 mM NaCl and D3: in buffer containing 300 mM NaCl). (E) ITC result showing that Myo7a CMF binds to USH1C PDZ3 with a K_d ∼11 μM.

Fig. S4.

Sequence alignment of CMF of class VII myosins from difference species. The secondary structure elements are labeled according to the Myo7b CMF structure. Residues that are identical and highly similar are indicated in red and yellow boxes, respectively. The residues involved in USH1C C-terminal tail binding and the PDZ3 domain binding are highlighted with blue and cyan stars. Missense mutations are highlighted with circles and color coded as that in Fig. 5.

Fig. 5.

Missense mutations in Myo7a CMF found in USH1B patients. (A) Ribbon representation of the Myo7a CMF structural model. The 20 missense mutation sites in USH1B patients are highlighted with spheres and colored in magenta, green, yellow, and pink, corresponding to the four categories. A de novo mutation found in Myo7b (W1642C) is shown in red. (B) Schematic cartoon diagram showing the distributions of the USH1B mutations mapped on to the structural model of Myo7a CMF. (C) Summary of impacts of some USH1B mutations on the Myo7a CMF/USH1C PDZ3 interaction. The matching mutations were also made in Myo7b CMF corresponding to each of the USH1B mutations, and their impacts on USH1C PDZ3 binding are measured.

Fig. 6.

Myo7b/ANKS4B/USH1C tripartite complex formation. (A) Schematic diagram showing the detailed interaction network governing the formation of the Myo7b/ANKS4B/USH1C and Myo7a/USH1G/USH1C tripartite complexes. (B and C) Analytical gel-filtration chromatography (B) together with SDS/PAGE analysis (C) showing that the Myo7b NCMF, the full-length ANKS4B, and the full-length USH1C form a stable tripartite complex. (D) Representative pull-down experiments showing that mutations disrupting or weakening each binary interaction can affect the Myo7b/ANKS4B/USH1C tripartite complex formation (Left for ANKS4B WT and Right for ANKS4B F275E). (E) Quantification of the amount of Myo7b NCMF pulled down in the assays shown in D. The data are derived from three different batches of experiments, and the error bars are expressed as mean ± SD. *P < 0.05; ****P < 0.0001; n.s., nonsignificant; t test.

Fig. S5.

Summary of the binding sites used by FERM domains to recognize targets as seen in complex structures. Schematic cartoon (Left) and ribbon diagrams (Right) of the FERM domain of Myo7b CMF are used to illustrate target binding sites of FERM domains. The 10 different sites are shown with numbers in circles. The dashed circle (site 10) indicates that the binding site is located at the back side of the F1 lobe. The circles are filled with color showing the frequencies seen in the crystal structures of FERM domains with their different targets as detailed in Table S1.