Cargo recognition mechanism of myosin X revealed by the structure of its tail MyTH4-FERM tandem in complex with the DCC P3 domain

Abstract

Myosin X (MyoX), encoded by Myo10, is a representative member of the MyTH4-FERM domain-containing myosins, and this family of unconventional myosins shares common functions in promoting formation of filopodia/stereocilia structures in many cell types with unknown mechanisms. Here, we present the structure of the MyoX MyTH4-FERM tandem in complex with the cytoplasmic tail P3 domain of the netrin receptor DCC. The structure, together with biochemical studies, reveals that the MyoX MyTH4 and FERM domains interact with each other, forming a structural and functional supramodule. Instead of forming an extended β-strand structure in other FERM binding targets, DCC_P3 forms a single α-helix and binds to the αβ-groove formed by β5 and α1 of the MyoX FERM F3 lobe. Structure-based amino acid sequence analysis reveals that the key polar residues forming the inter-MyTH4/FERM interface are absolutely conserved in all MyTH4-FERM tandem-containing proteins, suggesting that the supramodular nature of the MyTH4-FERM tandem is likely a general property for all MyTH4-FERM proteins.

Fig. 1.

Characterization of the MyoX_MF/DCC_P3 interaction. (A and B) The domain organizations of MyoX and DCC. The boundary of MyoX_MF is indicated. The MyoX_MF/DCC_P3 interaction is indicated by a two-way arrow. The sequence alignment of the P3 motif of DCC and neogenin from different species is included. The residues involved in the MyoX_MF/DCC_P3 interaction are indicated by triangle ups. The residues substituted with Ala to test their role in binding to MyoX_MF are labeled by arrows. (C) The ITC curves show the interactions between MyoX_MF and the different DCC cytoplasmic fragments. The MyoX_MF construct with 36-residue deletion in the FERM domain is indicated as MyoX_MFΔ36. (D) The dissociation constants of the binding reactions of various forms of MyoX_MF and DCC obtained from ITC-based assays. The boundaries for the DCC fragments are residues 1321–1445 for P2–P3, 1370–1445 for the extended P3, and 1409–1445 for P3.

Fig. 2.

Ribbon representation of the MyoX_MF/DCC_P3 complex structure. The color coding of the domains is used throughout the entire manuscript except as otherwise indicated. The linker region contains the residues of the N-terminal part of DCC_P3 (residues 1409–1421). A disordered loop in the F3 lobe and a truncated region (marked by a red arrow) in the F2 lobe are indicated by dashed lines.

Fig. 3.

The MyoX MyTH4 domain. (A) Ribbon diagram view of MyoX MyTH4. In this drawing, the six-helix bundle core and peripheral regions of MyoX MyTH4 are colored in gray and blue, respectively. (B) The VHS and ENTH domains each contain a similar six-helix bundle (colored in gray) core as MyoX MyTH4 does. (C) Surface analysis of MyoX MyTH4 reveals a highly positively charged surface. The figure is drawn using electrostatistic surface charge potential map. (D) Combined ribbon and stick models showing the residues forming the negatively charged surface shown in C. The molecules in C and D are drawn with the same orientation.

Fig. 4.

Comparison of the structure of MyoX FERM with those of other FERM domains. (A and B) MyoX FERM and other FERM domains, including radixin (PDB ID code 1GC6), merlin (1H4R), protein 4.1 (2HE7), and FAK (2AEH) are overlapped by superimposing their F1 lobes. The borders between each pair of the three lobes are indicated with red lines. The MyoX FERM domain is colored in green while other FERM domains are in gray. The αβ-groove in the F3 lobe is indicated by red ovals. In B, the superimposed F1 lobes are omitted for better appreciation of the conformational differences of the F2 lobes in different FERM domains. The schematic diagram shows the approximately 30° rotation of the MyoX F2 lobe. (C) Structural based sequence alignment of the αβ-grooves from MyoX, talin, and radixin. The conserved residues are labeled with yellow boxes. The residues of which side chains are involved in their respective target interactions are marked by triangles. The two residues highlighted in Fig. 5 C and D are also circled.

Fig. 5.

The hydrophobic interaction between MyoX_MF and DCC_P3. The interface residues in DCC_P3 (A) and the F3 lobe of MyoX (B) are presented as in the stick mode. The DCC_P3 binding surface of the F3 lobe is illustrated in A with the conserved hydrophobic residues colored in yellow. (C and D) The structural comparison of the αβ-grooves between MyoX and radixin (C) and between MyoX and talin (D). The F3 lobes of radixin and talin are colored in gray. The hydrophobic residues (corresponding to the DCC-binding residues in MyoX) in radixin and talin are drawn in the explicit atomic model for comparison. His288 in radixin and Trp359 in talin are highlighted by red circles.

Fig. 6.

The MyTH4/FERM interface of MyoX. (A) The stereo view of the MyTH4/F1 interface. The residues involved in the MyTH4/F1 interaction are displayed in the explicit stick model. Hydrogen bonds are indicated by dashed lines. (B) The interactions between GFP-tagged MyoX_MF and its mutants with DCC_P3 were assayed by GST pulldown assay. Note that weak background level interaction between GST and MyoX_MF could be detected. (C) Sequence alignment of MyTH4 domains from 24 MyTH4–FERM tandems showing the conserved residues in the MyTH4/FERM interface. As for the names of the MyTH4 domains, the first two lowercase letters represents their species (“hs” for human, “dr” for Danio rerio, “dm” for Drosophila melanogaster, “dd” for Dictyostelium discoideum, “ce” for Caenorhabditis elegans, and “at” for Arabidopsis thaliana), and the following upper cases represent the protein names, including MYO (myosin), PKHH (pleckstrin homology domain containing, family H), HUM (heavy chain of an unconventional myosin), and KCBP (kinesin-like calmodulin binding protein). For proteins containing two MyTH4–FERM tandems, the first and second MyTH4 are discriminated by suffixes (_N and _C, respectively).