X-ray crystal structure of a TRPM assembly domain reveals an antiparallel four-stranded coiled-coil

Abstract

Transient receptor potential (TRP) channels comprise a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Earlier studies showed that members of the TRPM subclass possess a self-assembling tetrameric C-terminal cytoplasmic coiled-coil domain that underlies channel assembly and trafficking. Here, we present the high-resolution crystal structure of the coiled-coil domain of the channel enzyme TRPM7. The crystal structure, together with biochemical experiments, reveals an unexpected four-stranded antiparallel coiled-coil architecture that bears unique features relative to other antiparallel coiled-coils. Structural analysis indicates that a limited set of interactions encode assembly specificity determinants and uncovers a previously unnoticed segregation of TRPM assembly domains into two families that correspond with the phylogenetic divisions seen for the complete subunits. Together, the data provide a framework for understanding the mechanism of TRPM channel assembly and highlight the diversity of forms found in the coiled-coil fold.

Figure 1. Structure of the TRPM channel coiled-coil assembly domain

A, Cartoon of TRPM family subunits. Conserved TRP domain (green box) and coiled-coil domain (red box) are indicated. Kinase domains of TRPM6 and TRPM7 and the TRPM2 NUTD9 motif are shown as ovals. B, Sequence alignment TRPM coiled-coil domains. Positions of the coiled-coil heptad repeat (abcdefg) are indicated below the alignment. Coiled-coil residues occupying the ‘a’ and ‘d’ positions are denoted by blue and red, respectively. Layer position for each residue in TRPM7cc is indicated above the alignment. Depicted sequences are as follows: rat TRPM7 1230–1282 (XM_001056331), rat TRPM6 1173–1225 (XP_219747), human TRPM1 1183–1235 (NP_002411), human TRPM3 1244–1296 (Q9HCF6), rat TRPM8 1055–1104 (NP_599198), rat TRPM2 1155–1207 (BAE72117), mouse TRPM4 1144–1196 (AAH96475), and mouse TRPM5 1071–1126 (AAI33713). C, TRPM7cc MAD experimental density map contoured at 1.0 σ. α-helix backbone and heavy atoms positions are indicated as green line trace and orange spheres, respectively. D, Final refined structure (sticks) and 2Fo-Fc composite-omit map built by random omission of 5% of model. Maps are contoured at 1.0 σ. E, (Left) TRPM7cc ribbon diagram. Pairs of similarly oriented helices are shown as blue/light blue and orange/light orange. Side chains are shown in stick representation. N- and C-terminal ends of the subunits are indicated. (Right) Axial view of TRPM7cc.

Figure 2. Core packing in the TRPM channel Coiled-Coil Domain

A, Coiled-coil core hydrophobic layers. van der Waals spheres depicting the sidechains of the ‘a’ (blue) and ‘d’ (red) layers on a ribbon backbone (gray) are shown. Layer numbers and N- and C-terminal ends of the coiled-coil are indicated. B, Geometry of individual coiled-coil layers. Ball-and-stick representations show each layer of the core. ‘a’ and ‘d’ positions are colored as in A. Layer numbers are indicated in black. C, Helical wheel representations for the different antiparallel homotetrameric coiled-coil core-packing arrangements. Heptad repeat positions are labeled a-g. Core-forming positions are highlighted in green and solid or dashed lines link positions lying in the same layer. D, Sausage diagram of TRPM7cc and GCN4-E20C ‘a-d’ core antiparallel coiled-coils. Cross-sections of the helical arrangements are shown for the center and ends of the coils.

Figure 3. TRPM7cc electrostatic interactions

A, TRPM7cc Network1 and Network 2. B, TRPM7cc Network3a, Network 3b, and intrasubunit Interaction 1. C, Closeup view of Network 1 and the sidechain-backbone hydrogen bond of Layer 6 (Thr1274). In all panels sidechains are shown as ball-and stick, hydrogen bonds are indicated as dotted lines, and residue carbon atoms and labels are color coded to indicate the subunit of origin.

Figure 4. Structure-based comparison of TRPM coiled-coils

A, Comparative interaction mapping of TRPM subtypes. Column labels identify residue positions involved in the ‘a-d’ core layers and electrostatic networks. Filled boxes indicate conserved positions. Shaded boxes indicate nonconserved residues that remain capable of supporting interactions similar to those observed in TRPM7cc. White boxes indicate nonconserved positions. B, TRPM channel phylogentic comparison.

Figure 5. TRPM7cc solution properties

A, TRPM7cc Circular dichroism (CD) spectrum at 4°C. B, Temperature dependence of the TRPM7cc circular dichroism signal at 222 nm. C, TRPM7cc stoichiometry determined by Superdex 75 (Amersham Biosciences) size exclusion chromatography. Horizontal axis shows elution volume, V_E, relative to the void elution volume of blue dextran (V₀) (ml). Dotted lines indicate predicted elution volume of tetrameric and monomeric peptides. (Inset) standard curve used to calculate molecular weight of eluted peptides. TRPM7cc molecular weights are 26.7 kD, observed; 6.4 kD, expected monomer; 25.6 kD, expected tetramer. D, Sedimentation equilibrium of TRPM7cc. Equilibrium distribution of TRPM7cc (open circles) measured by absorbance at 245 nm as a function of radial distance at 20,000 rpm and 4°C. Raw data are shown relative to predicted distribution curves for monomeric (red), dimeric (yellow), trimeric (green), and tetrameric (blue), species. Residuals for the tetrameric distribution are shown.

Figure 6. TRPM7cc helices are arranged antiparallel in solution

A, Schematic diagrams of expected disulfide bond species starting with the tetramers shown on the top and following subunit exchange to form the heteromeric species on the bottom for antiparallel (left) and parallel (right) orientations. Parallel tetramers will form only homodimeric disulfide-bonded species (right), whereas antiparallel tetramers will form homomeric and antiparallel heterodimeric disulfide bonded pairs (left). B, TRPM7cc disulfide exchange assay shows presence of antiparallel helices. Top, reduced starting mixture of N-TRPM7cc and C-TRPM7cc tetramers show single peaks for the monomeric, reduced peptides. Second from top, oxidation of reduced tetramers immediately after mixing (0 hr) shows two homomeric species, N-N and C-C. Bottom two traces, disulfide-bonded antiparallel heterodimer, N-C, appears after subunit exchange (2 hr, and 48 hr).

Figure 7. Comparisons between TRPM7 and Kv7 channel assembly domains

A, Cartoon model of TRPM7 showing the relationship between the antiparallel assembly domain, transmembrane domains, and dimeric kinase domains. For display purposes, the kinase domain dimer is shown linked to the C-terminal ends of antiparallel assembly domain strands. The other arrangement, kinase dimers linked to parallel strands is also possible. Presently available data do not favor one arrangement over the other. B, Cartoon model of a Kv7 channel. The parallel coiled-coil assembly domain, 1OVC , is shown as ribbons. In both panels only two of the four transmembrane domains are shown.