Structural and biophysical analyses of the skeletal dihydropyridine receptor β subunit β1a reveal critical roles of domain interactions for stability

Abstract

Excitation-contraction (EC) coupling in skeletal muscle requires a physical interaction between the voltage-gated calcium channel dihydropyridine receptor (DHPR) and the ryanodine receptor Ca²⁺ release channel. Although the exact molecular mechanism that initiates skeletal EC coupling is unresolved, it is clear that both the α₁ and β subunits of DHPR are essential for this process. Here, we employed a series of techniques, including size-exclusion chromatography-multi-angle light scattering, differential scanning fluorimetry, and isothermal calorimetry, to characterize various biophysical properties of the skeletal DHPR β subunit β_1a Removal of the intrinsically disordered N and C termini and the hook region of β_1a prevented oligomerization, allowing for its structural determination by X-ray crystallography. The structure had a topology similar to that of previously determined β isoforms, which consist of SH3 and guanylate kinase domains. However, transition melting temperatures derived from the differential scanning fluorimetry experiments indicated a significant difference in stability of ∼2-3 °C between the β_1a and β_2a constructs, and the addition of the DHPR α_1s I-II loop (α-interaction domain) peptide stabilized both β isoforms by ∼6-8 °C. Similar to other β isoforms, β_1a bound with nanomolar affinity to the α-interaction domain, but binding affinities were influenced by amino acid substitutions in the adjacent SH3 domain. These results suggest that intramolecular interactions between the SH3 and guanylate kinase domains play a role in the stability of β_1a while also providing a conduit for allosteric signaling events.

Keywords: X-ray crystallography; dihydropyridine receptor (DHPR); excitation-contraction coupling (E-C coupling); protein structure; skeletal muscle.

Figure 1.

Schematic representation of the DHPR subunits. a, diagram outlining the skeletal DHPR subunits. b, the domain architecture of β_1a illustrating a split SH3 domain (gray), which contains a polyproline binding site (diagonal stripes). The GK domain is shown in pink. The N and C termini (orange and cyan) and the hook region (purple) are intrinsically disordered. Modified β_1a constructs (β_1a-core, β_1a-SH3/GK, and β_1a-hook) are illustrated.

Figure 2.

The solution molecular mass(es) of β_1a constructs. Full-length β_1a (a), β_1a-core (b), β_1a-SH3/GK (c), and β_1a-hook (d) constructs were analyzed by SEC-MALS. Proteins (0.1 mg) were applied to an analytical Superdex 200 size exclusion column. They were eluted in 20 mm Tris-HCl at pH 8.0 and 150 mm potassium chloride at room temperature. Samples were reduced with 1 mm dithiothreitol prior to application. The elution profile was monitored by the change in refractive index (continuous blue line). The molecular masses (kDa; secondary axis) corresponding to peaks are shown as discrete points. Bovine serum albumin (non-monomeric) was analyzed as a standard.

Figure 3.

X-ray crystal structure of DHPR β_1a-SH3/GK complexed with AID peptide. a, cartoon representation of the DHPR β_1a-SH3/GK complexed with AID. The split architecture of the SH3 domain is shown in gray with its labeled RT-loop highlighted. The RT-loop is sandwiched between the α₁ (pink) and α₂ helices (green), which are involved in the occlusion of the polyproline binding site. The GK domain is displayed in orange, and the DHPR I-II AID peptide binding ligand is in blue. The yellow shading denotes the putative polyproline binding site. b, close-up of the interaction between the AID peptide (blue) and β_1a-SH3/GK (gray) highlighting contributing residues facilitating AID binding.

Figure 4.

Thermal denaturation measurements of β_SH3/GK constructs in the absence and presence of AID peptide. a, amino acid composition of β_SH3/GK constructs (black and red numbers denote β_1a and β_2a, respectively). b, temperature denaturation curves for selected β_SH3/GK constructs with and without AID. c, summary of transition melting temperatures for the constructs β_1a-SH3/GK, β_2a-SH3/GK, β_{2a-SH3(β1aRT)GK}, and β_1aSH3/β2aGK. Each data set represents two independent sets of quadruplicate measurements. Paired Student's t test analyses for ± AID were performed relative to β_1a-SH3/GK. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 5.

DHPR-β isoforms have high sequence identity and structural similarity. The primary sequences of the mouse protein cores, which matched the primary sequence of solved crystal structures (β_1a, A2A4545_58–454; β₂, Q8CC26_68–485; β₃, P54285_16–375; and β₄, Q8R0S4_48–398), were aligned pairwise using ClustalO. The boundaries of the core were defined by Simple Molecular Architecture Research Tool (SMART) and encompassed the VGCC domain through to the guanylate kinase domain. a, β; b, β_SH3/GK; c, β SH3 domain; d, β GK domain. The sequence identity is shown as a percentage beside the arrows linking each isoform.

Figure 6.

ITC curves for β_SH3/GK constructs titrated with AID. ITC isotherms and curves for the constructs β_1a-SH3/GK (a), β_2a-SH3/GK (b), β_{2a-SH3(β1aRT)GK} (c), and β_1aSH3/β2aGK (d). Binding and thermodynamic parameters are displayed in Table 2.

Figure 7.

Structural comparisons between β_1a isoforms. A, backbone superposition of β_1a with structures that have been crystallized with and without AID peptide. The color key corresponds to β isoforms. B, overlay of β X-ray crystal structures showing the SH3 domain, the α₁ and α₂ helices, and the RT-loop. The β_1a structure is depicted in red. C, the X-ray crystal structure of the SH3 domain of rabbit β_2a (PDB code 1t3l). The α₂ helix and the RT-loop are highlighted in magenta and green, respectively, and interact through a salt bridge involving the side chains of Glu⁷⁶ and Lys¹²⁴ (shown). These structural elements occlude the polyproline binding site, which is displayed as a pale orange line. The sequence alignment of the α₂ helix and the RT-loop is displayed for all β-subunit isoforms with the arrows denoting charged residues involved in a salt bridge that is absent in the β_1a RT-loop.