Structure of a complex between a voltage-gated calcium channel beta-subunit and an alpha-subunit domain

Abstract

Voltage-gated calcium channels (Ca(V)s) govern muscle contraction, hormone and neurotransmitter release, neuronal migration, activation of calcium-dependent signalling cascades, and synaptic input integration. An essential Ca(V) intracellular protein, the beta-subunit (Ca(V)beta), binds a conserved domain (the alpha-interaction domain, AID) between transmembrane domains I and II of the pore-forming alpha(1) subunit and profoundly affects multiple channel properties such as voltage-dependent activation, inactivation rates, G-protein modulation, drug sensitivity and cell surface expression. Here, we report the high-resolution crystal structures of the Ca(V)beta2a conserved core, alone and in complex with the AID. Previous work suggested that a conserved region, the beta-interaction domain (BID), formed the AID-binding site; however, this region is largely buried in the Ca(V)beta core and is unavailable for protein-protein interactions. The structure of the AID-Ca(V)beta2a complex shows instead that Ca(V)beta2a engages the AID through an extensive, conserved hydrophobic cleft (named the alpha-binding pocket, ABP). The ABP-AID interaction positions one end of the Ca(V)beta near the intracellular end of a pore-lining segment, called IS6, that has a critical role in Ca(V) inactivation. Together, these data suggest that Ca(V)betas influence Ca(V) gating by direct modulation of IS6 movement within the channel pore.

Figure 1

Structure of the Ca_Vβ_2a–Ca_V1.2 AID complex. a, Ribbon diagram of the complex. Dashed lines indicate regions absent from the structures. SH3 and nucleotide kinase (NK) domains are shown in green and blue, respectively. The AID is shown in red. Ca_Vβ_2a α-helices are labelled. Variable regions V1, V2 and V3 are indicated. The Ca_Vβ_2a unbound structure is similar to that shown here for the complex. The arrow indicates where the AID connects to transmembrane segment IS6. b, Sequence alignment of representatives of each Ca_Vβ isoform. The top sequence shows residues 40–425 of rat Ca_vβ_2a. Numbers on the right denote each line’s terminal residue. Shading denotes residues identical among isoforms. The two Ca_vβ_2a domains used for crystallization are indicated in green and blue, respectively. Secondary structure elements are indicated: α, α-helix; η, 3₁₀ helix; β, β-strand. Dashed lines indicate residues present in the crystallized constructs but absent in the electron density. Location of the V2 and part of the V3 regions are shown. Asterisks identify residues that contribute side-chain contacts to the AID-binding pocket; diamonds mark side chains with direct hydrogen bonds to the AID.

Figure 2

Structural comparisons between PSD-95 (gold) and Ca_Vβ_2a (blue). a, Superposition of Ca_Vβ_2a and PSD-95 nucleotide kinase domains (RMSD_Cα = 3.9 Å). The dashed circle indicates the guanosine-monophosphate (GMP)-binding domain present in PSD-95 but absent in Ca_Vβ_2a. The guanosine monophosphate molecule bound to PSD-95 is displayed in space-filling representation. Nucleotide kinase (NK) and SH3 domains are indicated. The relative change in SH3 domain orientation is indicated. b, Superposition of PSD-95 and Ca_Vβ_2a SH3 domains (RMSD_Cα = 1.6 Å). Position of the polyproline ligand from a superposition with the Sem5 SH3 domain (Protein Data Bank code 2SEM) (RMSD_Cα = 1.8 Å) is shown in space-filling representation. The Sem5 SH3 is not shown. The DALI server generated the superpositions (http://www.ebi.ac.uk/dali/).

Figure 3

Features of the AID–Ca_Vβ_2a interaction and location of the previously described BID. a, Sequence alignment of AID domains (Ca_V1.2 residues 428–445) and neighbouring residues. The positions of the last transmembrane segment of transmembrane domain I (IS6) and the first transmembrane segment of transmembrane domain II (IIS1) are shown. Secondary structure of the AID from the co-crystal structure is indicated (red). Dashed lines indicate residues absent from the electron density. Asterisks identify side-chain contacts with Ca_Vβ_2a closer than 4 Å. b, Position of the previously described BID (residues 212–252; yellow),. Residues previously proposed to mediate AID–BID interactions (P224, P228, P234, Y239) are indicated and have relative accessibilities of 1.4%, 0%, 0% and 32.4%. Putative PKC sites S225, S235 and S345 are also shown (magenta) and have relative accessibilities of 8.8%, 0% and 35.4%, respectively. S345 accessibility reduces to 12% in the complex. Accessibility values are relative to a tripeptide, Gly-X-Gly. c, The left panel shows F_o−F_c electron density, contoured at 2σ, for the AID–Ca_Vβ_2a complex before building the AID. The right panel shows final 2F_o−F_c density, contoured at 1σ, for the AID from the refined AID–Ca_Vβ_2a structure (right). In both panels the final AID model is shown.

Figure 4

AID–ABP interactions. a, Surface representation of the Ca_Vβ_2a ABP, bound to the AID. The AID (gold) is shown in stick representation. Y437 and W440 are white. Ca_Vβ_2a residues that contribute hydrophobic (blue) and hydrogen bond (red) side-chain contacts to the AID are labelled. Select residues of the AID are labelled to orient the reader. b, c, Slices through the AID–ABP interaction at AID positions Y437 and W440 (gold). Labels indicate the AID residues.

Figure 5

Cartoon of proposed model for how Ca_Vβ affects Ca_Vα₁ gating. a, Orientation of Ca_Vβ_2a with respect to the I–II loop (red), pore-forming subunit, and connection to IS6. In Ca_Vβ_2a, variable region 1 (V1) is tethered to the membrane. The I–II loop between the AID N terminus and IS6 is depicted as a helix. Ca_Vβ_2a SH3 and nucleotide kinase domains are coloured green and blue, respectively. The arrow indicates that Ca_Vβ couples to IS6 movements (rotations, translations or both). b, View from the opposite side of a. The groove between SH3 and nucleotide kinase domains (demarcated by the arrow) and two flexible Ca_vβ_2a regions, the 275–284 loop and variable region 2 (V2), are on the same Ca_Vβ face, opposite the ABP. These regions may interact with other pore-forming subunit cytoplasmic domains.