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September 24, 2004

Structural Basis of the α₁-β Subunit Interaction of Voltage-gated Ca²⁺ Channels

Y-h. Chen, M-h. Li, Y. Zhang, L-l. He, Y. Yamada, A. Fitzmaurice, Y. Shen, H. Zhang, L. Tong, and J. Yang
Department of Biological Sciences, Columbia University, New York, NY

High voltage-activated (HVA) Ca²⁺ channels in cell membranes control diverse biological processes, such as muscle contraction and hormone release. They are composed of α₁, α₂-δ, β, and sometimes γ, subunits. The proper expression and function of HVA Ca²⁺ channels are critically dependent on the β subunit, which binds directly to the α interaction domain (AID) in α₁, presumably through the β interaction domain (BID). We have solved the crystal structure of the conserved core region of β₃, alone and in complex with AID, and of β₄ alone. The structures show that the β core contains two common protein-interacting domains (a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain) and that AID does not bind to BID. These represent the first crystal structures of a Ca²⁺ channel subunit or domain and suggest that β may be a multi-functional protein.

There are four subfamilies of Ca²⁺ channel β subunits (β₁-β₄). Each β subunit contains five regions, with the three middle regions forming a core that can perform most functions of full-length β. The crystal structure of the β₃ core in complex with a 49-amino acid segment of α₁, which contains the 18-amino acid α interaction domain (AID) (at 2.6 Å-resolution), shows that the three regions comprising the β core are: an Src homology 3 (SH3) domain, a guanylate kinase (GK) domain, and a “HOOK” region that connects them (Figure 1a). The SH3 and GK domains interact intramolecularly. The AID forms an α helix and binds to a hydrophobic (water-fearing) groove in the GK domain. The interactions between AID and β are extensive, involving hydrophobic interactions, hydrogen bonds, and an ion pair (Figure 1b). These interactions form the basis for the high affinity binding between α₁ and β. Most of the amino acids involved in the interactions are conserved in both subunits. The structure also reveals that the β interaction domain (BID) spans all three regions of the SH3-HOOK-GK motif and is thus crucial for the intramolecular interaction between the SH3 and GK domains, as well as their structural integrity. But, it is not directly involved in binding AID (Figure 1a).

The overall structure of the unliganded (unbound) β₃ core is very similar to that of the β₃ core bound to AID, indicating that AID binding does not change the β subunit structure. Also, as predicted by the high amino acid similarity, the structure of the β₄ core is nearly identical to that of the β₃ core.

The β subunit SH3 domain (β-SH3) is structurally similar to canonical SH3 domains, but, unlike the continuous configuration of the latter, β-SH3 is split, with a separate, flexible HOOK region inserted between the first four β strands and the last one. Furthermore, although β-SH3 possesses several amino acids critical for binding PXXP motifs (molecules that typically bind to SH3 domains), it is unlikely to interact with PXXP-containing proteins because the binding site is shielded by part of the HOOK. Likewise, although the overall structure of the β subunit GK domain (β-GK) is similar to that of the yeast GK domain, it is catalytically inactive. Thus, the function of the β subunit SH and GK domains remains to be determined.

Interestingly, the SH3-HOOK-GK motif is also a common structural signature of a family of proteins called membrane-associated guanylate kinase homologs (MAGUKs). Many members of this family, including PSD95, are concentrated at the boundaries between neurons, or “synapses,” as are HVA Ca²⁺ channels, and play a central role in clustering and organizing both pre- and post-synaptic ion channels and neurotransmitter receptors. Electrophysiological studies indicate that the PSD95 SH3-HOOK-GK and PSD95_GK do not interact with HVA Ca²⁺ channels (Figure 2a). A comparison of the structure of PSD95_GK and β_GK clearly reveals why: The AID-binding pocket is completely blocked in PDS95_GK (Figure 2b). Our study thus provides a structural basis for the specificity of Ca²⁺ channel β subunits and MAGUK proteins.

BEAMLINE
X4A

FUNDING
National Institutes of Health
The McKnight Foundation
The EJLB Foundation
The American Heart Association

PUBLICATION
Y-h. Chen, M-h. Li, Y. Zhang, L-l. He, Y. Yamada, A. Fitzmaurice, Y. Shen, H. Zhang, L. Tong, and J. Yang. “Structural basis of the α₁-β interaction of voltage-gated Ca²⁺ channels”. Nature 429, 675-680 (2004).

FOR MORE INFORMATION
Prof. Jian Yang
Department of Biological Sciences
Columbia University
Email: Jy160@columbia.edu