Alanine-Scanning Mutagenesis Defines a Conserved Energetic Hotspot in the CaVα1 AID-CaVβ Interaction Site that Is Critical for Channel Modulation

Filip Van Petegem; Karl E. Duderstadt; Kimberly A. Clark; Michelle Wang; Daniel L. Minor

doi:10.1016/j.str.2007.11.010

Alanine-Scanning Mutagenesis Defines a Conserved Energetic Hotspot in the Ca_Vα₁ AID-Ca_Vβ Interaction Site that Is Critical for Channel Modulation

Filip Van Petegem, Karl E. Duderstadt, Kimberly A. Clark, Michelle Wang, Daniel L. Minor

University of California San Francisco

Research output: Contribution to journal › Article › peer-review

69 Scopus citations

Abstract

Voltage-gated calcium channels (Ca_Vs) are large, multisubunit complexes that control cellular calcium entry. Ca_V pore-forming (Ca_Vα₁) and cytoplasmic (Ca_Vβ) subunits associate through a high-affinity interaction between the Ca_Vα₁ α interaction domain (AID) and Ca_Vβ α binding pocket (ABP). Here we analyze AID-ABP interaction thermodynamics using isothermal titration calorimetry. We find that commensurate with their strong sequence similarity, all Ca_V1 and Ca_V2 AID peptides bind Ca_Vβ with similar nanomolar affinities. Although the AID-ABP interface encompasses 24 side chains, alanine-scanning mutagenesis reveals that the binding energy is focused in two complementary hotspots comprising four deeply conserved residues. Electrophysiological experiments show that hotspot interaction disruption prevents trafficking and functional modulation of Ca_V1.2 by Ca_Vβ. Together, the data support the primacy of the AID-ABP interface for Ca_Vα₁-Ca_Vβ association, underscore the idea that hotspots dominate protein-protein interaction affinities, and uncover a target for strategies to control cellular excitability by blocking Ca_Vα₁-Ca_Vβ complex formation.

Original language	English
Pages (from-to)	280-294
Number of pages	15
Journal	Structure
Volume	16
Issue number	2
DOIs	https://doi.org/10.1016/j.str.2007.11.010
State	Published - 12 Feb 2008
Externally published	Yes

Keywords

PROTEINS
SIGNALING

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10.1016/j.str.2007.11.010

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title = "Alanine-Scanning Mutagenesis Defines a Conserved Energetic Hotspot in the CaVα1 AID-CaVβ Interaction Site that Is Critical for Channel Modulation",

abstract = "Voltage-gated calcium channels (CaVs) are large, multisubunit complexes that control cellular calcium entry. CaV pore-forming (CaVα1) and cytoplasmic (CaVβ) subunits associate through a high-affinity interaction between the CaVα1 α interaction domain (AID) and CaVβ α binding pocket (ABP). Here we analyze AID-ABP interaction thermodynamics using isothermal titration calorimetry. We find that commensurate with their strong sequence similarity, all CaV1 and CaV2 AID peptides bind CaVβ with similar nanomolar affinities. Although the AID-ABP interface encompasses 24 side chains, alanine-scanning mutagenesis reveals that the binding energy is focused in two complementary hotspots comprising four deeply conserved residues. Electrophysiological experiments show that hotspot interaction disruption prevents trafficking and functional modulation of CaV1.2 by CaVβ. Together, the data support the primacy of the AID-ABP interface for CaVα1-CaVβ association, underscore the idea that hotspots dominate protein-protein interaction affinities, and uncover a target for strategies to control cellular excitability by blocking CaVα1-CaVβ complex formation.",

keywords = "PROTEINS, SIGNALING",

author = "{Van Petegem}, Filip and Duderstadt, {Karl E.} and Clark, {Kimberly A.} and Michelle Wang and Minor, {Daniel L.}",

note = "Funding Information: We thank F. Findeisen for comments on the manuscript and for the ssCa V β 2a clone and M. Cho for expert molecular biology assistance. This work was supported by grants to D.L.M. from NIH-NHLBI and the American Heart Association and to F.V.P. from the American Heart Association. D.L.M. is an AHA Established Investigator. ",

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AU - Van Petegem, Filip

AU - Duderstadt, Karl E.

AU - Clark, Kimberly A.

AU - Wang, Michelle

AU - Minor, Daniel L.

N1 - Funding Information: We thank F. Findeisen for comments on the manuscript and for the ssCa V β 2a clone and M. Cho for expert molecular biology assistance. This work was supported by grants to D.L.M. from NIH-NHLBI and the American Heart Association and to F.V.P. from the American Heart Association. D.L.M. is an AHA Established Investigator.

PY - 2008/2/12

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N2 - Voltage-gated calcium channels (CaVs) are large, multisubunit complexes that control cellular calcium entry. CaV pore-forming (CaVα1) and cytoplasmic (CaVβ) subunits associate through a high-affinity interaction between the CaVα1 α interaction domain (AID) and CaVβ α binding pocket (ABP). Here we analyze AID-ABP interaction thermodynamics using isothermal titration calorimetry. We find that commensurate with their strong sequence similarity, all CaV1 and CaV2 AID peptides bind CaVβ with similar nanomolar affinities. Although the AID-ABP interface encompasses 24 side chains, alanine-scanning mutagenesis reveals that the binding energy is focused in two complementary hotspots comprising four deeply conserved residues. Electrophysiological experiments show that hotspot interaction disruption prevents trafficking and functional modulation of CaV1.2 by CaVβ. Together, the data support the primacy of the AID-ABP interface for CaVα1-CaVβ association, underscore the idea that hotspots dominate protein-protein interaction affinities, and uncover a target for strategies to control cellular excitability by blocking CaVα1-CaVβ complex formation.

AB - Voltage-gated calcium channels (CaVs) are large, multisubunit complexes that control cellular calcium entry. CaV pore-forming (CaVα1) and cytoplasmic (CaVβ) subunits associate through a high-affinity interaction between the CaVα1 α interaction domain (AID) and CaVβ α binding pocket (ABP). Here we analyze AID-ABP interaction thermodynamics using isothermal titration calorimetry. We find that commensurate with their strong sequence similarity, all CaV1 and CaV2 AID peptides bind CaVβ with similar nanomolar affinities. Although the AID-ABP interface encompasses 24 side chains, alanine-scanning mutagenesis reveals that the binding energy is focused in two complementary hotspots comprising four deeply conserved residues. Electrophysiological experiments show that hotspot interaction disruption prevents trafficking and functional modulation of CaV1.2 by CaVβ. Together, the data support the primacy of the AID-ABP interface for CaVα1-CaVβ association, underscore the idea that hotspots dominate protein-protein interaction affinities, and uncover a target for strategies to control cellular excitability by blocking CaVα1-CaVβ complex formation.

KW - PROTEINS

KW - SIGNALING

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Alanine-Scanning Mutagenesis Defines a Conserved Energetic Hotspot in the Ca_Vα₁ AID-Ca_Vβ Interaction Site that Is Critical for Channel Modulation

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