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Zn2+-binding and molecular determinants of tetramerization in voltage-gated K+ channels

Nature Structural Biology volume 6, pages 38–43 (1999)Cite this article

Abstract

The N-terminal, cytoplasmic tetramerization domain (T1) of voltage-gated K+ channels encodes molecular determinants for subfamily-specific assembly of α-subunits into functional tetrameric channels. Crystal structures of T1 tetramers from Shaw and Shaker subfamilies reveal a common four-layered scaffolding. Within layer 4, on the hypothetical membrane-facing side of the tetramer, the Shaw T1 tetramer contains four zinc ions; each is coordinated by a histidine and two cysteines from one monomer and by one cysteine from an adjacent monomer. The amino acids involved in coordinating the Zn2+ ion occur in a HX5CX20CC sequence motif that is highly conserved among all Shab, Shaw and Shal subfamily members, but is not found in Shaker subfamily members. We demonstrate by coimmunoprecipitation that a few characteristic residues in the subunit interface are crucial for subfamily-specific tetramerization of the T1 domains.

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Figure 1: a, The Shaw T1 tetramer drawn in MOLSCRIPT37 and rendered using POVRAY version 3.0.
Figure 2: a, The zinc binding in the Shaw T1 tetramer.
Figure 3: a, Sequence alignments for T1 domains have been made for Shaw (23 sequences), Shaker (55 sequences), Shab (25 sequences), and Shal (14 sequences). Sequences in each subfamily were aligned using CLUSTALW (Complete sequences are at http://sbl.salk.edu/current_res/kv_seq.html).
Figure 4: Coimmunoprecipitation analysis between the epitope tagged, wild-type N-terminal construct (1nT1; labeled 1nT1-tag in the Figure) of Shaker and the mutant or chimeric forms of Shaker T1 domain (1T1; labeled Test in the Figure) for assembly test.

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Acknowledgements

We thank J. Greenwald and R. Robinson for help in data collection at SSRL; K. Turbedsky for analytical centrifugation; M. Park for amino acid sequencing; G. Louie and T. Pollard for discussions and comments on the manuscript; P. Biggin for sequence alignment advice and web page construction; and W. Kwiatkowski for rendering figures. Supported in part by the Lucille P. Markey Charitable Trust (K.B.) and the American Heart Association (K.B., A.K.), the Chapman Foundation (K.B.), the Hoffman Foundation (A.K.), and an NIH training grant (K.B., M.H.N.) administered through the University of California, San Diego. S.C. is a recipient of the Klingenstein Fellowship Award in Neuroscience. SSRL is funded by the Department of Energy, Office of Basic Energy Science. This work is supported by grants from the NIH (to P.J.P. and S.C.).

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Authors and Affiliations

  1. Structural Biology Labratory, The Salk Institute, La Jolla, 92037, California, USA

    Kathryn A. Bixby, Max H. Nanao, Andreas Kreusch & Senyon Choe

  2. Department of Chemistry and Biochemistry, University of California, San Diego, 92037, California, USA

    Kathryn A. Bixby & Senyon Choe

  3. Department of Biology, University of California, San Diego, 92037, California, USA

    Max H. Nanao & Senyon Choe

  4. Division of Neuroscience, Baylor College of Medicine, Houston, 77030, Texas, USA

    N. Vivienne Shen & Paul J. Pfaffinger

  5. Stanford Synchotron Radiation Labratory, Stanford, 94309, California, USA

    Henry Bellamy

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Correspondence to Senyon Choe.

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Bixby, K., Nanao, M., Shen, N. et al. Zn2+-binding and molecular determinants of tetramerization in voltage-gated K+ channels. Nat Struct Mol Biol 6, 38–43 (1999). https://doi.org/10.1038/4911

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