NHE1/CaM complex structure 1 Structure of human Na + /H + exchanger NHE1 regulatory region in complex with CaM and Ca 2+ Stefan Köster 1 , Tea Pavkov-Keller 1,2 , Werner Kühlbrandt 1 and Özkan Yildiz 1 * 1 Max Planck Institute of Biophysics, Department of Structural Biology, Max von Laue Str. 3, 60438 Frankfurt am Main, Germany 2 Institute of Molecular Biosciences, Structural Biology, University of Graz, Humboldtstrasse 50/3, 8010 Graz, Austria Running title: NHE1/CaM complex structure To whom correspondence should be addressed: Özkan Yildiz, Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany, Tel.: +49 – 69 – 6303 – 3051; Fax: +49 – 69 – 6303 – 3002; E-mail: Oezkan.Yildiz@biophys.mpg.de Keywords: Calcium-binding proteins; Calmodulin; Cellular regulation; Crystal structure; Membrane transport; pH regulation; Sodium proton exchange; Structural biology Background: The human Na + /H + exchanger NHE1 is activated through binding of calmodulin. Results: X-ray structure of NHE1 regulatory region in complex with calmodulin and calcium Conclusion: The complex structure serves as a basis for a transport regulatory model. Significance: Complex structure improves our understanding of the medically important NHE1 exchanger. SUMMARY The ubiquitous mammalian Na + /H + - exchanger NHE1 has critical functions in regulating intracellular pH, salt concentration and cellular volume. The regulatory C-terminal domain of NHE1 is linked to the ion- translocating N-terminal membrane domain, and acts as a scaffold for signalling complexes. A major interaction partner is calmodulin (CaM), which binds to two neighbouring regions of NHE1 in a strongly Ca 2+ dependent manner. Upon CaM binding, NHE1 is activated by a shift in sensitivity towards alkaline intracellular pH. Here we report the 2.23 Å crystal structure of the NHE1 CaM binding region (NHE1 CaMBR ) in complex with CaM and Ca 2+ . The C- and N- lobes of CaM bind the first and second helix of NHE1 CaMBR , respectively. Both the NHE1 helices and Ca 2+ -bound CaM are elongated, as confirmed by small angle X-ray scattering analysis. Our X-ray structure sheds new light on the molecular mechanisms of the phosphorylation-dependent regulation of NHE1 and enables us to propose a model of how Ca 2+ regulates NHE1 activity The sodium/proton exchangers (NHEs) of the solute carrier 9 (SLC9) family are secondary transporters found in a wide variety of tissues of all animal species and have homologues in all kingdoms of life (1,2). Plasma membrane NHEs use the chemical energy of the Na + gradient across the plasma membrane for electroneutral counter- transport of H + (3,4). So far ten different mammalian NHE isoforms of 25-70% amino acid identity have been identified and characterized (2,5). The type-1 Na + /H + exchanger NHE1 (6) is ubiquitous in the plasma membrane of virtually all mammalian cells, where it regulates intracellular pH (pH i ), salt concentration and cell volume (4,7). NHE1 is therefore critical for the control and maintenance of some of the most fundamental processes in cellular physiology, including cell growth and differentiation (7). For human health and disease, NHE1 plays crucial roles in heart hypertrophy (8,9), cardiac ischemia (10) and hypertension (11). NHE1 has two functional modules: an N- terminal ion translocation domain of ~500 amino acids with 12 or 14 predicted transmembrane helices, and a regulatory C-terminal, cytoplasmic domain of ~300 amino acids (12,13). The http://www.jbc.org/cgi/doi/10.1074/jbc.M111.286906 The latest version is at JBC Papers in Press. Published on September 19, 2011 as Manuscript M111.286906 Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on November 14, 2016 http://www.jbc.org/ Downloaded from by guest on November 14, 2016 http://www.jbc.org/ Downloaded from by guest on November 14, 2016 http://www.jbc.org/ Downloaded from by guest on November 14, 2016 http://www.jbc.org/ Downloaded from