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Allosteric mechanism of water-channel gating by Ca2+–calmodulin

Nature Structural & Molecular Biology volume 20, pages 1085–1092 (2013)Cite this article

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Abstract

Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudoatomic structure of full-length mammalian aquaporin-0 (AQP0, Bos taurus) in complex with CaM, using EM to elucidate how this signaling protein modulates water-channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits.

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Figure 1: Purification and pseudoatomic model of the AQP0–CaM complex determined by EM.
Figure 2: Hydrophobic interactions involved in AQP0–CaM complex formation.
Figure 3: Calmodulin restricts the dynamics of AQP0.
Figure 4: CaM binding closes the AQP0 cytoplasmic constriction site gate II (CSII).
Figure 5: Mechanism of Ca2+–CaM regulation of AQP0.

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Acknowledgements

The authors would like to thank M. Sarhan (Howard Hughes Medical Institute (HHMI), Janelia Farm Research Campus) for help with ITC and D. Shi (HHMI, Janelia Farm Research Campus) for help with various aspects of EM. Research in the laboratory of J.E.H. is supported by US National Institutes of Health (NIH) National Eye Institute grant EY5661 (J.E.H.). Research by D.M.C. was supported by the NIH National Library of Medicine Biomedical Informatics Research Training Program Award, no. LM007443. Research by S.L.R. was supported by the Ruth L. Kirschtein National Research Service Award from NIH. Research in the laboratory of D.J.T. is supported by NIH National Institute of Neurological Disorders–National Institute of General Medical Sciences grant GM86685 and US National Science Foundation grant CHE-0750175 (D.J.T.). M.H. is supported by a fellowship from the German Academy of Sciences Leopoldina. This work was supported in part by NIH grant R01 GM079233 (T.G.). Research in the laboratory of T.G. is funded by the HHMI (T.G.).

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  1. Steve L Reichow and Daniel M Clemens: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA

    Steve L Reichow & Tamir Gonen

  2. Department of Physiology and Biophysics, University of California, Irvine, Irvine, California, USA

    Daniel M Clemens, Karin L Németh-Cahalan & James E Hall

  3. Department of Chemistry, University of California, Irvine, Irvine, California, USA

    J Alfredo Freites, Matthias Heyden & Douglas J Tobias

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Contributions

S.L.R., D.M.C., J.E.H. and T.G. conceived of and designed the experiments for this work. All authors contributed to data analysis and preparation of the manuscript. S.L.R. performed protein purification, EM and ITC binding studies on the AQP0–CaM complexes. D.M.C., J.A.F., M.H. and D.J.T. performed setup and analysis of molecular dynamics simulations. D.M.C. and K.L.N.-C. performed oocyte permeability measurements, construction of oocyte expression constructs and analysis of oocyte permeability data.

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Correspondence to James E Hall or Tamir Gonen.

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Integrated supplementary information

Supplementary Figure 1 Purification of the cross-linked AQP0–CaM complex.

Schematic of crosslinking and purification of the AQP0–CaM complex (EDC/NHS – crosslinking reagents; IEX – ion exchange chromatography; SEC – size exclusion chromatography). (b and c) Separation of AQP0–CaM from unreacted AQP0 by IEX. (UV(280nm) – blue; conductivity – red). Selected fractions for all steps indicated in grey. (d) Separation of the AQP0–CaM from free CaM by SEC. (inset) Molecular weight calibration showing elution of the AQP0–CaM complex at ~180 KDa; blue dot. (e) SDS-PAGE (lane 1) CaM alone, treated with EDC/NHS resulted in two bands, corresponding to CaM ~13 KDa and the crosslinked CaM dimer (CaM)2 ~22 KDa. (lane 2) AQP0 alone, treated with EDC/NHS resulted in at least four bands, corresponding to the AQP0 monomer ~26 KDa, the crosslinked dimer (AQP0)2 ~52 KDa, trimer (AQP0)3 ~80 KDa, and tetramer (AQP0)4 ~110 KDa. (lane 3) Crude crosslinking products following addition of activated CaM to AQP0 (corresponding to step 2 in (a)). (lane 4) Flow-through from IEX #1. (lanes 5 and 6) Fractions from IEX #1 containing unreacted AQP0 and crosslinked AQP0–CaM products, respectively. (lane 7) Flow-through from IEX #2. (lanes 8 and 9) Fraction from IEX #2 containing unreacted AQP0 and crosslinked AQP0–CaM products, respectively. (lane 10) SEC peak fraction containing AQP0–CaM. Note lanes 3, 6, 9 and 10 contain two unique bands at ~39 KDa and ~65 KDa correspond to the crosslinked 1:1 AQP0–CaM complex and the 2:1 (AQP0)2–CaM denatured complexes.

Supplementary Figure 2 Construction of the pseudoatomic model of AQP0–CaM derived by EM.

(Step 1) The crystallographic structure of AQP0 (PDB 2B6P)29 was chosen for the transmembrane domain of the complex. (Step 2) The cytoplasmic C-terminal domains (residues 223-263) of AQP0 were removed. (Step 3) The AQP0 transmembrane domain was computationally fit into the EM map using Chimera44. (Step 4) The α-helical region of the AQP0CBD (residues 227–241) was threaded onto the two anti-parallel α-helices (Helix A and Helix B) in the ptGAD–CaM structure (PDB 1NWD)45. The AQP0CBD residue L234 occupies the site equivalent to the major hydrophobic anchoring residue in the ptGAD–CaM complex (W485)45. Positioning L234 at this site accommodated connectivity between the AQP0CBD and the transmembrane domain. A different hydrophobic residue within the AQP0CBD (such as L227 and/or V230) may also act as a primary anchor, but this would require a CaM conformation that is unique from the ptGAD–CaM complex. (Step 5) The resulting structure of CaM bound to two AQP0CBD α-helices was placed into each vacant lobe in the EM map, with the N-termini of the AQP0CBD α-helices facing the last transmembrane α-helix in AQP0. (Step 6) Linker domains (residues 223–226) were modeled connecting the transmembrane domain of AQP0 to each AQP0CBD α-helix (labeled, and shown in grey). The final model was subjected to energy minimization to remove steric interactions. A 25 Å map calculated from this model gave a crosscorrelation of 0.95 with the experimental map.

Supplementary Figure 3 Isothermal titration calorimetry (ITC) of CaM binding toAQP0CBD peptides.

(a) (top panels) Raw heats of binding obtained by ITC when CaM was mixed with AQP0CBD peptides (residue 223–242 from the cow sequence shown in Figure 2a) corresponding to the wildtype and alanine point mutations made at the conserved hydrophobic residues indicated. (bottom panels) Binding isotherms fitted to the raw data using two-state and single-state binding models as indicated. (b) Table of thermodynamic parameters obtained by fitting the ITC data to a two-state or single-state binding model (N = number of binding sites, Ka = association constant, ΔH = change in enthalpy, -TΔS = change in entropy, ΔG = Gibb's free energy, subscript 1 and 2 refer to the first and second binding step for data fit to a two-state model.

Supplementary Figure 4 Superposition of starting models for molecular dynamics simulations.

(a) Superposition showing the starting conformations of AQP0 for used for MD simulations. Two MD simulations were generated, one using AQP0 in complex with Calmodulin (AQP0Cam-bound; blue) and the second using AQP0 alone (AQP0CaM-free; yellow). For the CaMfree system, a starting conformation of the AQP0 tetramer (residues 5 to 239) in the absence of CaM was created by deleting the CaM coordinates from the AQP0–CaM complex. In this way, the starting conformations of AQP0 in the two simulations were identical (r.m.s.d. = 0.0 Å). (b) Zoom view, showing that for both systems, the CSII sites of AQP0 were unaltered from the original 2B6P model. Note that CaM is not shown in this overlay.

Supplementary Figure 5 Immunoblot analysis of AQP0 membrane expression in Xenopus oocytes.

Immunoblot analysis of AQP0 membrane expression in Xenopus oocytes.Full-length gel showing immunoblot analysis of AQP0 constructs corresponding to Figure 4e, (inset) within the main text. Each lane corresponds to the membrane fraction for cells of that were uninjected (UI) or injected with RNA for wildtype AQP0 (Y149) and AQP0 point mutants (G149), (L149) and (S149). Samples were separated on SDS-PAGE and blotted with AQP0 antibody (H-44).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 628 kb)

Dynamics of AQP0 CSII gating residue Tyr149.

The AQP0 protein is depicted as grey cartoon with the CSII residues (Tyr149 and Phe75) displayed as blue sticks and water molecules displayed as red and white atoms. Note the movement of Tyr149 (lower right) out of the pore that coincides with a rush of water molecules across the CSII gate. (MOV 4762 kb)

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Reichow, S., Clemens, D., Freites, J. et al. Allosteric mechanism of water-channel gating by Ca2+–calmodulin. Nat Struct Mol Biol 20, 1085–1092 (2013). https://doi.org/10.1038/nsmb.2630

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