Rebuilding a macromolecular membrane complex at the atomic scale: Case of the Kir6.2 potassium channel coupled to the muscarinic acetylcholine receptor M2

Ion channel‐coupled receptors (ICCR) are artificial proteins built from a G protein‐coupled receptor and an ion channel. Their use as molecular biosensors is promising in diagnosis and high‐throughput drug screening. The concept of ICCR was initially validated with the combination of the muscarinic receptor M2 with the inwardly rectifying potassium channel Kir6.2. A long protein engineering phase has led to the biochemical characterization of the M2‐Kir6.2 construct. However, its molecular mechanism remains to be elucidated. In particular, it is important to determine how the activation of M2 by its agonist acetylcholine triggers the modulation of the Kir6.2 channel via the M2‐Kir6.2 linkage. In the present study, we have developed and validated a computational approach to rebuild models of the M2‐Kir6.2 chimera from the molecular structure of M2 and Kir6.2. The protocol was first validated on the known protein complexes of the μ‐opioid Receptor, the CXCR4 receptor and the Kv1.2 potassium channel. When applied to M2‐Kir6.2, our protocol produced two possible models corresponding to two different orientations of M2. Both models highlights the role of the M2 helices I and VIII in the interaction with Kir6.2, as well as the role of the Kir6.2 N‐terminus in the channel opening. Those two hypotheses will be explored in a future experimental study of the M2‐Kir6.2 construct. Proteins 2014; 82:1694–1707. © 2014 Wiley Periodicals, Inc.

[1]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[2]  Gabriel Waksman,et al.  Structure of the outer membrane complex of a type IV secretion system , 2009, Nature.

[3]  Z. Weng,et al.  Protein–protein docking benchmark version 3.0 , 2008, Proteins.

[4]  Michel Bouvier,et al.  Restructuring G-Protein- Coupled Receptor Activation , 2012, Cell.

[5]  M. Paetzel,et al.  Crystal structure of a bacterial signal Peptide peptidase. , 2008, Journal of molecular biology.

[6]  R. Jockers,et al.  Asymmetry of GPCR oligomers supports their functional relevance. , 2011, Trends in pharmacological sciences.

[7]  F. Ashcroft,et al.  Functional analysis of a structural model of the ATP‐binding site of the KATP channel Kir6.2 subunit , 2005, The EMBO journal.

[8]  R. Stevens,et al.  Structure of the human k-opioid receptor in complex with JDTic , 2012 .

[9]  F. Ashcroft,et al.  Involvement of the N‐terminus of Kir6.2 in coupling to the sulphonylurea receptor , 1999, The Journal of physiology.

[10]  R. Abagyan,et al.  Structures of the CXCR4 Chemokine GPCR with Small-Molecule and Cyclic Peptide Antagonists , 2010, Science.

[11]  C. Notredame,et al.  Tcoffee add igs: a web server for computing, evaluating and combining multiple sequence alignments , 2003, Nucleic Acids Res..

[12]  M. Karplus,et al.  Effective energy function for proteins in solution , 1999, Proteins.

[13]  D. Rodríguez,et al.  Characterization of the homodimerization interface and functional hotspots of the CXCR4 chemokine receptor , 2012, Proteins.

[14]  A. Kruse,et al.  Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist , 2011, Nature.

[15]  Jeffrey J. Gray,et al.  Protein-protein docking with simultaneous optimization of rigid-body displacement and side-chain conformations. , 2003, Journal of molecular biology.

[16]  D. Ritchie,et al.  Evaluation of Protein Docking Predictions Using Hex 3.1 in CAPRI Rounds 1{2 , 2002 .

[17]  O. Ernst,et al.  Engineering of an Artificial Light-Modulated Potassium Channel , 2012, PloS one.

[18]  Dynamics and stability of the metal binding domains of the Menkes ATPase and their interaction with metallochaperone HAH1. , 2012, Biochemistry.

[19]  M. Karplus,et al.  Simulation of activation free energies in molecular systems , 1996 .

[20]  Jianpeng Ma,et al.  Structure of the full-length Shaker potassium channel Kv1.2 by normal-mode-based X-ray crystallographic refinement , 2010, Proceedings of the National Academy of Sciences.

[21]  R. Stevens,et al.  High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein–Coupled Receptor , 2007, Science.

[22]  Xiao Tao,et al.  Structural basis of PIP2 activation of the classical inward rectifier K+ channel Kir2.2 , 2011, Nature.

[23]  Sergey Lyskov,et al.  The RosettaDock server for local protein–protein docking , 2008, Nucleic Acids Res..

[24]  Jianpeng Ma,et al.  CHARMM: The biomolecular simulation program , 2009, J. Comput. Chem..

[25]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[26]  Andrey Tovchigrechko,et al.  GRAMM-X public web server for protein–protein docking , 2006, Nucleic Acids Res..

[27]  C. Nichols,et al.  Octameric Stoichiometry of the KATP Channel Complex , 1997, The Journal of general physiology.

[28]  Lydia N. Caro,et al.  β2-Adrenergic Ion-Channel Coupled Receptors as Conformational Motion Detectors , 2011, PloS one.

[29]  Marc F Lensink,et al.  Blind predictions of protein interfaces by docking calculations in CAPRI , 2010, Proteins.

[30]  D. Baker,et al.  Multipass membrane protein structure prediction using Rosetta , 2005, Proteins.

[31]  Patrick Scheerer,et al.  Crystal structure of the ligand-free G-protein-coupled receptor opsin , 2008, Nature.

[32]  Zhiping Weng,et al.  Protein–protein docking benchmark version 4.0 , 2010, Proteins.

[33]  F. Ashcroft,et al.  Identification of residues contributing to the ATP binding site of Kir6.2 , 2003, The EMBO journal.

[34]  D. Higgins,et al.  T-Coffee: A novel method for fast and accurate multiple sequence alignment. , 2000, Journal of molecular biology.

[35]  S. Rasmussen,et al.  Crystal Structure of the β2Adrenergic Receptor-Gs protein complex , 2011, Nature.

[36]  K. Kunjilwar,et al.  Association and Stoichiometry of KATP Channel Subunits , 1997, Neuron.

[37]  S. Wodak,et al.  Docking and scoring protein complexes: CAPRI 3rd Edition , 2007, Proteins.

[38]  L. Pardo,et al.  Crystal structure of the μ-opioid receptor bound to a morphinan antagonist , 2012, Nature.

[39]  M. Karplus,et al.  CHARMM: A program for macromolecular energy, minimization, and dynamics calculations , 1983 .

[40]  Z. Weng,et al.  ZDOCK: An initial‐stage protein‐docking algorithm , 2003, Proteins.

[41]  Roderick MacKinnon,et al.  Crystal Structure of the Mammalian GIRK2 K+ Channel and Gating Regulation by G Proteins, PIP2, and Sodium , 2011, Cell.

[42]  Zaida Luthey-Schulten,et al.  MultiSeq: unifying sequence and structure data for evolutionary analysis , 2006, BMC Bioinformatics.

[43]  Jonathan A. Javitch,et al.  Structure of the Human Dopamine D3 Receptor in Complex with a D2/D3 Selective Antagonist , 2010, Science.

[44]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[45]  Manfred Burghammer,et al.  Structure of bovine rhodopsin in a trigonal crystal form. , 2003, Journal of molecular biology.

[46]  Bryan L. Roth,et al.  Structure of the human kappa opioid receptor in complex with JDTic , 2012, Nature.

[47]  J. Dupuis,et al.  Coupling ion channels to receptors for biomolecule sensing. , 2008, Nature nanotechnology.

[48]  Hyeon Joo,et al.  OPM database and PPM web server: resources for positioning of proteins in membranes , 2011, Nucleic Acids Res..