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Identification and Structural Characterization of an Intermediate in the Folding of the Measles Virus X Domain*

  • Daniela Bonetti
    Affiliations
    Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche “A. Rossi Fanelli,” Sapienza Università di Roma, 00185 Rome, Italy
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  • Carlo Camilloni
    Affiliations
    Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom

    Department of Chemistry and Institute for Advanced Study, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany
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  • Lorenzo Visconti
    Affiliations
    Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche “A. Rossi Fanelli,” Sapienza Università di Roma, 00185 Rome, Italy
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  • Sonia Longhi
    Affiliations
    Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS, UMR 7257, 13288 Marseille, France

    CNRS, AFMB UMR 7257, 13288 Marseille, France
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  • Maurizio Brunori
    Affiliations
    Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche “A. Rossi Fanelli,” Sapienza Università di Roma, 00185 Rome, Italy
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  • Michele Vendruscolo
    Affiliations
    Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom
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  • Stefano Gianni
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche “A. Rossi Fanelli,” Sapienza Università di Roma, 00185 Rome, Italy

    Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom
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  • Author Footnotes
    * This work was supported by the Italian Ministero dell'Istruzione dell'Università e della Ricerca (Progetto di Interesse “Invecchiamento”; to S. G.) and by Sapienza Università di Roma (C26A155S48; to S. G.). The authors declare that they have no conflicts of interest with the contents of this article.
    This article contains supplemental Figs. S1 and S2.
Open AccessPublished:March 21, 2016DOI:https://doi.org/10.1074/jbc.M116.721126
      Although most proteins fold by populating intermediates, the transient nature of such states makes it difficult to characterize their structures. In this work we identified and characterized the structure of an intermediate of the X domain of phosphoprotein (P) of measles virus. We obtained this result by a combination of equilibrium and kinetic measurements and NMR chemical shifts used as structural restraints in replica-averaged metadynamics simulations. The structure of the intermediate was then validated by rationally designing four mutational variants predicted to affect the stability of this state. These results provide a detailed view of an intermediate state and illustrate the opportunities offered by a synergistic use of experimental and computational methods to describe non-native states at atomic resolution.

      Introduction

      A major goal in protein folding studies lies in identifying intermediates and in characterizing their structure (
      • Capaldi A.P.
      • Kleanthous C.
      • Radford S.E.
      Im7 folding mechanism: misfolding on a path to the native state.
      ,
      • Capaldi A.P.
      • Shastry M.C.
      • Kleanthous C.
      • Roder H.
      • Radford S.E.
      Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate.
      ,
      • Gianni S.
      • Ivarsson Y.
      • De Simone A.
      • Travaglini-Allocatelli C.
      • Brunori M.
      • Vendruscolo M.
      Structural characterization of a misfolded intermediate populated during the folding process of a PDZ domain.
      ,
      • Matouschek A.
      • Kellis Jr., J.T.
      • Serrano L.
      • Bycroft M.
      • Fersht A.R.
      Transient folding intermediates characterized by protein engineering.
      ,
      • Parker M.J.
      • Spencer J.
      • Clarke A.R.
      An integrated kinetic analysis of intermediates and transition states in protein folding reactions.
      ,
      • Religa T.L.
      • Markson J.S.
      • Mayor U.
      • Freund S.M.
      • Fersht A.R.
      Solution structure of a protein denatured state and folding intermediate.
      ,
      • Wildegger G.
      • Kiefhaber T.
      Three-state model for lysozyme folding: triangular folding mechanism with an energetically trapped intermediate.
      ,
      • Gruebele M.
      An intermediate seeks instant gratification.
      ). This task is often challenging because, as marginally stable, folding intermediates are elusive and can be detected only transiently during the folding reaction. The combination of experimental and theoretical approaches represents a powerful strategy to address this problem, providing the possibility to unveil the molecular structure of folding intermediates and to define their role in the reaction mechanism.
      The viral polymerase complex of paramyxoviruses is composed by the protein L and by the phosphoprotein P (
      • Bourhis J.M.
      • Canard B.
      • Longhi S.
      Structural disorder within the replicative complex of measles virus: functional implications.
      ,
      • Bourhis J.M.
      • Johansson K.
      • Receveur-Bréchot V.
      • Oldfield C.J.
      • Dunker K.A.
      • Canard B.
      • Longhi S.
      The C-terminal domain of measles virus nucleoprotein belongs to the class of intrinsically disordered proteins that fold upon binding to their physiological partner.
      ,
      • Sedlmeier R.
      • Neubert W.
      The replicative complex of paramyxoviruses: structure and function.
      ). In measles virus (a member of the Morbillivirus genus), the C-terminal region of phosphoprotein P is a globular domain of 49 amino acids, called X domain (XD),
      The abbreviations used are: XD
      X domain
      D
      denatured state
      I
      intermediate state
      N
      native state.
      composed of three α-helices organized as an anti-parallel bundle (
      • Johansson K.
      • Bourhis J.-M.
      • Campanacci V.
      • Cambillau C.
      • Canard B.
      • Longhi S.
      Crystal structure of the measles virus phosphoprotein domain responsible for the induced folding of the C-terminal domain of the nucleoprotein.
      ). Previous investigations on measles virus XD have suggested this domain to be structurally heterogeneous, populating at least two alternative conformations under native conditions (
      • D'Urzo A.
      • Konijnenberg A.
      • Rossetti G.
      • Habchi J.
      • Li J.
      • Carloni P.
      • Sobott F.
      • Longhi S.
      • Grandori R.
      Molecular basis for structural heterogeneity of an intrinsically disordered protein bound to a partner by combined ESI-IM-MS and modeling.
      ). This feature is consistent with the findings of Kingston et al. (
      • Kingston R.L.
      • Gay L.S.
      • Baase W.S.
      • Matthews B.W.
      Structure of the nucleocapsid-binding domain from the mumps virus polymerase; an example of protein folding induced by crystallization.
      ), who suggested that the native state of XD from mumps virus (a Rubulavirus member) represents an example of folding induced by crystal packing effects. Notably, the lack of a unique stable three-dimensional structure is not a feature unique to mumps virus XD, being also shared by the corresponding domains from other rubulaviruses that were found to populate in solution a structural continuum ranging from stable α-helical bundles to largely disordered (
      • Yegambaram K.
      • Bulloch E.M.
      • Kingston R.L.
      Protein domain definition should allow for conditional disorder.
      ). Because of such heterogeneity, XD represents an ideal candidate for folding studies, as it is likely to populate partially structured states at equilibrium, displaying an overall stability comparable with that of the native conformation.
      In this work we characterized the folding pathway of XD, and present the high resolution structure of an intermediate while characterizing its mechanistic role in time-resolved kinetic experiments. The occurrence of a folding intermediate is supported by the observation of bi-phasic (un)folding transitions in discharge-induced temperature-jump experiments as well as by all-atom replica-averaged metadynamics simulations with NMR chemical shift restraints. In an effort to further address the structure of the intermediate state, we designed rationally and characterized experimentally mutational variants that selectively tune the stability of this state with respect to that of the native state.

      Experimental Procedures

      Experiments were performed on a fluorescent pseudo-wild type XD variant Y480W, which was previously produced and characterized. All proteins were expressed and purified as described (
      • Dosnon M.
      • Bonetti D.
      • Morrone A.
      • Erales J.
      • di Silvio E.
      • Longhi S.
      • Gianni S.
      Demonstration of a folding after binding mechanism in the recognition between the measles virus NTAIL and X domains.
      ). All reagents were of analytical grade.

      Circular Dichroism Equilibrium Experiments

      Circular dichroism equilibrium denaturation experiments were carried out on a JASCO circular dichroism (CD) spectropolarimeter (Jasco, Inc., Easton, MD). CD spectra were recorded between 200 and 250 nm using a quartz cuvette with a light path of 1 cm, at different urea concentrations. For each urea concentration three spectra were averaged. Protein concentration was typically 10 μm, and the buffer used was 50 mm sodium phosphate, 300 mm NaCl at pH 7.2. The experiments were performed at 25 °C.

      Fluorescence Equilibrium Experiments

      Fluorescence equilibrium denaturation experiments were carried out using a Fluoromax single photon counting spectrofluorometer (Jobin-Yvon, Edison, NJ). Emission spectra were recorded between 300 and 400 nm using a quartz cuvette with a light path of 1 cm, at different urea concentrations. The excitation wavelength was 280 nm. Protein concentration was typically 5 μm, and the buffer used was 50 mm sodium phosphate, 300 mm NaCl at pH 7.2. The experiments were performed at 25 °C.

      Temperature-jump Fluorescence Spectroscopy

      Kinetic (un)folding experiments were performed using a Hi-Tech PTJ-64 capacitor-discharge T-jump apparatus (Hi-Tech, Salisbury, UK). Temperature was rapidly changed with a jump-size of 9 °C, from 16 °C to 25 °C. The fluorescence change of N-acetyltryptophanamide (NATA) was used in control measurements. Degassed samples were slowly pumped through the 0.5 × 2-mm quartz flow cell before data acquisition. Usually 10 individual traces were averaged. The excitation wavelength was 296 nm, and the fluorescence emission was collected using a 320-nm cut-off glass filter. Protein concentration was typically 100 μm, and the buffer used was 50 mm sodium phosphate, 300 mm NaCl at pH 7.2.

      Replica-averaged Metadynamics Simulations

      Replica-averaged metadynamics simulations (
      • Camilloni C.
      • Cavalli A.
      • Vendruscolo M.
      Replica-averaged metadynamics.
      ,
      • Camilloni C.
      • Vendruscolo M.
      Statistical mechanics of the denatured state of a protein using replica-averaged metadynamics.
      ) were performed using GROMACS compiled with PLUMED and ALMOST (
      • Fu B.
      • Sahakyan A.B.
      • Camilloni C.
      • Tartaglia G.G.
      • Paci E.
      • Caflisch A.
      • Vendruscolo M.
      • Cavalli A.
      ALMOST: an all atom molecular simulation toolkit for protein structure determination.
      ). The system was simulated using the Amber03W force field (
      • Best R.B.
      • Mittal J.
      Protein simulations with an optimized water model: cooperative helix formation and temperature-induced unfolded state collapse.
      ) in explicit TIP4P05 water (
      • Abascal J.L.
      • Vega C.
      A general purpose model for the condensed phases of water: TIP4P/2005.
      ). Van der Waals and short-range electrostatic interactions were cut off at 0.9 nm, whereas long range electrostatic interaction was treated with the Particle Mesh Ewald method and a mesh size of 0.12 nm (
      • Essmann U.
      • Perera L.
      • Berkowitz M.L.
      • Darden T.
      • Lee H.
      • Pedersen L.G.
      A smooth particle mesh Ewald method.
      ). The isothermal-isobaric ensemble was enforced using the Bussi thermostat (
      • Bussi G.
      • Donadio D.
      • Parrinello M.
      Canonical sampling through velocity rescaling.
      ) at 300 K and the Parrinello-Rahman barostat (
      • Parrinello M.
      • Rahman A.
      Polymorphic transitions in single crystals: a new molecular dynamics method.
      ) at 1 bar. The starting conformation for XD was taken from the 2K9D NMR solution structure (
      • Bernard C.
      • Gely S.
      • Bourhis J.M.
      • Morelli X.
      • Longhi S.
      • Darbon H.
      Interaction between the C-terminal domains of N and P proteins of measles virus investigated by NMR.
      ). The structure was solvated with 5845 water molecules and 3 chloride ions.
      Replica-averaged metadynamic simulations were performed using chemical shifts as replica-averaged restraints and bias-exchange metadynamics (
      • Camilloni C.
      • Vendruscolo M.
      Statistical mechanics of the denatured state of a protein using replica-averaged metadynamics.
      ). Four replicas of the system were simulated in parallel with a restraint applied on the square difference between the CamShift (
      • Kohlhoff K.J.
      • Robustelli P.
      • Cavalli A.
      • Salvatella X.
      • Vendruscolo M.
      Fast and accurate predictions of protein NMR chemical shifts from interatomic distances.
      ) back-calculated NMR chemical shifts for the Hα and HN hydrogens and the experimental data using a force constant of 6 and 3 kJ/(mol ppm2), respectively. Each of the four replicas is bias along one of the following four collective variables: α-helical content, radius of gyration, root mean square deviation from the crystal structure calculated over the Cα carbons, and number of contacts among the heavy atoms of the hydrophobic residues. Gaussian deposition was performed with an initial rate of 0.125 kJ/mol/ps, a bias factor of 10, and with σ values set to 0.2, 0.005, 0.1, and 0.25, respectively. Each replica was been evolved for 180 ns, with exchange trials every 50 ps.

      Author Contributions

      D. B. and L. V. conducted the experimental work. C. C. determined the structure of the intermediate. D. B, C. C., L. V., S. L., M. B., M. V., and S. G, analyzed and discussed the data. S. G. conceived the idea of the project and wrote the main draft of the manuscript, which was later edited by all the authors.

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