Letter | Published:

Open-to-closed transition in apo maltose-binding protein observed by paramagnetic NMR

Nature volume 449, pages 10781082 (25 October 2007) | Download Citation


Large-scale domain rearrangements in proteins have long been recognized to have a critical function in ligand binding and recognition, catalysis and regulation1,2,3,4,5. Crystal structures have provided a static picture of the apo (usually open) and holo usually closed) states. The general question arises as to whether the apo state exists as a single species in which the closed state is energetically inaccessible and interdomain rearrangement is induced by ligand or substrate binding, or whether the predominantly open form already coexists in rapid equilibrium with a minor closed species. The maltose-binding protein (MBP), a member of the bacterial periplasmic binding protein family6, provides a model system for investigating this problem because it has been the subject of extensive studies by crystallography7,8, NMR9,10,11 and other biophysical techniques11,12,13. Here we show that although paramagnetic relaxation enhancement (PRE) data for the sugar-bound form are consistent with the crystal structure of holo MBP, the PRE data for the apo state are indicative of a rapidly exchanging mixture (ns to μs regime) of a predominantly (95%) open form (represented by the apo crystal structure) and a minor (5%) partially closed species. Using ensemble simulated annealing refinement against the PRE data we are able to determine a 〈r-6〉 ensemble average structure of the minor apo species and show that it is distinct from the sugar-bound state.

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  1. 1.

    & Steitz, T. Space-filling models of kinase clefts and conformational change. Science 204, 375–380 (1979)

  2. 2.

    , & Structural mechanisms for domain movements in proteins. Biochemistry 33, 6739–6749 (1994)

  3. 3.

    & A database of macromolecular motions. Nucleic Acids Res. 26, 4280–4290 (1998)

  4. 4.

    & Systematic analysis of domain motions in proteins from conformational change: new results on citrate synthase and T4 lysozyme. Proteins Struct. Funct. Genet. 30, 144–154 (1998)

  5. 5.

    et al. Linkage between dynamics and catalysis in a thermophilic–mesophilic enzyme pair. Nat. Struct. Mol. Biol. 11, 945–949 (2004)

  6. 6.

    & Jr. Structural, functional and evolutionary relationships among extracellular solute-binding receptors of bacteria. Microbiol. Rev. 57, 320–346 (1993)

  7. 7.

    , , & Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of maltodextrin binding protein involved in active transport and chemotaxis. Biochemistry 31, 10657–10663 (1992)

  8. 8.

    & &. Rodseth, L. E. Extensive features of tight oligosaccharide binding revealed in high-resolution structures of the maltodextrin transport/chemosensory receptor. Structure 5, 997–1015 (1997)

  9. 9.

    et al. Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: differences in solution and crystal forms of maltodextrin binding protein loaded with β-cyclodextrin. J. Mol. Biol. 295, 1265–1273 (2000)

  10. 10.

    et al. Ligand-induced structural changes to maltodextrin-binding protein as studied by solution NMR spectroscopy. J. Mol. Biol. 309, 961–974 (2001)

  11. 11.

    , & The energetic cost of domain reorientation in maltose-binding protein as studied by NMR and fluorescence spectroscopy. Proc. Natl Acad. Sci. USA 100, 12700–12705 (2003)

  12. 12.

    , , , & Two modes of ligand binding in maltose-binding protein of Escherichia coli: electron paramagnetic resonance study of ligand-induced global conformational changes by site-directed spin labeling. J. Biol. Chem. 272, 17615–17622 (1997)

  13. 13.

    et al. Model of maltose-binding protein/chemoreceptor complex supports intrasubunit signaling mechanism. Proc. Natl Acad. Sci. USA 96, 939–944 (1999)

  14. 14.

    & Detecting transient intermediates in macromolecular binding by paramagnetic NMR. Nature 440, 1227–1230 (2006)

  15. 15.

    , & Visualization of transient encounter complexes in protein-protein association. Nature 444, 383–386 (2006)

  16. 16.

    & R-factor, free R and complete cross-validation for dipolar coupling refinement of NMR structures. J. Am. Chem. Soc. 121, 9008–9012 (1999)

  17. 17.

    , & Ensemble approach for NMR structure refinement against 1H paramagnetic relaxation enhancement data arising from a flexible paramagnetic group attached to a macromolecules. J. Am. Chem. Soc. 126, 5879–5896 (2004)

  18. 18.

    & Using conjoined rigid body/torsion angle simulated annealing to determine the relative orientation of covalently linked protein domains from dipolar couplings. J. Magn. Reson. 154, 329–335 (2002)

  19. 19.

    , & Prediction of charge-induced molecular alignment of biomolecules dissolved in dilute liquid-crystalline phases. Biophys. J. 86, 3444–3460 (1994)

  20. 20.

    NMR characterization of the dynamics of biomacromolecules. Chem. Rev. 104, 3623–3640 (2004)

  21. 21.

    & Structural evidence for a dominant role of nonpolar interactions in the binding of a transport/chemosensory receptor to its highly polar ligands. Biochemistry 41, 706–712 (2002)

  22. 22.

    , & Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules. J. Magn. Reson. 184, 185–195 (2007)

  23. 23.

    , & Measurement of residual dipolar couplings of macromolecules aligned in a nematic phase of a colloidal suspension of rod-shaped viruses. J. Am. Chem. Soc. 120, 10571–10572 (1998)

  24. 24.

    , & Using Xplor-NIH for NMR molecular structure determination. Progr. NMR Spectrosc. 48, 47–62 (2006)

  25. 25.

    & Reweighted atomic densities to represent ensembles of NMR structures. J. Biomol. NMR 23, 221–225 (2002)

  26. 26.

    , , , & Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)

  27. 27.

    , , , & Thermodynamic characterization of the reversible, two-state unfolding of maltose binding protein, a large two-domain protein. Biochemistry 36, 5020–5028 (1997)

  28. 28.

    , , & Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biologial macromolecules in solution. Proc. Natl Acad. Sci. USA 94, 12366–12371 (1997)

  29. 29.

    & TROSY triple-resonance four-dimensional NMR spectroscopy of a 46 ns tumbling protein. J. Am. Chem. Soc. 121, 2571–2575 (1999)

  30. 30.

    , & Dipolar couplings in macromolecular structure determination. Methods Enzymol. 339, 127–174 (2001)

  31. 31.

    , & Evaluation of cross-relaxation effects and measurement of one-bond couplings in proteins with short transverse relaxation times. J. Magn. Reson. 143, 184–196 (2000)

  32. 32.

    , & Domain orientation in β-cyclodextrin-loaded maltose binding protein: diffusion anisotropy measurements confirm the results of a dipolar coupling study. J. Biomol. NMR 20, 83–88 (2001)

  33. 33.

    & Internal coordinates for molecular dynamics and minimization in structure determination and refinement. J. Magn. Reson. 152, 288–302 (2001)

  34. 34.

    , , & Determination of three-dimensional structures of proteins by simulated annealing with interproton distance restraints: application to crambin, potato carboxypeptidase inhibitor and barley serine proteinase inhibitor 2. Protein Eng. 2, 27–38 (1988)

  35. 35.

    & χ1 rotamer populations and angles of mobile surface side chains are accurately predicted by a torsion angle database potential of mean force. J. Am. Chem. Soc. 124, 2866–2867 (2002)

  36. 36.

    , & Intramolecular domain–domain association/dissociation and phosphoryl transfer in the mannitol transporter of Escherichia coli are not coupled. Proc. Natl Acad. Sci. USA 104, 3153–3158 (2007)

  37. 37.

    , & CRYSOL: a program to evaluate X-ray scattering of biological macromolecules from atomic coordinates. J. Appl. Cryst. 28, 768–773 (1995)

  38. 38.

    The PyMol Molecular Graphics System, 〈〉 (DeLano Scientific, San Carlos, CA, 2002)

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We thank J. Iwahara, A. Grishaev and A. Szabo for helpful discussions; and A. Grishaev and L. Guo for assistance with SAXS data collection and processing. Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science. BioCAT is a National Institutes of Health-supported Research Center. This work was supported by funds from the Intramural Program of the NIH, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and the AIDS Targeted Antiviral program of the Office of the Director of the NIH (to G.M.C.).

An ensemble of 50 sets of coordinates for the equilibrium mixture of major (95%) open and minor (5%) partly closed species of MBP, together with the PRE experimental restraints, are deposited in the Protein Data Bank with accession codes 2V93 and R2V93MR, respectively.

Author information


  1. Laboratory of Chemical Physics, Building 5, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA

    • Chun Tang
    •  & G. Marius Clore
  2. Division of Computational Bioscience, Building 12A, Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892-5624, USA

    • Charles D. Schwieters


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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to G. Marius Clore.

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    Supplementary Information

    The file contains Supplementary Figures S1-S17 and Supplementary Table S1 with Legends, Supplementary Notes and additional references.

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