Abstract
This chapter describes nuclear magnetic resonance (NMR) methods that can be used to determine the structures of protein complexes. Many of these techniques are also applicable to other systems (e.g., protein-nucleic acid complexes). In the first section, we discuss methodologies for optimizing the sample conditions for the study of complexes. This is followed by a description of the methods that can be used to map interfaces when a full structure determination of the complex is not appropriate or not possible. We then describe experimental approaches for resonance assignment in complexes, these are essentially the same as those for isolated proteins. Subheading 6. describes the different types of so-called X-filtered NMR experiments that have been devised to separate and selectively observe either inter- or intramolecular structural information. These filtered NMR experiments are then exploited in the experimental strategies for structure determination of either protein complexes or homodimeric proteins. This is followed by a description of the calculation of their structures. Finally, we present case studies from three projects carried out in our laboratory, where we successfully used the methods presented in this chapter.
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References
Lepre, C. A. and Moore, J. M. (1998) Microdrop screening: a rapid method to optimize solvent conditions for NMR spectroscopy of proteins. J. Biomol. NMR 12, 493–499.
Bagby, S., Tong, K. I., and Ikura, M. (2001) Optimization of protein solubility and stability for protein nuclear magnetic resonance. Methods Enzymol. 339, 20–41.
Eftink, M. R. (1997) Fluorescence methods for studying equilibrium macromolecule-ligand interactions. Methods Enzymol. 278, 221–257.
Lofas, S. and Johnsson, B. (1990) A novel hydrogel matrix on gold surfaces in surface-plasmon resonance sensors for fast and efficient covalent immobilization of ligands. J. Chem. Soc. Chem. Commun. 21, 1526–1528.
Laue, T. M. and Stafford, W. F. (1999) Modern applications of analytical ultracentrifugation. Annu. Rev. Biophys. Biomol. Struct. 28, 75–100.
Leavitt, S. and Freire, E. (2001) Direct measurement of protein binding energetics by isothermal titration calorimetry. Curr. Opin. Struct. Biol. 11, 560–566.
Hubbard, S. J. (1998) The structural aspects of limited proteolysis of native proteins. Biochim. Biophys. Acta. 1382, 191–206.
Emerson, S. D., Madison, V. S., Palermo, R. E., Waugh, D. S., Scheffler, J. E., Tsao, K. L., et al. (1995) Solution structure of the Ras-binding domain of C-Raf-1 and identification of its Ras interaction surface. Biochemistry 34, 6911–6918.
Shuker, S. B., Hajduk, P. J., Meadows, R. P., and Fesik, S. W. (1996) Discovering high-affinity ligands for proteins: SAR by NMR. Science 274, 1531–1534.
Hubbard, S. J. and Thornton, J. M. (1993) NACCESS. Department of Biochemistry and Molecular Biology, University College London, London.
Clore, G. M. and Gronenborn, A. M. (1982) Theory and applications of the transferred nuclear Overhauser effect to the study of the conformations of small ligands bound to proteins. J. Magn. Reson. 48, 402–417.
Clore, G. M. and Gronenborn, A. M. (1983) Theory of the time-dependent transferred nuclear Overhauser effect—applications to structural-analysis of ligand protein complexes in solution. J. Magn. Reson. 53, 423–442.
Blommers, M. J. J., Stark, W., Jones, C. E., Head, D., Owen, C. E., and Jahnke, W. (1999) Transferred cross-correlated relaxation complements transferred NOE: structure of an IL-4R-derived peptide bound to STAT-6. J. Am. Chem. Soc. 121, 1949–1953.
Takahashi, H., Nakanishi, T., Kami, K., Arata, Y., and Shimada, I. (2000) A novel NMR method for determining the interfaces of large protein-protein complexes. Nat. Struct. Biol. 7, 220–223.
Dominguez, C., Boelens, R., and Bonvin, A. (2003) HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J. Am. Chem. Soc. 125, 1731–1737.
Clore, G. M. and Schwieters, C. D. (2003) Docking of protein-protein complexes on the basis of highly ambiguous intermolecular distance restraints derived from H-1(N)/N-15 chemical shift mapping and backbone N-15-H-1 residual dipolar couplings using conjoined rigid body/torsion angle dynamics. J. Am. Chem. Soc. 125, 2902–2912.
Markley, J. L. (1975) Observation of histidine residues in proteins by nuclear magnetic resonance spectroscopy. Acc. Chem. Res. 8, 70–80.
Pelton, J. G., Torchia, D. A., Meadow, N. D., and Roseman, S. (1993) Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated III(Glc), a signal-transducing protein from Escherichia-coli, using 2-dimensional heteronuclear NMR techniques. Protein Sci. 2, 543–558.
Gooley, P. R., Johnson, B. A., Marcy, A. I., Cuca, G. C., Salowe, S. P., Hagmann, W. K., et al. (1993) Secondary structure and zinc ligation of human recombinant short-form stromelysin by multidimensional heteronuclear NMR. Biochemistry 32, 13,098–13,108.
Yamazaki, T., Nicholson, L. K., Torchia, D. A., Wingfield, P., Stahl, S. J., Kaufman, J. D., et al. (1994) NMR and X-Ray evidence that the HIV protease catalytic aspartyl groups are protonated in the complex formed by the protease and a nonpeptide cyclic urea-based inhibitor. J. Am. Chem. Soc. 116, 10,791–10,792.
Qin, J., Clore, G. M., and Gronenborn, A. M. (1996) Ionization equilibria for sidechain carboxyl groups in oxidized and reduced human thioredoxin and in the complex with its target peptide from the transcription factor NF kappa B. Biochemistry 35, 7–13.
Jeng, M. F. and Dyson, H. J. (1996) Direct measurement of the aspartic acid 26 pK(a) for reduced Escherichia coli thioredoxin by C-13 NMR. Biochemistry 35, 1–6.
Otting, G., Liepinsh, E., and Wuthrich, K. (1991) Protein hydration in aqueous-solution. Science 254, 974–980.
Grzesiek, S. and Bax, A. (1993) Measurement of amide proton-exchange rates and NOEs with water in C-13/N-15-enriched calcineurin-B. J. Biomol. NMR 3, 627–638.
Clore, G. M., Bax, A., Omichinski, J. G., and Gronenborn, A. M. (1994) Localization of bound water in the solution structure of a complex of the erythroid transcription factor GATA-1 with DNA. Structure 2, 89–94.
Grzesiek, S., Bax, A., Nicholson, L. K., Yamazaki, T., Wingfield, P., Stahl, S. J., et al. (1994) NMR evidence for the displacement of a conserved interior water molecule in HIV protease by a nonpeptide cyclic urea-based inhibitor. J. Am. Chem. Soc. 116, 1581, 1582.
Jasanoff, A., Wagner, G., and Wiley, D. C. (1998) Structure of a trimeric domain of the MHC class II-associated chaperonin and targeting protein Ii. EMBO J. 17, 6812–6818.
Kogler, H., Sorensen, O. W., Bodenhausen, G., and Ernst, R. R. (1983) Low-pass J-filters—suppression of neighbor peaks in heteronuclear relayed correlation spectra. J. Magn. Reson. 55, 157–163.
Ikura, M. and Bax, A. (1992) Isotope-filtered 2D NMR of a protein peptide complex—study of a skeletal-muscle myosin light chain kinase fragment bound to calmodulin. J. Am. Chem. Soc. 114, 2433–2440.
Lee, W., Revington, M. J., Arrowsmith, C., and Kay, L. E. (1994) A pulsed-field gradient isotope-filtered 3D C-13 HMQC-NOESY experiment for extracting intermolecular NOE contacts in molecular-complexes. FEBS Lett. 350, 87–90.
Vuister, G. W., Kim, S. J., Wu, C., and Bax, A. (1994) 2D and 3D NMR-study of phenylalanine residues in proteins by reverse isotopic labeling. J. Am. Chem. Soc. 116, 9206–9210.
Otting, G., Senn, H., Wagner, G., and Wuthrich, K. (1986) Editing of 2D H-1-NMR spectra using X half-filters—combined use with residue-selective N-15 labeling of proteins. J. Magn. Reson. 70, 500–505.
Otting, G. and Wuthrich, K. (1989) Extended heteronuclear editing of 2D H-1-NMR spectra of isotope-labeled proteins, using the X(omega-1, omega-2) double half filter. J. Magn. Reson. 85, 586–594.
Otting, G. and Wuthrich, K. (1990) Heteronuclear filters in 2-dimensional [H-1, H-1] NMR-spectroscopy—combined use with isotope labeling for studies of macromolecular conformation and intermolecular interactions. Q. Rev. Biophys. 23, 39–96.
Wider, G., Weber, C., and Wuthrich, K. (1991) Proton proton Overhauser effects of receptor-bound cyclosporine-A observed with the use of a heteronuclear-resolved half-filter experiment. J. Am. Chem. Soc. 113, 4676–4678.
Folmer, R. H. A., Hilbers, C. W., Konings, R. N. H., and Hallenga, K. (1995) A C-13 double-filtered NOESY with strongly reduced artifacts and improved sensitivity. J. Biomol. NMR 5, 427–432.
Folkers, P. J. M., Folmer, R. H. A., Konings, R. N. H., and Hilbers, C. W. (1993) Overcoming the ambiguity problem encountered in the analysis of nuclear Overhauser magnetic-resonance spectra of symmetrical dimer proteins. J. Am. Chem. Soc. 115, 3798, 3799.
Burgering, M., Boelens, R., and Kaptein, R. (1993) Observation of intersubunit NOEs in a dimeric P22 Mnt repressor mutant by a time-shared [N-15,C-13] double half-filter technique. J. Biomol. NMR 3, 709–714.
Zwahlen, C., Legault, P., Vincent, S. J. F., Greenblatt, J., Konrat, R., and Kay, L. E. (1997) Methods for measurement of intermolecular NOEs by multinuclear NMR spectroscopy: application to a bacteriophage lambda N-peptide/boxB RNA complex. J. Am. Chem. Soc. 119, 6711–6721.
Mueller, L., Kumar, A., and Ernest, R. R. (1975) Two-dimensional carbon-13 NMR spectroscopy. J. Chem. Phys. 63, 5490, 5491.
Melacini, G. (2000) Separation of intra-and intermolecular NOEs through simultaneous editing and J-compensated filtering: a 4D quadrature-free constant-time J-resolved approach. J. Am. Chem. Soc. 122, 9735–9738.
Hwang, T. L. and Shaka, A. J. (1995) Water suppression that works—excitation sculpting using arbitrary wave-forms and pulsed-field gradients. J. Magn. Reson. Ser. A 112, 275–279.
Solomon, I. and Bloembergen, N. (1956) Nuclear magnetic interactions in the HF molecule. J. Chem. Phys. 25, 261–266.
Krugh, T. R. (1976) in Spin Labeling: Theory and Applications (Berliner, L. J., ed.). Academic Press, New York, pp. 339–372.
Kosen, P. A. (1989) Spin labeling of proteins. Methods Enzymol. 177, 86–121.
Gillespie, J. R. and Shortle, D. (1997) Characterization of long-range structure in the denatured state of staphylococcal nuclease, 1: paramagnetic relaxation enhancement by nitroxide spin labels. J. Mol. Biol. 268, 158–169.
Gillespie, J. R. and Shortle, D. (1997) Characterization of long-range structure in the denatured state of staphylococcal nuclease,2: distance restraints from paramagnetic relaxation and calculation of an ensemble of structures. J. Mol. Biol. 268, 170–184.
Battiste, J. L. and Wagner, G. (2000) Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear Overhauser effect data. Biochemistry 39, 5355–5365.
Gaponenko, V., Altieri, A. S., Li, J., and Byrd, R. A. (2002) Breaking symmetry in the structure determination of (large) symmetric protein dimers. J. Biomol. NMR 24, 143–148.
Ogura, K., Terasawa, H., and Inagaki, F. (1996) An improved double-tuned and isotope-filtered pulse scheme based on a pulsed field gradient and a wide-band inversion shaped pulse. J. Biomol. NMR 8, 492–498.
Wider, G., Weber, C., Traber, R., Widmer, H., and Wuthrich, K. (1990) Use of a double-half-filter in 2-dimensional 1H nuclear-magnetic-resonance studies of receptor-bound cyclosporine. J. Am. Chem. Soc. 112, 9015, 9016.
Skrynnikov, N. R., Goto, N. K., Yang, D. W., Choy, W. Y., Tolman, J. R., Mueller, G.A., et al. (2000) Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: differences in solution and crystal forms of maltodextrin binding protein loaded with beta-cyclodextrin. J. Mol. Biol. 295, 1265–1273.
Walters, K. J., Matsuo, H., and Wagner, G. (1997) A simple method to distinguish intermonomer nuclear Overhauser effects in homodimeric proteins with C-2 symmetry. J. Am. Chem. Soc. 119, 5958, 5959.
Xia, Y. L., Sze, K. H., and Zhu, G. (2000) Transverse relaxation optimized 3D and 4D N-15/N-15 separated NOESY experiments of N-15 labeled proteins. J. Biomol. NMR 18, 261–268.
Venters, R. A., Metzler, W. J., Spicer, L. D., Mueller, L., and Farmer, B. T. (1995) Use of H-1(N)-H-1(N) NOEs to determine protein global folds in perdeuterated proteins. J. Am. Chem. Soc. 117, 9592, 9593.
Mal, T. K., Matthews, S. J., Kovacs, H., Campbell, I. D., and Boyd, J. (1998) Some NMR experiments and a structure determination employing a {N-15,H-2} enriched protein. J. Biomol. NMR 12, 259–276.
Walters, K. J., Dayie, K. T., Reece, R. J., Ptashne, M., and Wagner, G. (1997) Structure and mobility of the PUT3 dimer. Nat. Struct. Biol. 4, 744–750.
Ferentz, A. E., Opperman, T., Walker, G. C., and Wagner, G. (1997) Dimerization of the UmuD’ protein in solution and its implications for regulation of SOS mutagenesis. Nat. Struct. Biol. 4, 979–983.
Caffrey, M., Cai, M. L., Kaufman, J., Stahl, S. J., Wingfield, P. T., Covell, D. G., et al. (1998) Three-dimensional solution structure of the 44 kDa ectodomain of SIV gp41. EMBO J. 17, 4572–4584.
Nooren, I. M. A., Kaptein, R., Sauer, R. T., and Boelens, R. (1999) The tetramerization domain of the Mnt repressor consists of two right-handed coiled coils. Nat. Struct. Biol. 6, 755–759.
Jasanoff, A. (1998) An asymmetric deuterium labeling strategy to identify interprotomer and intraprotomer NOEs in oligomeric proteins. J. Biomol. NMR 12, 299–306.
Nilges, M. (1993) A calculation strategy for the structure determination of symmetrical dimers by H-1-NMR. Proteins 17, 297–309.
O’Donoghue, S. I., Junius, F. K., and King, G. F. (1993) Determination of the structure of symmetrical coiled-coil proteins from NMR dta—application of the leucine-zipper proteins Jun and Gcn4. Protein Eng. 6, 557–564.
O’Donoghue, S. I., Chang, X. Q., Abseher, R., Nilges, M., and Led, J. J. (2000) Unraveling the symmetry ambiguity in a hexamer: calculation of the R-6 human insulin structure. J. Biomol. NMR 16, 93–108.
Nilges, M. (1995) Calculation of protein structures with ambiguous distance restraints-automated assignment of ambiguous NOE crosspeaks and disulfide connectivities. J. Mol. Biol. 245, 645–660.
Pervushin, K., Riek, R., Wider, G., and Wuthrich, K. (1997) Attenuated T-2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc. Natl. Acad. Sci. USA 94, 12,366–12,371.
Riek, R., Wider, G., Pervushin, K., and Wuthrich, K. (1999) Polarization transfer by cross-correlated relaxation in solution NMR with very large molecules. Proc. Natl. Acad. Sci. USA 96, 4918–4923.
Linge, J. P., O’Donoghue, S. I., and Nilges, M. (2001) Automated assignment of ambiguous NOEs with ARIA. 339, 71–90.
Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R.W., et al. (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921.
Herrmann, T., Guntert, P., and Wuthrich, K. (2002) Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. J. Mol. Biol. 319, 209–227.
O’Donoghue, S. I., King, G. F., and Nilges, M. (1996) Calculation of symmetric multimer structures from NMR data using a priori knowledge of the monomer structure, co-monomer restraints, and interface mapping: the case of leucine zippers. J. Biomol. NMR 8, 193–206.
Nilges, M. and O’Donoghue, S. I. (1998) Ambiguous NOEs and automated NOE assignment. Prog. Nucl. Magn. Reson. Spectrosc. 32, 107–139.
Means, G. E. and Feeney, R. E. (1995) Reductive alkylation of proteins. Anal. Biochem. 224, 1–16.
Nielsen, P. R., Nietlispach, D., Mott, H. R., Callaghan, J., Bannister, A., Kouzarides, et al. (2002) Structure of the HP1 chromodomain bound to histone H3 methylated at lysine 9. Nature 416, 103–107.
Morreale, A., Venkatesan, M., Mott, H. R., Owen, D., Nietlispach, D., Lowe, P. N., et al. (2000) Structure of Cdc42 bound to the GTPase binding domain of PAK. Nat. Struct. Biol. 7, 384–388.
Brasher, S. V., Smith, B. O., Fogh, R. H., Nietlispach, D., Thiru, A., Nielsen, P. R., et al. (2000) The structure of mouse HP1 suggests a unique mode of single peptide recognition by the shadow chrome domain dimer. EMBO J. 19, 1587–1597.
Grzesiek, S. and Bax, A. (1993) Amino-acid type determination in the sequential assignment procedure of uniformly C-13/N-15-enriched proteins. J. Biomol. NMR 3, 185–204.
Hwang, T. L., Kadkhodaei, M., Mohebbi, A., and Shaka, A. J. (1992) Coherent and incoherent magnetization transfer in the rotating frame. Magn. Reson. Chem. 30, S24–S34.
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Nietlispach, D., Mott, H.R., Stott, K.M., Nielsen, P.R., Thiru, A., Laue, E.D. (2004). Structure Determination of Protein Complexes by NMR. In: Downing, A.K. (eds) Protein NMR Techniques. Methods in Molecular Biology™, vol 278. Humana Press. https://doi.org/10.1385/1-59259-809-9:255
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