Abstract
Combinations of experimentally derived data from nuclear magnetic resonance spectroscopy and analyses of molecular dynamics trajectories increasingly allow us to obtain a detailed description of the molecular mechanisms by which proteins function in signal transduction. This chapter provides an introduction into these two methodologies, illustrated by example of a small GTPase–effector interaction. It is increasingly becoming clear that new insights are provided by the combination of experimental and computational methods. Understanding the structural and protein dynamical contributions to allostery will be useful for the engineering of new binding interfaces and protein functions, as well as for the design/in silico screening of chemical agents that can manipulate the function of small GTPase–protein interactions in diseases such as cancer.
Key words
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Swain, J. F. and Gierasch, L. M. (2006) The changing landscape of protein allostery. Curr Opin Struct Biol. 16, 102–8.
Gunasekaran, K., Ma, B., and Nussinov, R. (2004) Is allostery an intrinsic property of all dynamic proteins? Proteins 57, 433–443.
Smock, R. G. and Gierasch, L. M. (2009) Sending signals dynamically. Science 324, 198–203.
Cooper, A. and Dryden, D. T. (1984) Allostery without conformational change. A plausible model. Eur Biophys J. 11, 103–9.
Gianni, S., Walma, T., Arcovito, A., Calosci, N., Bellelli, A., et al. (2006) Demonstration of long-range interactions in a PDZ domain by NMR, kinetics, and protein engineering. Structure 14, 1801–9.
Riek, R., Fiaux, J., Bertelsen, E.B., Horwich, A.L. and Wüthrich, K. (2002) Solution NMR techniques for large molecular and supramolecular structures. J. Am. Chem. Soc. 124, 12144–12153.
Kay, L. E. (2005) NMR studies of protein structure and dynamics. J. Magn. Reson. 173, 193–207.
Palmer III, A.G. (2001) NMR probes of molecular dynamics: overview and comparison with other techniques. Annu. Rev. Biophys. Biomol. Struct. 30, 129–55.
Nirmala, N. R. and Wagner, G. (1988) Measurement of 13C relaxation times in proteins by two-dimensional heteronuclear 1H-13C correlation spectroscopy. J. Am. Chem. Soc. 110, 7557–7558.
Kay, L. E., Torchia, D. A. and Bax, A. (1989) Backbone dynamics of proteins as studied by nitrogen-15 inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry 28, 8972–8979.
Clore, G. M., Driscoll, P. C., Wingfield, P. T. and Gronenborn, A. M. (1990) Analysis of the backbone dynamics of interleukin-1 beta using two-dimensional inverse detected heteronuclear nitrogen-15-proton NMR spectroscopy. Biochemistry 29, 7387–7401.
Palmer III, A. G., Rance, M. and Wright, P. E. (1991) Intramolecular motions of a zinc finger DNA-binding domain from Xfin characterized by proton-detected natural abundance carbon-13 heteronuclear NMR spectroscopy. J. Am. Chem. Soc. 113, 4371–4380.
Peng, J. W. and Wagner, G. (1992) Mapping of the spectral densities of nitrogen-hydrogen bond motions in Eglin c using heteronuclear relaxation experiments. Biochemistry 31, 8571–8586.
Buck, M., Boyd, J., Redfield, C., MacKenzie, D. A., Jeenes, D. J., et al. (1995) Structural determinants of protein dynamics: Analysis of 15N relaxation measurements for mainchain and sidechain nuclei of hen egg-white lysozyme. Biochemistry 34, 4041–4055.
Palmer, A. G., Hochstrasser, R. A., Millar, D. P., Rance, M., Wright, P. E. (1993) Characterization of amino-acid sidechain dynamics in a zinc-finger peptide using C-13 NMR spectroscopy and time-resolved fluorescence spectroscopy. J. Am. Chem. Soc. 115, 6333–45.
Nicholson, L. K., Kay, L. E., Baldisseri, D. M., Arango, J., Young, P. E., et al. (1992) Dynamics of methyl groups in proteins as studied by proton-detected C-13 NMR spectroscopy – application to the leucine residues of staphylococcal nuclease. Biochemistry 31, 5253–63.
Muhandriram, D. R., Yamazaki, T., Sykes, B. D., and Kay, L. E. (1995) Measurement of H-2 T1 and T1p relaxation times in uniformly C-13 labeled and fractionally H-2 labeled proteins in solution. J. Am. Chem. Soc. 117, 11536–44.
MaCammon, J. A., Gelin, B. R., and Karplus, M. (1977) Dynamics of folded proteins. Nature 267, 585–590.
Klepeis, J. L., Lindorff-Larsen, K., Dror, R. O., and Shaw, D. E. (2009) Long-timescale molecular dynamics simulations of protein structure and function. Curr Opin Struct Biol. 19, 120–7.
Vetter, I. R. and Wittinghofer, A. (2001) The guanine nucleotide-binding switch in three dimensions. Science. 294, 1299–304.
Spoerner, M., Herrmann, C., Vetter, I. R., Kalbitzer, H. R., and Wittinghofer, A. (2001) Dynamic properties of the Ras switch I region and its importance for binding to effectors. Proc Natl Acad Sci 98, 4944–9.
Ford, B., Skowronek, K., Boykevisch, S., Bar-Sagi, D., and Nassar, N. (2005) Structure of the G60A mutant of Ras: implications for the dominant negative effect. J. Biol Chem. 280, 25697–705.
Heo, W. D. and Meyer, T. (2003) Switch-of-function mutants based on morphology classification of Ras superfamily small GTPases. Cell. 113, 315–28.
Hatley, M. E., Lockless, S.W., Gibson, S.K., Gilman, A.G., and Ranganathan, R. (2003) Allosteric determinants in guanine nucleotide-binding proteins. Proc Natl Acad Sci 100, 14445–50.
Abankwa, D., Hanzal-Bayer, M., Ariotti, N., Plowman, S. J., Gorfe, A. A., et al. (2008) A novel switch region regulates H-ras membrane orientation and signal output. EMBO J. 27, 727–35.
Edreira, M. M., Li, S., Hochbaum, D., Wong, S., Gorfe, A. A., et al. (2009) Phosphorylation-induced conformational changes in Rap1b: allosteric effects on switch domains and effector loop. J Biol Chem. 284, 27480–6.
Modha, R., Campbell, L. J., Nietlispach, D., Buhecha, H. R., Owen, D., et al. (2008) The Rac1 polybasic region is required for interaction with its effector PRK1. J Biol Chem. 283, 1492–500.
Pasqualato, S., Renault, L., and Cherfils, J. (2002) Arf, Arl, Arp and Sar proteins: a family of GTP-binding proteins with a structural device for ‘front-back’ communication. EMBO Rep. 3, 1035–41.
Buhrman, G., Holzapfel, G., Fetics, S., and Mattos, C. (2010) Allosteric modulation of Ras positions Q61 for a direct role in catalysis. Proc Natl Acad Sci USA. 107, 4931–6.
Fenton, A. W. (2008) Allostery: an illustrated definition for the ‘second secret of life’. Trends Biochem Sci. 33, 420–5.
Farrow, N. A., Muhandiram, R., Singer, A. U., Pascal, S. M., Kay, C. M., et al. (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33, 5984–6003.
Lipari, G. and Szabo, A. (1982) Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity. J. Am. Chem. Soc. 104, 4546–4559.
Clore, G. M., Szabo, A., Bax, A., Kay, L. E., Driscoll, P. C. et al. (1990) Deviations from the simple two parameter model free approach to the interpretation of 15N nuclear magnetic relaxation of proteins. J. Am. Chem. Soc. 112, 4989–4991.
Hall, J. B. and Fushman, D. (2003) Characterization of the overall and local dynamics of a protein with intermediate rotational anisotropy: differentiating between conformational exchange and anisotropic diffusion in the B3 domain of protein G. J. Biomol. NMR 27, 261–275.
Walker, O., Varadan, R. and Fushman, D. (2004) Efficient and accurate determination of the overall rotational diffusion tensor of a molecule from 15N relaxation data using computer program ROTDIF. J. Magn. Reson. 168, 336–345.
Bouguet-Bonnet, S. and Buck, M. (2008) Compensatory and long-range changes in ps-ns mainchain dynamics upon complex formation. 15N relaxation analysis of the free and bound states of the ubiquitin-like domain of human plexin-B1 and the small GTPase Rac1. J. Mol. Biol. 377, 1474–1487.
Akerud, T., Thulin, E., Van Etten, R. L. and Akke, M. (2002) Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: a model for changes in dynamics upon target binding. J. Mol. Biol. 322, 137–52.
Spyracopoulos, L., Lewis, M. J. and Saltibus, L. F. (2005) Main chain and side chain dynamics of the ubiquitin conjugating enzyme variant human Mms2 in the free and ubiquitin-bound States. Biochemistry 44, 8770–81.
Boehr, D. D., McElheny, D., Dyson, H. J. and Wright, P. E. (2006) The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313, 1638–42.
Finerty, P. J., Mittermaier, A. K., Muhandiram, R., Kay, L. E. and Forman-Kay, J. D. (2005) NMR dynamics-derived insights into the binding properties of a peptide interacting with an SH2 domain. Biochemistry 44, 694–703.
Tong, Y., Bagheri-Hamaneh, M., Penachioni, J. Y., Hota, P.K., Kim, S., et al. (2009) Structure and Function of the Intracellular Region of the Plexin-B1 Transmembrane Receptor. J. Biol. Chem. 284, 35962–35972.
Ma, B., Kumar, S., Tsai, C. J., and Nussinov, R. (1999) Folding funnels and binding mechanisms. Protein Eng. 12, 713–20.
Tong, Y, Chugha, P., Hota, PK., Li, M., Alviani, RS., et al. (2007) Binding of Rac1, Rnd1 and RhoD to a novel Rho GTPase interaction motif destabilizes dimerization of the plexin-B1 Effector domain. J.Biol.Chem. 282, 37215–37224.
Henzler-Wildman, K. A., Lei, M., Thai, V., Kerns, S. J., Karplus, M., et al. (2007) A hierarchy of timescales in protein dynamics is linked to enzyme catalysis. Nature 450, 913–6.
Gizachew, D. and Oswald, R. E. (2001) Concerted motion of a protein–peptide complex: backbone dynamics studies of a 15N-labeled peptide derived from P21-activated kinase bound to Cdc42Hs, GMPPCP. Biochemistry 40, 14368–14375.
Tong, Y., Hota, P. K., Bagheri Hamaneh, M., and Buck, M. (2008) Insights into Oncogenic Mutations of plexin-B1 based on the Solution Structure of the Rho GTPase Binding Domain. Structure 16, 246–258.
Wang, H., Hota, P. K., Tong, Y., Li, B. Shen, L., et al. (2011) Structural Basis of Rho GTPase Rnd1 Binding to Plexin RBDs J. Biol. Chem. 286, 26093–2610.
James, C. P., Rosemary, B., Wei, W., James, G., Emad, T., et al. (2005) Scalable molecular dynamics with NAMD. Journal of Computational Chemistry 26, 1781–1802.
MacKerell, A. D., Jr, Bashford, D., Bellott, M., Dunbrack, R. I., Jr, Evanseck, J., et al. (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586–3616.
Buck, M., Bouguet-Bonnet, S., Pastor, R.W., and MacKerell, A. D. (2006) Importance of the CMAP correction to the CHARMM22 protein force field: Dynamics of Hen Lysozyme. Biophys.J. 90, L36-L39.
Zerbetto, M., Polimeno, A., and Meirovitch, E. (2009) General theoretical/computational tool for interpreting NMR spin relaxation in proteins. J Phys Chem B 113, 13613–25.
Best, R. B., Clarke, J., and Karplus, M. (2005) What contributions to protein side-chain dynamics are probed by NMR experiments? A molecular dynamics simulation analysis. J Mol Biol. 349, 185–203.
Hota, P. and Buck, M. (2009) Thermodynamic characterization of two homologous protein complexes: Association of the semaphorin receptor plexin-B1 Rho GTPase binding domain with Rnd1 and active Rac1. Protein Science 18, 1060–1071.
Ichiye, T. and Karplus, M. (1991) Collective motions in proteins: a covariance analysis of atomic fluctuations in molecular dynamics and normal mode simulations. Proteins 11, 205–217.
Kong, Y. and Karplus, M. (2009) Signaling pathways of PDZ2 domain: a molecular dynamics interaction correlation analysis. Proteins 74, 145–54.
Gorfe, A.A., Grant, B.J., and McCammon, J. A. (2008) Mapping the nucleotide and isoform-dependent structural and dynamical features of Ras proteins. Structure 16, 885–96.
Lukman, S., Grant, B. J., Gorfe, A. A., Grant, G. H., and McCammon, J. A. (2010) The Distinct Conformational Dynamics of K-Ras and H-Ras A59G. PLoS Comput Biol. 6, pii: e1000922.
Gohlke, H., Kuhn, L. A., and Case, D. A. (2004) Change in protein flexibility upon complex formation: analysis of Ras-Raf using molecular dynamics and a molecular framework approach. Proteins 56, 322–37.
Lee, J., Natarajan, M., Nashine, V. C., Socolich, M., Vo, T., et al. (2008) Surface sites for engineering allosteric control in proteins. Science 322, 438–42.
Chennubhotla, C. and Bahar, I. (2007) Signal propagation in proteins and relation to equilibrium fluctuations. PLoS Comput Biol. 3, 1716–26.
Moritsugu, K., Kurkal-Siebert, V., and Smith, J. C. (2009) REACH coarse-grained normal mode analysis of protein dimer interaction dynamics. Biophys J. 97, 1158–67.
Dobbins, S. E., Lesk, V. I., and Sternberg, M. J. (2008) Insights into protein flexibility: The relationship between normal modes and conformational change upon protein–protein docking. Proc Natl Acad Sci USA. 105, 10390–5.
Lange, O. F., Lakomek, N. A., Farès, C., Schröder, G. F., Walter, K. F., et al. (2008) Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science. 320, 1471–5.
O’Connor, C. and Kovrigin, E. L. Global conformational dynamics in ras. Biochemistry 47, 10244–6.
Korzhnev, D. M. and Kay, L. E. (2008) Probing invisible, low-populated States of protein molecules by relaxation dispersion NMR spectroscopy: an application to protein folding. Acc Chem Res. 41, 442–51.
Palmer, A. G. 3rd and Massi, F. (2006) Characterization of the dynamics of biomacromolecules using rotating-frame spin relaxation NMR spectroscopy. Chem Rev. 106, 1700–19.
Ho, B. K. and Agard, D. A. (2010) Conserved tertiary couplings stabilize elements in the PDZ fold, leading to characteristic patterns of domain conformational flexibility. Protein Sci. 19, 398–411.
Acknowledgments
We thank Shufen Cao, Dr. Prasanta K. Hota, and other members of the Buck laboratory for insightful discussion, as well as Dr. Aron Fenton for editorial help. Some of the molecular dynamics calculations were carried out by Dr. Mehdi Bagheri Hamaneh at the Case Western Reserve High Performance Cluster and at Lonestar (Austin, TX, USA) via a TeraGrid award (to M. B.). The work of M. B. is supported by the NIH grants 1R01GM092851, 1K02HL084384, and 1R01GM73071, which included an ARRA supplement.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Zhang, L., Bouguet-Bonnet, S., Buck, M. (2012). Combining NMR and Molecular Dynamics Studies for Insights into the Allostery of Small GTPase–Protein Interactions. In: Fenton, A. (eds) Allostery. Methods in Molecular Biology, vol 796. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-334-9_13
Download citation
DOI: https://doi.org/10.1007/978-1-61779-334-9_13
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-61779-333-2
Online ISBN: 978-1-61779-334-9
eBook Packages: Springer Protocols