Skip to main content
SpringerLink
Log in

Structural determination of biomolecular interfaces by nuclear magnetic resonance of proteins with reduced proton density

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Abstract

Protein interactions are important for understanding many molecular mechanisms underlying cellular processes. So far, interfaces between interacting proteins have been characterized by NMR spectroscopy mostly by using chemical shift perturbations and cross-saturation via intermolecular cross-relaxation. Although powerful, these techniques cannot provide unambiguous estimates of intermolecular distances between interacting proteins. Here, we present an alternative approach, called REDSPRINT (REDduced/Standard PRoton density INTerface identification), to map protein interfaces with greater accuracy by using multiple NMR probes. Our approach is based on monitoring the cross-relaxation from a source protein (or from an arbitrary ligand that need not be a protein) with high proton density to a target protein (or other biomolecule) with low proton density by using isotope-filtered nuclear Overhauser spectroscopy (NOESY). This methodology uses different isotropic labeling for the source and target proteins to identify the source-target interface and also determine the proton density of the source protein at the interface for protein-protein or protein-ligand docking. Simulation indicates significant gains in sensitivity because of the resultant relaxation properties, and the utility of this technique, including a method for direct determination of the protein interface, is demonstrated for two different protein–protein complexes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Bermejo GA, Llinás M (2008) Deuterated protein folds obtained directly from unassigned nuclear overhauser effect data. J Am Chem Soc 130:3797–3805

    Article  Google Scholar 

  • Bottini N, Musumeci L, Alonso A, Rahmouni S, Nika K, Rostamkhani M, Macmurray J, Meloni GF, Lucarelli P, Pellecchia M, Eisenbarth GS, Comings D, Mustelin T (2004) A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genet 36:337–338

    Article  Google Scholar 

  • Breeze AL (2000) Isotope-filtered NMR methods for the study of biomolecular structure and interactions. Prog NMR Spectrosc 36:323–372

    Article  Google Scholar 

  • Burz D, Dutta K, Cowburn D, Shekhtman A (2006a) In-cell NMR for protein-protein interactions (STINT-NMR). Nat Protoc 1:146–152

    Article  Google Scholar 

  • Burz D, Dutta K, Cowburn D, Shekhtman A (2006b) Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR). Nat Methods 3:91–93

    Article  Google Scholar 

  • Clore GM (2000) Accurate and rapid docking of protein-protein complexes on the basis of intermolecular nuclear overhauser enhancement data and dipolar couplings by rigid body minimization. Proc Natl Acad Sci USA 97:9021–9025

    Article  ADS  Google Scholar 

  • Cornilescu G, Marquardt JL, Ottiger M, Bax A (1998) Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid crystalline phase. J Amer Chem Soc 120:6836–6837

    Article  Google Scholar 

  • Das R, Andre I, Shen Y, Wu Y, Lemak A, Bansal S, Arrowsmith CH, Szyperski T, Baker D (2009) Simultaneous prediction of protein folding and docking at high resolution. Proc Natl Acad Sci USA 106:18978–18983

    Article  ADS  Google Scholar 

  • Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293

    Article  Google Scholar 

  • Dominguez C, Boelens R, Bonvin A (2003) HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J Am Chem Soc 125:1731–1737

    Article  Google Scholar 

  • Eichmuller C, Tollinger M, Krautler B, Konrat R (2001) Mapping the ligand binding site at protein side-chains in protein-ligand complexes through NOE difference spectroscopy. J Biomol NMR 20:195–202

    Article  Google Scholar 

  • Ferrage F, Zoonens M, Warschawski DE, Popot JL, Bodenhausen G (2003) Slow diffusion of macromolecular assemblies by a new pulsed field gradient NMR method. J Am Chem Soc 125:2541–2545

    Article  Google Scholar 

  • Ferrage F, Zoonens M, Warschawski DE, Popot JL, Bodenhausen G (2004) Slow diffusion of macromolecular assemblies measured by a new pulsed field gradient NMR method (vol 125, pg 2541, 2003). J Am Chem Soc 126:5654

    Article  Google Scholar 

  • Fiaux J, Bertelsen EB, Horwich AL, Wuthrich K (2002) NMR analysis of a 900 K GroEL GroES complex. Nature 418:207–211

    Article  ADS  Google Scholar 

  • Foster MP, Wuttke DS, Clemens KR, Jahnke W, Radhakrishnan I, Tennant L, Reymond M, Chung J, Wright PE (1998) Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by the three amino-terminal zinc finger domains from transcription factor IIIA. J Biomol NMR 12:51–71

    Article  Google Scholar 

  • Frueh DP, Ito T, Li JS, Wagner G, Glaser SJ, Khaneja N (2005) Sensitivity enhancement in NMR of macromolecules by application of optimal control theory. J Biomol NMR 32:23–30

    Article  Google Scholar 

  • Fushman D, Cowburn D (2003) Characterization of inter-domain orientations in solution using the NMR relaxation approach. In: Krishna NR, Berliner LJ (eds) Biological Magnetic Resonance. Kluwer Academic, New York, pp 53–77

    Google Scholar 

  • Fushman D, Xu R, Cowburn D (1999) Direct determination of changes of interdomain orientation on ligation: use of the orientational dependence of 15N NMR relaxation in Abl SH(32). Biochemistry 38:10225–10230

    Article  Google Scholar 

  • Fushman D, Varadan R, Assfalg M, Walker O (2004) Determining domain orientation in macromolecules by using spin-relaxation and residual dipolar coupling measurements. Prog NMR Spectrosc 44:189–214

    Article  Google Scholar 

  • Gaponenko V, Altieri AS, Li J, Byrd RA (2002) Breaking symmetry in the structure determination of (large) symmetric protein dimers. J Biomol NMR 24:143–148

    Article  Google Scholar 

  • Gardner KH, Kay LE (1998) The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins. Annu Rev Biophys Biomol Struct 27:357–406

    Article  Google Scholar 

  • Ghose R, Shekhtman A, Goger M, Ji H, Cowburn D (2001) A novel, specific interaction involving the Csk SH3 domain and its natural ligand. Nat Struct Biol 8:998–1004

    Article  Google Scholar 

  • Gross JD, Gelev VM, Wagner G (2003) A sensitive and robust method for obtaining intermolecular NOEs between side chains in large protein complexes. J Biomol NMR 25:235–242

    Article  Google Scholar 

  • Guiles RD, Sarma S, Digate RJ, Banville D, Basus VJ, Kuntz ID, Waskell L (1996) Pseudocontact shifts used in the restraint of the solution structures of electron transfer complexes. Nat Struct Biol 3:333–339

    Article  Google Scholar 

  • Hamel DJ, Dahlquist FW (2005) The contact interface of a 120 kD CheA-CheW complex by methyl TROSY interaction spectroscopy. J Am Chem Soc 127:9676–9677

    Article  Google Scholar 

  • Horst R, Fenton WA, Englander SW, Wuthrich K, Horwich AL (2007) Folding trajectories of human dihydrofolate reductase inside the GroEL GroES chaperonin cavity and free in solution. Proc Natl Acad Sci USA 104:20788–20792

    Article  ADS  Google Scholar 

  • Ikura M, Bax A (1992) Isotope-filtered 2D NMR of a protein-peptide complex: study of a skeletal muscule myosin light chain kinase fragment bound to calmodulin. J Am Chem Soc 114:2433–2440

    Article  Google Scholar 

  • Jayalakshmi V, Krishna NR (2002) Complete relaxation and conformational exchange matrix (CORCEMA) analysis of intermolecular saturation transfer effects in reversibly forming ligand-receptor complexes. J Mag Res 155:106–118

    Article  ADS  Google Scholar 

  • Johnson BA, Blevins RA (1994) NMRView: a computer program for the visualization and analysis of NMR data. J Biomol NMR 4:603–614

    Article  Google Scholar 

  • Kiihne SR, Creemers AF, De Grip WJ, Bovee-Geurts PH, Lugtenburg J, De Groot HJ (2005) Selective interface detection: mapping binding site contacts in membrane proteins by NMR spectroscopy. J Am Chem Soc 127:5734–5735

    Article  Google Scholar 

  • Kumar A, Ernst RR, Wüthrich K (1980) A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem Biophys Res Comm 95:1–6

    Article  Google Scholar 

  • Macura S, Ernst RR (1980) Elucidation of cross relaxation in liquids by two-dimensional NMR-spectroscopy. Mol Phys 41:95–117

    Article  ADS  Google Scholar 

  • Matsuda T, Ikegami T, Nakajima N, Yamazaki T, Nakamura H (2004) Model building of a protein-protein complexed structure using saturation transfer and residual dipolar coupling without paired intermolecular NOE. J Biomol NMR 29:325–338

    Article  Google Scholar 

  • Muralidharan V, Dutta K, Cho J, Vila-Perello M, Raleigh DP, Cowburn D, Muir TW (2006) Solution structure and folding characteristics of the C-terminal SH3 domain of c-Crk-II. Biochemistry 45:8874–8884

    Article  Google Scholar 

  • Neuhaus D, Williamson MP (2000) The nuclear overhauser effect in structural and conformational analysis. John Wiley & Sons, New York

    Google Scholar 

  • Ollerenshaw JE, Tugarinov V, Kay LE (2003) Methyl TROSY: explanation and experimental verification. Mag Res Chem 41:843–852

    Article  Google Scholar 

  • Otting G, Wuthrich K (1990) Heteronuclear filters in two-dimensional [1H, 1H]-NMR spectroscopy: combined use with isotope labelling for studies of macromolecular conformation and intermolecular interactions. Q Rev Biophys 23:39–96

    Article  Google Scholar 

  • Pintacuda G, Park AY, Keniry MA, Dixon NE, Otting G (2006) Lanthanide labeling offers fast NMR approach to 3D structure determinations of protein-protein complexes. J Am Chem Soc 128:3696–3702

    Article  Google Scholar 

  • Rain JC, Selig L, De Reuse H, Battaglia V, Reverdy C, Simon S, Lenzen G, Petel F, Wojcik J, Schachter V, Chemama Y, Labigne A, Legrain P (2001) The protein-protein interaction map of Helicobacter pylori. Nature 409:211–215

    Article  ADS  Google Scholar 

  • Ryabov Y, Fushman D (2007) Structural assembly of multidomain proteins and protein complexes guided by the overall rotational diffusion tensor. J Am Chem Soc 129:7894–7902

    Article  Google Scholar 

  • Shekhtman A, Ghose R, Goger M, Cowburn D (2002) NMR structure determination and investigation using a reduced proton (REDPRO) labeling strategy for proteins. FEBS Lett 524:177–182

    Article  Google Scholar 

  • Shimada I, Ueda T, Matsumoto M, Sakakura M, Osawa M, Takeuchi K, Nishida N, Takahashi H (2009) Cross-saturation and transferred cross-saturation experiments. Prog Nucl Magn Reson Spectrosc 54:123–140

    Article  Google Scholar 

  • Staley JP, Kim PS (1994) Formation of a native-like subdomain in a partially folded intermediate of bovine pancreatic trypsin inhibitor. Protein Sci 3:1822–1832

    Article  Google Scholar 

  • Sui XG, Xu YQ, Giovannelli JL, Ho NT, Ho C, Yang DW (2005) Mapping protein-protein interfaces on the basis of proton density difference. Ang Chem Int Ed 44:5141–5144

    Article  Google Scholar 

  • Swanson KA, Kang RS, Stamenova SD, Hicke L, Radhakrishnan I (2003) Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor downregulation. EMBO J 22:4597–4606

    Article  Google Scholar 

  • Takahashi H, Nakanishi T, Kami K, Arata Y, Shimada I (2000) A novel NMR method for determining the interfaces of large protein-protein complexes. Nat Stru Biol 7:220–223

    Article  Google Scholar 

  • Takahashi H, Miyazawa M, Ina Y, Fukunishi Y, Mizukoshi Y, Nakamura H, Shimada I (2006) Utilization of methyl proton resonances in cross-saturation measurement for determining the interfaces of large protein-protein complexes. J Biomol NMR 34:167–177

    Article  Google Scholar 

  • Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR, Lockshon D, Narayan V, Srinivasan M, Pochart P, Qureshi-Emili A, Li Y, Godwin B, Conover D, Kalbfleisch T, Vijayadamodar G, Yang M, Johnston M, Fields S, Rothberg JM (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403:623–627

    Article  ADS  Google Scholar 

  • Van Dijk ADJ, Boelens R, Bonvin A (2005) Data-driven docking for the study of biomolecular complexes. FEBS J 272:293–312

    Article  Google Scholar 

  • Williams DC Jr, Cai M, Suh JY, Peterkofsky A, Clore GM (2005) Solution NMR structure of the 48-kDa IIαMannose-HPr complex of the Escherichia coli mannose phosphotransferase system. J Biol Chem 280:20775–20784

    Article  Google Scholar 

  • Wolff K, Vendruscolo M, Porto M (2008) Stochastic reconstruction of protein structures from effective connectivity profiles. PMC Biophys 1:5

    Article  Google Scholar 

  • Xu Y, Zheng Y, Fan J, Yang D (2006) A new strategy for structure determination of large proteins in solution without deuteration. Nat Meth 33:931–937

    Article  Google Scholar 

  • Yamazaki T, Forman-Kay JD, Kay LE (1993) 2-Dimensional NMR experiments for correlating 13Cβ and 1H-δ/ε chemical-shifts of aromatic residues in 13C-labeled proteins via scalar couplings. J Am Chem Soc 115:11054–11055

    Article  Google Scholar 

  • You J, Cohen RE, Pickart CM (1999) Construct for high-level expression and low misincorporation of lysine for arginine during expression of pET-encoded eukaryotic proteins in Escherichia coli. Biotechn 27:950–954

    Google Scholar 

  • Zangger K, Oberer M, Keller W, Sterk H (2003) X-filtering for a range of coupling constants: application to the detection of intermolecular NOEs. J Mag Res 160:97–106

    Article  ADS  Google Scholar 

  • Zwahlen C, Legault P, Vincent SJF, Greenblatt J, Konrat R, Kay LE (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

    Article  Google Scholar 

Download references

Acknowledgments

We thank Geoffrey Bodenhausen for his careful reading of a version of the manuscript. Supported by grant GM 47021 from the National Institute of Health. AS was supported by grant 1-06-CD-23 from the American Diabetes Association. FF thanks the French Ministry of Foreign Affairs for a Lavoisier fellowship.

Author information

Author notes

  1. Fabien Ferrage

    Present address: Département de chimie, CNRS–UMR 7203 and Ecole normale supérieure, 24, rue Lhomond, 75231, Paris Cedex 05, France

  2. Alexander Shekhtman

    Present address: State University of New York at Albany, 1400 Washington Ave., Albany, NY, 12222, USA

Authors and Affiliations

  1. New York Structural Biology Center, 89 Convent Avenue, New York, NY, 10027, USA

    Fabien Ferrage, Kaushik Dutta, Alexander Shekhtman & David Cowburn

Authors
  1. Fabien Ferrage
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kaushik Dutta
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alexander Shekhtman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David Cowburn
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David Cowburn.

Electronic supplementary material

Below is the link to the electronic supplementary material.

(DOCX 11 kb)

(PDF 5295 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ferrage, F., Dutta, K., Shekhtman, A. et al. Structural determination of biomolecular interfaces by nuclear magnetic resonance of proteins with reduced proton density. J Biomol NMR 47, 41–54 (2010). https://doi.org/10.1007/s10858-010-9409-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-010-9409-9

Keywords

Search

Navigation