Skip to main content
SpringerLink
Log in

Determination of ligand binding modes in weak protein–ligand complexes using sparse NMR data

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

Abstract

We describe a general approach to determine the binding pose of small molecules in weakly bound protein–ligand complexes by deriving distance constraints between the ligand and methyl groups from all methyl-containing residues of the protein. We demonstrate that using a single sample, which can be prepared without the use of expensive precursors, it is possible to generate high-resolution data rapidly and obtain the resonance assignments of Ile, Leu, Val, Ala and Thr methyl groups using triple resonance scalar correlation data. The same sample may be used to obtain Met εCH3 assignments using NOESY-based methods, although the superior sensitivity of NOESY using [U-13C,15N]-labeled protein makes the use of this second sample more efficient. We describe a structural model for a weakly binding ligand bound to its target protein, DsbA, derived from intermolecular methyl-to-ligand nuclear Overhauser enhancements, and demonstrate that the ability to assign all methyl resonances in the spectrum is essential to derive an accurate model of the structure. Once the methyl assignments have been obtained, this approach provides a rapid means to generate structural models for weakly bound protein–ligand complexes. Such weak complexes are often found at the beginning of programs of fragment based drug design and can be challenging to characterize using X-ray crystallography.

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

  • Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr Sect D Biol Crystallogr 66:213–221. doi:10.1107/S0907444909052925

    Article  Google Scholar 

  • Adams LA, Sharma P, Mohanty B, Ilyichova OV, Mulcair MD, Williams ML, Gleeson EC, Totsika M, Doak BC, Caria S, Rimmer K, Horne J, Shouldice SR, Vazirani M, Headey SJ, Plumb BR, Martin JL, Heras B, Simpson JS, Scanlon MJ (2015) Application of fragment-based screening to the design of inhibitors of Escherichia coli DsbA. Angew Chem Int Ed Engl 54:2179–2184. doi:10.1002/anie.201410341

    Article  Google Scholar 

  • Ayala I, Sounier R, Use N, Gans P, Boisbouvier J (2009) An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein. J Biomol NMR 43:111–119. doi:10.1007/S10858-008-9294-7

    Article  Google Scholar 

  • Battye TG, Kontogiannis L, Johnson O, Powell HR, Leslie AG (2011) iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr Sect D Biol Crystallogr 67:271–281. doi:10.1107/S0907444910048675

    Article  Google Scholar 

  • Breeze AL (2000) Isotope-filtered NMR methods for the study of biomolecular structure and interactions. Prog Nucl Magn Reson Spectrosc 36:323–372. doi:10.1016/S0079-6565(00)00020-0

    Article  Google Scholar 

  • Chao FA, Shi L, Masterson LR, Veglia G (2012) FLAMEnGO: a fuzzy logic approach for methyl group assignment using NOESY and paramagnetic relaxation enhancement data. J Magn Reson 214:103–110. doi:10.1016/J.Jmr.2011.10.008

    Article  ADS  Google Scholar 

  • Cowieson NP, Aragao D, Clift M, Ericsson DJ, Gee C, Harrop SJ, Mudie N, Panjikar S, Price JR, Riboldi-Tunnicliffe A, Williamson R, Caradoc-Davies T (2015) MX1: a bending-magnet crystallography beamline serving both chemical and macromolecular crystallography communities at the Australian Synchrotron. J Synchrotron Radiat 22:187–190. doi:10.1107/S1600577514021717

    Article  Google Scholar 

  • Danley DE (2006) Crystallization to obtain protein–ligand complexes for structure-aided drug design. Acta Crystallogr Sect D Biol Crystallogr 62:569–575. doi:10.1107/S0907444906012601

    Article  Google Scholar 

  • De Vries SJ, van Dijk M, Bonvin AMJJ (2010) The HADDOCK web server for data-driven biomolecular docking. Nat Protoc 5:883–897. doi:10.1038/Nprot.2010.32

    Article  Google Scholar 

  • Doak BC, Morton CJ, Simpson JS, Scanlon MJ (2013) Design and evaluation of the performance of an NMR screening fragment library. Aust J Chem 66:1465–1472

    Article  Google Scholar 

  • Dominguez C, Boelens R, Bonvin AMJJ (2003) HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J Am Chem Soc 125:1731–1737. doi:10.1021/Ja026939x

    Article  Google Scholar 

  • Edfeldt FNB, Folmer RHA, Breeze AL (2011) Fragment screening to predict druggability (ligandability) and lead discovery success. Drug Discov Today 16:284–287. doi:10.1016/J.Drudis.2011.02.002

    Article  Google Scholar 

  • Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr Sect D Biol Crystallogr 60:2126–2132. doi:10.1107/S0907444904019158

    Article  Google Scholar 

  • Erlanson DA, Fesik SW, Hubbard RE, Jahnke W, Jhoti H (2016) Twenty years on: the impact of fragments on drug discovery. Nat Rev Drug Discov 15:605–619. doi:10.1038/nrd.2016.109

    Article  Google Scholar 

  • Evans P (2006) Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr 62:72–82. doi:10.1107/S0907444905036693

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Fielding L (2003) NMR methods for the determination of protein–ligand dissociation constants. Curr Top Med Chem 3:39–53. doi:10.2174/1568026033392705

    Article  MathSciNet  Google Scholar 

  • Gardner KH, Kay LE (1997) Production and incorporation of N-15, C-13, H-2 (H-1-delta 1 methyl) isoleucine into proteins for multidimensional NMR studies. J Am Chem Soc 119:7599–7600. doi:10.1021/Ja9706514

    Article  Google Scholar 

  • Gossert AD, Hiller S, Fernandez C (2011) Automated NMR resonance assignment of large proteins for protein–ligand interaction studies. J Am Chem Soc 133:210–213. doi:10.1021/ja108383x

    Article  Google Scholar 

  • Goto NK, Gardner KH, Mueller GA, Willis RC, Kay LE (1999) A robust and cost-effective method for the production of Val, Leu, Ile (delta 1) methyl-protonated N-15-, C-13-, H-2-labeled proteins. J Biomol NMR 13:369–374. doi:10.1023/A:1008393201236

    Article  Google Scholar 

  • Guan JY, Keizers PHJ, Liu WM, Lohr F, Skinner SP, Heeneman EA, Schwalbe H, Ubbink M, Siegal G (2013) Small-molecule binding sites on proteins established by paramagnetic NMR spectroscopy. J Am Chem Soc 135:5859–5868. doi:10.1021/Ja401323m

    Article  Google Scholar 

  • Guddat LW, Bardwell JC, Glockshuber R, Huber-Wunderlich M, Zander T, Martin JL (1997) Structural analysis of three His32 mutants of DsbA: support for an electrostatic role of His32 in DsbA stability. Protein Sci 6:1893–1900. doi:10.1002/pro.5560060910

    Article  Google Scholar 

  • Guntert P, Mumenthaler C, Wuthrich K (1997) Torsion angle dynamics for NMR structure calculation with the new program DYANA. J Mol Biol 273:283–298. doi:10.1006/jmbi.1997.1284

    Article  Google Scholar 

  • Guo C, Tugarinov V (2010) Selective 1H–13C NMR spectroscopy of methyl groups in residually protonated samples of large proteins. J Biomol NMR 46:127–133. doi:10.1007/s10858-009-9393-0

    Article  Google Scholar 

  • Hajduk PJ (2006) Puzzling through fragment-based drug design. Nat Chem Biol 2:658–659. doi:10.1038/nchembio1206-658

    Article  Google Scholar 

  • Hajduk PJ, Greer J (2007) A decade of fragment-based drug design: strategic advances and lessons learned. Nat Rev Drug Discov 6:211–219. doi:10.1038/nrd2220

    Article  Google Scholar 

  • Hajduk PJ, Mack JC, Olejniczak ET, Park C, Dandliker PJ, Beutel BA (2004) SOS-NMR: a saturation transfer NMR-based method for determining the structures of protein–ligand complexes. J Am Chem Soc 126:2390–2398. doi:10.1021/Ja039480v

    Article  Google Scholar 

  • Hann MM, Keseru GM (2012) Finding the sweet spot: the role of nature and nurture in medicinal chemistry. Nat Rev Drug Discov 11:355–365. doi:10.1038/nrd3701

    Article  Google Scholar 

  • Hartshorn MJ, Murray CW, Cleasby A, Frederickson M, Tickle IJ, Jhoti H (2005) Fragment-based lead discovery using X-ray crystallography. J Med Chem 48:403–413. doi:10.1021/jm0495778

    Article  Google Scholar 

  • Hoch JC, Maciejewski M, Mobli M, Schuyler AD, Stern AS (2012) Nonuniform sampling in multidimensional NMR, vol 1. Wiley, New York

    Google Scholar 

  • Hopkins AL, Groom CR, Alex A (2004) Ligand efficiency: a useful metric for lead selection. Drug Discov Today 9:430–431. doi:10.1016/S1359-6446(04)03069-7

    Article  Google Scholar 

  • Hopkins AL, Keseru GM, Leeson PD, Rees DC, Reynolds CH (2014) The role of ligand efficiency metrics in drug discovery. Nat Rev Drug Discov 13:105–121. doi:10.1038/nrd4163

    Article  Google Scholar 

  • Hyberts SG, Arthanari H, Wagner G (2012) Applications of non-uniform sampling and processing. Top Curr Chem 316:125–148. doi:10.1007/128_2011_187

    Article  Google Scholar 

  • Hyberts SG, Robson SA, Wagner G (2013) Exploring signal-to-noise ratio and sensitivity in non-uniformly sampled multi-dimensional NMR spectra. J Biomol NMR 55:167–178. doi:10.1007/S10858-012-9698-2

    Article  Google Scholar 

  • Isaacson RL, Simpson PJ, Liu M, Cota E, Zhang X, Freemont P, Matthews S (2007) A new labeling method for methyl transverse relaxation-optimized spectroscopy NMR spectra of alanine residues. J Am Chem Soc 129:15428. doi:10.1021/Ja0761784

    Article  Google Scholar 

  • Ishima R (2015) Protein-inhibitor interaction studies using NMR. Appl NMR Spectrosc 1:143–181. doi:10.2174/9781608059621115010007

    Article  Google Scholar 

  • Iwahara J, Wojciak JM, Clubb RT (2001) Improved NMR spectra of a protein-DNA complex through rational mutagenesis and the application of a sensitivity optimized isotope-filtered NOESY experiment. J Biomol NMR 19:231–241. doi:10.1023/A:1011296112710

    Article  Google Scholar 

  • Joosten RP, Long F, Murshudov GN, Perrakis A (2014) The PDB_REDO server for macromolecular structure model optimization. IUCrJ 1:213–220. doi:10.1107/S2052252514009324

    Article  Google Scholar 

  • Keseru GM, Erlanson DA, Ferenczy GG, Hann MM, Murray CW, Pickett SD (2016) Design principles for fragment libraries: maximizing the value of learnings from Pharma Fragment-Based Drug Discovery (FBDD) programs for use in academia. J Med Chem. doi:10.1021/acs.jmedchem.6b00197

    Google Scholar 

  • Koradi R, Billeter M, Wuthrich K (1996) MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graph 14(51–55):29–32

    Google Scholar 

  • McPhillips TM, McPhillips SE, Chiu HJ, Cohen AE, Deacon AM, Ellis PJ, Garman E, Gonzalez A, Sauter NK, Phizackerley RP, Soltis SM, Kuhn P (2002) Blu-Ice and the distributed control system: software for data acquisition and instrument control at macromolecular crystallography beamlines. J Synchrotron Radiat 9:401–406

    Article  Google Scholar 

  • Mobli M, Maciejewski MW, Gryk MR, Hoch JC (2007) An automated tool for maximum entropy reconstruction of biomolecular NMR spectra. Nat Methods 4:467–468. doi:10.1038/Nmeth0607-467

    Article  Google Scholar 

  • Mobli M, Stern AS, Bermel W, King GF, Hoch JC (2010) A non-uniformly sampled 4D HCC(CO)NH-TOCSY experiment processed using maximum entropy for rapid protein sidechain assignment. J Magn Reson 204:160–164. doi:10.1016/J.Jmr.2010.02.012

    Article  ADS  Google Scholar 

  • Mohanty B, Serrano P, Pedrini B, Jaudzems K, Geralt M, Horst R, Herrmann T, Elsliger MA, Wilson IA, Wuthrich K (2010) Comparison of NMR and crystal structures for the proteins TM1112 and TM1367. Acta Crystallogr Sect F Struct Biol Cryst Commun 66:1381–1392. doi:10.1107/S1744309110020956

    Article  Google Scholar 

  • Mund M, Overbeck JH, Ullmann J, Sprangers R (2013) LEGO-NMR spectroscopy: a method to visualize individual subunits in large heteromeric complexes. Angew Chem Int Ed 52:11401–11405. doi:10.1002/anie.201304914

    Article  Google Scholar 

  • Murray CW, Rees DC (2009) The rise of fragment-based drug discovery. Nat Chem 1:187–192. doi:10.1038/Nchem.217

    Article  Google Scholar 

  • Murray CW, Verdonk ML, Rees DC (2012) Experiences in fragment-based drug discovery. Trends Pharmacol Sci 33:224–232. doi:10.1016/j.tips.2012.02.006

    Article  Google Scholar 

  • Neri D, Szyperski T, Otting G, Senn H, Wuthrich K (1989) Stereospecific nuclear magnetic-resonance assignments of the methyl-groups of valine and leucine in the DNA-binding domain of the 434-repressor by biosynthetically directed fractional C-13 labeling. Biochemistry 28:7510–7516. doi:10.1021/Bi00445a003

    Article  Google Scholar 

  • Ollerenshaw JE, Tugarinov V, Skrynnikov NR, Kay LE (2005) Comparison of 13CH3, 13CH2D, and 13CHD2 methyl labeling strategies in proteins. J Biomol NMR 33:25–41. doi:10.1007/s10858-005-2614-2

    Article  Google Scholar 

  • Otten R, Chu B, Krewulak KD, Vogel HJ, Mulder FAA (2010) Comprehensive and cost-effective NMR spectroscopy of methyl groups in large proteins. J Am Chem Soc 132:2952–2960. doi:10.1021/Ja907706a

    Article  Google Scholar 

  • Robertson IM, Spyracopoulos L, Sykes BD (2009) The evaluation of isotope editing and filtering for protein–ligand interaction elucidation by NMR. In: Proceedings of the NATO advanced study institute on biophysics and the challenges of emerging threats. Springer, Berlin, pp 101–119

  • Rovnyak D, Frueh DP, Sastry M, Sun ZY, Stern AS, Hoch JC, Wagner G (2004) Accelerated acquisition of high resolution triple-resonance spectra using non-uniform sampling and maximum entropy reconstruction. J Magn Reson 170:15–21. doi:10.1016/j.jmr.2004.05.016

    Article  ADS  Google Scholar 

  • Ruschak AM, Velyvis A, Kay LE (2010) A simple strategy for C-13, H-1 labeling at the Ile-gamma 2 methyl position in highly deuterated proteins. J Biomol NMR 48:129–135. doi:10.1007/S10858-010-9449-1

    Article  Google Scholar 

  • Schmitz C, Stanton-Cook MJ, Su XC, Otting G, Huber T (2008) Numbat: an interactive software tool for fitting Deltachi-tensors to molecular coordinates using pseudocontact shifts. J Biomol NMR 41:179–189. doi:10.1007/s10858-008-9249-z

    Article  Google Scholar 

  • Schuttelkopf AW, van Aalten DMF (2004) PRODRG: a tool for high-throughput crystallography of protein–ligand complexes. Acta Crystallogr Sect D Biol Crystallogr 60:1355–1363. doi:10.1107/S0907444904011679

    Article  Google Scholar 

  • Shah DM, Ab E, Diercks T, Hass MAS, van Nuland NAJ, Siegal G (2012) Rapid protein-ligand costructures from sparse NOE data. J Med Chem 55:10786–10790. doi:10.1021/Jm301396d

    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 

  • Sinha K, Jen-Jacobson L, Rule GS (2011) Specific labeling of threonine methyl groups for NMR studies of protein–nucleic acid complexes. Biochemistry 50:10189–10191. doi:10.1021/Bi201496d

    Article  Google Scholar 

  • Sprangers R, Velyvis A, Kay LE (2007) Solution NMR of supramolecular complexes: providing new insights into function. Nat Methods 4:697–703. doi:10.1038/nmeth1080

    Article  Google Scholar 

  • Stockman BJ, Dalvit C (2002) NMR screening techniques in drug discovery and drug design. Prog Nucl Magn Reson Spectrosc 41:187–231. doi:10.1016/s0079-6565(02)00049-3

    Article  Google Scholar 

  • Stoffregen MC, Schwer MM, Renschler FA, Wiesner S (2012) Methionine scanning as an NMR tool for detecting and analyzing biomolecular interaction surfaces. Structure 20:573–581. doi:10.1016/J.Str.2012.02.012

    Article  Google Scholar 

  • Tugarinov V, Kay LE (2003) Ile, Leu, and Val methyl assignments of the 723-residue malate synthase G using a new labeling strategy and novel NMR methods. J Am Chem Soc 125:13868–13878. doi:10.1021/Ja030345s

    Article  Google Scholar 

  • Tugarinov V, Kay LE (2004) An isotope labeling strategy for methyl TROSY spectroscopy. J Biomol NMR 28:165–172. doi:10.1023/B:Jnmr.0000013824.93994.1f

    Article  Google Scholar 

  • van Dijk AD, Boelens R, Bonvin AM (2005) Data-driven docking for the study of biomolecular complexes. FEBS J 272:293–312. doi:10.1111/j.1742-4658.2004.04473.x

    Article  Google Scholar 

  • Velyvis A, Ruschak AM, Kay LE (2012) An economical method for production of (2)H, (13)CH3-threonine for solution NMR studies of large protein complexes: application to the 670 kDa proteasome. PLoS One 7:e43725. doi:10.1371/journal.pone.0043725

    Article  ADS  Google Scholar 

  • Venditti V, Fawzi NL, Clore GM (2011) Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methyl-methyl nuclear overhauser enhancement spectroscopy. J Biomol NMR 51:319–328. doi:10.1007/S10858-011-9559-4

    Article  Google Scholar 

  • Wallach I, Lilien R (2009) The protein-small-molecule database, a non-redundant structural resource for the analysis of protein–ligand binding. Bioinformatics 25:615–620. doi:10.1093/Bioinformatics/Btp035

    Article  Google Scholar 

  • Ziarek JJ, Peterson FC, Lytle BL, Volkman BF (2011) Binding site identification and structure determination of protein–ligand complexes by NMR a semiautomated approach. Methods Enzymol 493:241–275. doi:10.1016/B978-0-12-381274-2.00010-8

    Article  Google Scholar 

Download references

Acknowledgments

We thank A/Prof. Frans Mulder (Aarhus) for providing the original C-TOCSY-CHD2 pulse sequence. B.M. thanks Prof. A. M. J. J. Bonvin for his helpful comments and suggestions on HADDOCK docking of protein–small molecule complexes, and Dr. Hiromasa Yagi for helpful discussions on PCS and PRE measurements. This work was supported in part by funding from the National Health and Medical Research Council of Australia (Grants 1009785, 1099151) and the Australian Research Council (FT110100925).

Authors contribution

Project conception: MJS, GFK, MM, BM; Experimental design: MJS, BM, MM, WB; Sample preparation: MLW, MV, BM, GW, BCD; NMR data acquisition: BM, MM, MLW, MJS; Crystal structure determination: MV, OI; Data analysis: BM, MJS, MM, MLW, DKC; Manuscript preparation: BM, MJS, MLW, DKC, MM, GFK.

Author information

Author notes

  1. Geqing Wang

    Present address: La Trobe Institute for Molecular Bioscience, La Trobe University, Bundoora, VIC, 3083, Australia

Authors and Affiliations

  1. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC, 3052, Australia

    Biswaranjan Mohanty, Martin L. Williams, Bradley C. Doak, Mansha Vazirani, Olga Ilyichova, Geqing Wang, Jamie S. Simpson, David K. Chalmers & Martin J. Scanlon

  2. Bruker Biospin GmbH, Silberstreifen, 76287, Rheinstetten, Germany

    Wolfgang Bermel

  3. Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4072, Australia

    Glenn F. King

  4. Centre for Advanced Imaging, The University of Queensland, St Lucia, QLD, 4072, Australia

    Mehdi Mobli

Authors
  1. Biswaranjan Mohanty
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Martin L. Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bradley C. Doak
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mansha Vazirani
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Olga Ilyichova
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Geqing Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wolfgang Bermel
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jamie S. Simpson
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. David K. Chalmers
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Glenn F. King
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Mehdi Mobli
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Martin J. Scanlon
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Mehdi Mobli or Martin J. Scanlon.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 2017 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mohanty, B., Williams, M.L., Doak, B.C. et al. Determination of ligand binding modes in weak protein–ligand complexes using sparse NMR data. J Biomol NMR 66, 195–208 (2016). https://doi.org/10.1007/s10858-016-0067-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-016-0067-4

Keywords

Search

Navigation