Abstract
DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect protein–DNA and protein–ATP interactions and identify the residue type establishing the intermolecular contacts. While conventional solid-state NMR can detect protein–DNA interactions in large oligomeric protein assemblies in favorable cases, it typically suffers from low signal-to-noise ratios. We show here, for the oligomeric DnaB helicase from Helicobacter pylori complexed with ADP and single-stranded DNA, that this limitation can be overcome by using DNP-enhanced spectroscopy. Interactions are established by DNP-enhanced 31P–13C polarization-transfer experiments followed by the recording of a 2D 13C–13C correlation experiment. The NMR spectra were obtained in less than 2 days and allowed the identification of residues of the motor protein involved in nucleotide binding.
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References
Abragam A (1961) The principles of nuclear magnetism. Clarendon Press, Oxford
Abragam A, Goldman M (1978) Principles of dynamic nuclear polarisation. Rep Prog Phys 41:395
Akbey Ü., Oschkinat H (2016) Structural biology applications of solid state MAS DNP NMR. J Magn Reson 269:213–224
Baldus M, Petkova AT, Herzfeld J, Griffin RG (1998) Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems. Mol Phys 95:1197–1207
Bauer T et al (2017) Line-broadening in low-temperature solid-state NMR spectra of fibrils. J Biomol NMR 67:51–61
Bazin A, Cherrier MV, Gutsche I, Timmins J, Terradot L (2015) Structure and primase-mediated activation of a bacterial dodecameric replicative helicase. Nucleic Acids Res 43:8564–8576
Bertini I et al (2012) NMR properties of sedimented solutes. Phys Chem Chem Phys 14:439–447
Carlomagno T (2014) Present and future of NMR for RNA–protein complexes: a perspective of integrated structural biology. J Magn Reson 241:126–136
Dominguez C, Schubert M, Duss O, Ravindranathan S, Allain F.H.T. (2011) Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy. Prog Nucl Magn Reson Spectrosc 58:1–61
Esadze A et al (2016) Changes in conformational dynamics of basic side chains upon protein–DNA association. Nucleic Acids Res 44:6961–6970
Frigyes D, Alber F, Pongor S, Carloni P (2001) Arginine–phosphate salt bridges in protein–DNA complexes: a Car–Parrinello study. J Mol Struct (Thoechem) 574:39–45
Gardiennet C et al (2012) A sedimented sample of a 59 kDa dodecameric helicase yields high-resolution solid-state NMR spectra. Angew Chem Int Ed 51:7855–7858
Itsathitphaisarn O, Wing RA., Eliason WK., Wang J, Steitz TA (2012) The hexameric helicase DnaB adopts a nonplanar conformation during translocation. Cell 151:267–277
Lange A, Luca S, Baldus M (2002) Structural constraints from proton-mediated rare-spin correlation spectroscopy in rotating solids. J Am Chem Soc 124:9704–9705
LeBowitz JH, McMacken R (1986) The Escherichia coli dnaB replication protein is a DNA helicase. J Biol Chem 261:4738–4748
Morag O, Abramov G, Goldbourt A (2014) Complete chemical shift assignment of the ssDNA in the filamentous bacteriophage fd reports on its conformation and on its interface with the capsid shell. J Am Chem Soc 136:2292–2301
Ravera E et al (2013) Dynamic nuclear polarization of sedimented solutes. J Am Chem Soc 135:1641–1644
Ravera E et al (2014) DNP-enhanced MAS NMR of bovine serum albumin sediments and solutions. J Phys Chem B 118:2957–2965
Sauvée C et al (2013) Highly efficient, water-soluble polarizing agents for dynamic nuclear polarization at high frequency. Angew Chem Int Ed 52:10858–10861
Siemer AB, McDermott AE (2008) Solid-state NMR on a type III antifreeze protein in the presence of ice. J Am Chem Soc 130:17394–17399
Singleton MR, Dillingham MS, Wigley DB (2007) Structure and mechanism of helicases and nucleic acid translocases. Annu Rev Biochem 76:23–50
Spies M (2012) DNA helicases and DNA motor proteins. Springer, New York
Stevens T et al (2011) A software framework for analysing solid-state MAS NMR data. J Biomol NMR 51:437–447
Takegoshi K, Nakamura S, Terao T (1999) 13C–13C polarization transfer by resonant interference recoupling under magic-angle spinning in solid-state NMR. Chem Phys Lett 307:295–302
Takegoshi K, Nakamura S, Terao T (2001) 13C–1H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem Phys Lett 344:631–637
Ulrich EL et al (2008) BioMagResBank. Nucleic Acids Res 36:D402-D408
Walker JE, Saraste M, Runswick MJ, Gay NJ (1982) Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1:945–951
Wiegand T et al (2016) Monitoring ssDNA binding to the DnaB helicase from Helicobacter pylori by solid-state NMR spectroscopy. Angew Chem Int Ed 55:14164–14168
Wu H, Finger LD, Feigon J (2005) Structure determination of protein–RNA complexes by NMR. In: Methods in enzymology, vol 394. Academic Press, Cambridge, pp 525–545
Yount RG, Babcock D, Ballantyne W, Ojala D (1971) Adenylyl imidiodiphosphate, an adenosine triphosphate analog containing a P–N–P linkage. BioChemistry 10:2484–2489
Acknowledgements
This work was supported by the Swiss National Science Foundation (Grant Nos. 200020_159707 and 200020_146757), the French ANR (ANR-14-CE09-0024B) and the ETH Career SEED-69 16-1. We cordially acknowledge the support of Laura Piveteau during one DNP session and Simon Widler for help with the expression of the protein.
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Wiegand, T., Liao, WC., Ong, T.C. et al. Protein–nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR. J Biomol NMR 69, 157–164 (2017). https://doi.org/10.1007/s10858-017-0144-3
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DOI: https://doi.org/10.1007/s10858-017-0144-3