Skip to main content
SpringerLink
Log in

High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

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

Abstract

The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail’s biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with 13C or 1H detection, have very narrow line widths (0.40–0.60 ppm for 13C, 0.11–0.15 ppm for 1H, and 0.46–0.64 ppm for 15N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The 1H-detected solid-state NMR 1H/15N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR 1H/15N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.

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

  • Akbey U, Rossum BJ, Oschkinat H (2012) Practical aspects of high-sensitivity multidimensional (1)(3)C MAS NMR spectroscopy of perdeuterated proteins. J Magn Reson 217:77–85. doi:10.1016/j.jmr.2012.02.015

    Article  ADS  Google Scholar 

  • Andreas LB et al (2015) Structure and mechanism of the influenza A M218-60 dimer of dimers. J Am Chem Soc 137:14877–14886. doi:10.1021/jacs.5b04802

    Article  Google Scholar 

  • Andronesi OC, Becker S, Seidel K, Heise H, Young HS, Baldus M (2005) Determination of membrane protein structure and dynamics by magic-angle-spinning solid-state NMR spectroscopy. J Am Chem Soc 127:12965–12974. doi:10.1021/ja0530164

    Article  Google Scholar 

  • Baker LA, Folkers GE, Sinnige T, Houben K, Kaplan M, van der Cruijsen EA, Baldus M (2015) Magic-angle-spinning solid-state NMR of membrane proteins. Methods Enzymol 557:307–328. doi:10.1016/bs.mie.2014.12.023

    Article  Google Scholar 

  • Baldus M, Petkova A, Herzfield J, Griffin R (1998) Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems. Mol Phys 95:1197–1207

    Article  ADS  Google Scholar 

  • Barbet-Massin E et al (2014) Rapid proton-detected NMR assignment for proteins with fast magic angle spinning. J Am Chem Soc 136:12489–12497. doi:10.1021/ja507382j

    Article  Google Scholar 

  • Behlau M, Mills DJ, Quader H, Kuhlbrandt W, Vonck J (2001) Projection structure of the monomeric porin OmpG at 6 A resolution. J Mol Biol 305:71–77. doi:10.1006/jmbi.2000.4284

    Article  Google Scholar 

  • Brown LS, Ladizhansky V (2015) Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy. Protein Sci 24:1333–1346. doi:10.1002/pro.2700

    Article  Google Scholar 

  • Cross TA, Ekanayake V, Paulino J, Wright A (2014) Solid state NMR: the essential technology for helical membrane protein structural characterization. J Magn Reson 239:100–109. doi:10.1016/j.jmr.2013.12.006

    Article  ADS  Google Scholar 

  • Das BB, Nothnagel HJ, Lu GJ, Son WS, Tian Y, Marassi FM, Opella SJ (2012) Structure determination of a membrane protein in proteoliposomes. J Am Chem Soc 134:2047–2056. doi:10.1021/ja209464f

    Article  Google Scholar 

  • De Zorzi R, Mi W, Liao M, Walz T (2016) Single-particle electron microscopy in the study of membrane protein structure. Microscopy 65:81–96. doi:10.1093/jmicro/dfv058

    Article  Google Scholar 

  • Devaux PF, Seigneuret M (1985) Specificity of lipid-protein interactions as determined by spectroscopic techniques. Biochim Biophys Acta 822:63–125

    Article  Google Scholar 

  • Ding Y, Yao Y, Marassi FM (2013) Membrane protein structure determination in membrana. Acc Chem Res 46:2182–2190. doi:10.1021/ar400041a

    Article  Google Scholar 

  • Ding Y, Fujimoto LM, Yao Y, Plano GV, Marassi FM (2015) Influence of the lipid membrane environment on structure and activity of the outer membrane protein Ail from Yersinia pestis. Biochim Biophys Acta 1848:712–720. doi:10.1016/j.bbamem.2014.11.021

    Article  Google Scholar 

  • Dolder M, Zeth K, Tittmann P, Gross H, Welte W, Wallimann T (1999) Crystallization of the human, mitochondrial voltage-dependent anion-selective channel in the presence of phospholipids. J Struct Biol 127:64–71. doi:10.1006/jsbi.1999.4141

    Article  Google Scholar 

  • Eddy MT et al (2012) Lipid dynamics and protein-lipid interactions in 2D crystals formed with the beta-barrel integral membrane protein VDAC1. J Am Chem Soc 134:6375–6387. doi:10.1021/ja300347v

    Article  Google Scholar 

  • Eddy MT et al (2015) Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR. J Biomol NMR 61:299–310. doi:10.1007/s10858-015-9903-1

    Article  Google Scholar 

  • Ernst M, Samoson A, Meier BH (2003) Low-power XiX decoupling in MAS NMR experiments. J Magn Reson 163:332–339

    Article  ADS  Google Scholar 

  • Findlay EJ, Barton PG (1978) Phase behavior of synthetic phosphatidylglycerols and binary mixtures with phosphatidylcholines in the presence and absence of calcium ions. BioChemistry 17:2400–2405

    Article  Google Scholar 

  • Fung BM, Khitrin AK, Ermolaev K (2000) An improved broadband decoupling sequence for liquid crystals and solids. J Magn Reson 142:97–101

    Article  ADS  Google Scholar 

  • Gennis RB (1989) Biomembranes : molecular structure and function. springer advanced texts in chemistry. Springer, New York

    Book  Google Scholar 

  • Hagn F, Etzkorn M, Raschle T, Wagner G (2013) Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. J Am Chem Soc 135:1919–1925. doi:10.1021/ja310901f

    Article  Google Scholar 

  • Hiller M, Krabben L, Vinothkumar KR, Castellani F, van Rossum BJ, Kuhlbrandt W, Oschkinat H (2005) Solid-state magic-angle spinning NMR of outer-membrane protein G from Escherichia coli. Chembiochem 6:1679–1684

    Article  Google Scholar 

  • Huber M, With O, Schanda P, Verel R, Ernst M, Meier BH (2012) A supplementary coil for (2)H decoupling with commercial HCN MAS probes. J Magn Reson 214:76–80. doi:10.1016/j.jmr.2011.10.010

    Article  ADS  Google Scholar 

  • Janiak MJ, Small DM, Shipley GG (1976) Nature of the Thermal pretransition of synthetic phospholipids: dimyristolyl- and dipalmitoyllecithin. BioChemistry 15:4575–4580

    Article  Google Scholar 

  • Lee AG (2003) Lipid-protein interactions in biological membranes: a structural perspective. Biochim Biophys Acta 1612:1–40

    Article  Google Scholar 

  • Lee AG (2011) Biological membranes: the importance of molecular detail. Trends Biochem Sci 36:493–500. doi:10.1016/j.tibs.2011.06.007

    Article  Google Scholar 

  • Lee J, Patel DS, Kucharska I, Tamm LK, Im W (2017) Refinement of OprH-LPS interactions by molecular simulations. Biophys J 112:346–355. doi:10.1016/j.bpj.2016.12.006

    Article  Google Scholar 

  • Leftin A, Brown MF (2011) An NMR database for simulations of membrane dynamics. Biochim Biophys Acta 1808:818–839. doi:10.1016/j.bbamem.2010.11.027

    Article  Google Scholar 

  • Li Y, Berthold DA, Frericks HL, Gennis RB, Rienstra CM (2007) Partial (13)C and (15)N chemical-shift assignments of the disulfide-bond-forming enzyme DsbB by 3D magic-angle spinning NMR spectroscopy. Chembiochem 8:434–442. doi:10.1002/cbic.200600484

    Article  Google Scholar 

  • Linser R et al (2011) Proton-detected solid-state NMR spectroscopy of fibrillar and membrane proteins. Angew Chem Int Ed Engl 50:4508–4512. doi:10.1002/anie.201008244

    Article  Google Scholar 

  • Mahalakshmi R, Marassi FM (2008) Orientation of the Escherichia coli outer membrane protein OmpX in phospholipid bilayer membranes determined by solid-state NMR. BioChemistry 47:6531–6538. doi:10.1021/bi800362b

    Article  Google Scholar 

  • Mahalakshmi R, Franzin CM, Choi J, Marassi FM (2007) NMR structural studies of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes. Biochim Biophys Acta 1768:3216–3224. doi:10.1016/j.bbamem.2007.08.008

    Article  Google Scholar 

  • Marassi FM, Ding Y, Schwieters CD, Tian Y, Yao Y (2015) Backbone structure of Yersinia pestis Ail determined in micelles by NMR-restrained simulated annealing with implicit membrane solvation. J Biomol NMR 63:59–65. doi:10.1007/s10858-015-9963-2

    Article  Google Scholar 

  • Maslennikov I, Choe S (2013) Advances in NMR structures of integral membrane proteins. Curr Opin Struct Biol 23:555–562. doi:10.1016/j.sbi.2013.05.002

    Article  Google Scholar 

  • Moraes I, Evans G, Sanchez-Weatherby J, Newstead S, Stewart PD (2014) Membrane protein structure determination—the next generation. Biochim Biophys Acta 1838:78–87. doi:10.1016/j.bbamem.2013.07.010

    Article  Google Scholar 

  • Morris GA, Freeman R (1979) Enhancement of nuclear magnetic resonance signals by polarization transfer. J Am Chem Soc 101:760–762. doi:10.1021/ja00497a058

    Article  Google Scholar 

  • Pauli J, Baldus M, van Rossum B, de Groot H, Oschkinat H (2001) Backbone and side-chain 13 C and 15 N signal assignments of the alpha-spectrin SH3 domain by magic angle spinning solid-state NMR at 17.6 T. Chembiochem 2:272–281. doi:10.1002/1439-7633(20010401)2:4<272::AID-CBIC272>3.0.CO;2-2

    Article  Google Scholar 

  • Pines A, Gibby MG, Waugh JS (1973) Proton-enhanced NMR of dilute spins in solids. J Chem Phys 59:569–590. doi:10.1063/1.1680061

    Article  ADS  Google Scholar 

  • Plesniak LA, Mahalakshmi R, Rypien C, Yang Y, Racic J, Marassi FM (2011) Expression, refolding, and initial structural characterization of the Y. pestis Ail outer membrane protein in lipids. Biochim Biophys Acta 1808:482–489. doi:10.1016/j.bbamem.2010.09.017

    Article  Google Scholar 

  • Rand RP, Chapman D, Larsson K (1975) Tilted hydrocarbon chains of dipalmitoyl lecithin become perpendicular to the bilayer before melting. Biophys J 15:1117–1124. doi:10.1016/S0006-3495(75)85888-7

    Article  Google Scholar 

  • Renault M, Tommassen-van Boxtel R, Bos MP, Post JA, Tommassen J, Baldus M (2012) Cellular solid-state nuclear magnetic resonance spectroscopy. Proc Natl Acad Sci USA 109:4863–4868. doi:10.1073/pnas.1116478109

    Article  ADS  Google Scholar 

  • Saurel O et al (2017) Local and global dynamics in klebsiella pneumoniae outer membrane protein a in lipid bilayers probed at atomic resolution. J Am Chem Soc. doi:10.1021/jacs.6b11565

    Google Scholar 

  • Shahid SA, Markovic S, Linke D, van Rossum BJ (2012) Assignment and secondary structure of the YadA membrane protein by solid-state MAS NMR. Sci Rep 2:803. doi:10.1038/srep00803

    Article  ADS  Google Scholar 

  • Shaka AJ, Keeler J, Frenkiel T, Freeman R (1983) An improved sequence for broadband decoupling: WALTZ-16. J Magn Reson 52:335–338. doi:10.1016/0022-2364(83)90207-X

    ADS  Google Scholar 

  • Szeverenyi NM, Sullivan MJ, Maciel GE (1982) Observation of spin exchange by two-dimensional fourier transform 13 C cross polarization-magic-angle spinning. J Magn Reson 47:462–475. doi:10.1016/0022-2364(82)90213-X

    ADS  Google Scholar 

  • Takamori S et al (2006) Molecular anatomy of a trafficking organelle. Cell 127:831–846. doi:10.1016/j.cell.2006.10.030

    Article  Google Scholar 

  • Takegoshi K, Nakamura S, Terao T (2001) 13 C-1H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem Phys Lett 344:631–637

    Article  ADS  Google Scholar 

  • Takegoshi K, Nakamura S, Terao T (2003) 13C–1 H dipolar-driven 13C–13 C recoupling without 13 C rf irradiation in nuclear magnetic resonance of rotating solids. J Chem Phys 118:2325–2341. doi:10.1063/1.1534105

    Article  ADS  Google Scholar 

  • Takeuchi K, Arthanari H, Shimada I, Wagner G (2015) Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR. J Biomol NMR 63:323–331. doi:10.1007/s10858-015-9991-y

    Article  Google Scholar 

  • Tang M, Comellas G, Mueller LJ, Rienstra CM (2010) High resolution (1)(3)C-detected solid-state NMR spectroscopy of a deuterated protein. J Biomol NMR 48:103–111. doi:10.1007/s10858-010-9442-8

    Article  Google Scholar 

  • Tardieu A, Luzzati V, Reman FC (1973) Structure and polymorphism of the hydrocarbon chains of lipids: a study of lecithin-water phases. J Mol Biol 75:711–733

    Article  Google Scholar 

  • Warschawski DE, Devaux PF (2005) 1 H-13C polarization transfer in membranes: a tool for probing lipid dynamics and the effect of cholesterol. J Magn Reson 177:166–171. doi:10.1016/j.jmr.2005.07.011

    Article  ADS  Google Scholar 

  • Yamashita S et al (2011) Structural insights into Ail-mediated adhesion in Yersinia pestis. Structure 19:1672–1682. doi:10.1016/j.str.2011.08.010

    Article  Google Scholar 

  • Yao Y, Ding Y, Tian Y, Opella SJ, Marassi FM (2013) Membrane protein structure determination: back to the membrane. Methods Mol Biol 1063:145–158. doi:10.1007/978-1-62703-583-5_8

    Article  Google Scholar 

  • Zhou HX, Cross TA (2013) Influences of membrane mimetic environments on membrane protein structures. Annu Rev Biophys 42:361–392. doi:10.1146/annurev-biophys-083012-130326

    Article  Google Scholar 

  • Zhou DH, Rienstra CM (2008) High-performance solvent suppression for proton detected solid-state NMR. J Magn Reson 192:167–172. doi:10.1016/j.jmr.2008.01.012

    Article  ADS  Google Scholar 

Download references

Acknowledgements

This research was supported by grants from the National Institutes of Health (GM 118186, GM 099986, and GM 066978) and by the Biotechnology Resource for Molecular Imaging of Proteins at UCSD supported by the National Institutes of Health (P41 EB 002031).

Author information

Authors and Affiliations

  1. Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA

    Yong Yao, Samit Kumar Dutta, L. Miya Fujimoto, Andrey A. Bobkov & Francesca M. Marassi

  2. Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0307, USA

    Sang Ho Park, Ratan Rai & Stanley J. Opella

Authors
  1. Yong Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Samit Kumar Dutta
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sang Ho Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ratan Rai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. L. Miya Fujimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Andrey A. Bobkov
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Stanley J. Opella
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Francesca M. Marassi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Francesca M. Marassi.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yao, Y., Dutta, S.K., Park, S.H. et al. High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes. J Biomol NMR 67, 179–190 (2017). https://doi.org/10.1007/s10858-017-0094-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-017-0094-9

Keywords

Search

Navigation