Letters to Editor: New NMR Assignment
Resonance assignments of the complex between TraN and the C-terminal domain of TraO from the conjugative plasmid pKM101.
Type IV secretion systems (T4SS) are sophisticated machines that deliver macromolecules across the cell envelope of Gram-negative bacteria and mediate conjugation (Christie et al., 2005). T4SS in E. coli is comprised of 12 proteins termed VirB1-11 and VirD4. The C-terminal domain of VirB9 forms a stable complex with full length VirB7. Here we report the NMR resonance assignments of the complex between the homologues from the pKM101 T4SS system for VirB7 (TraN) and the C-terminal domain of VirB9 (TraO). 2D/3D NMR experiments were used to obtain near complete sequence specific backbone resonance assignments of uniformly 13C, 15N-labelled TraN/unlabelled TraO or 13C, 15N-labelled TraO/unlabelled TraN. 100% of TraN and over 97% of TraO aliphatic side chain resonances were assigned. BMRB deposit: accession number 6936.
Reference: Christie et al. (2005) Annu. Rev. Microbiol., 59, 451-485.
Richard Harris1,2, Richard Bayliss1,3, Gabriel Waksman1,2,3,*, and Paul C. Driscoll1,2,*
1) Institute of Structural Molecular Biology, UCL/Birkbeck, Malet Street, WC1E 7HX, London, UK.
2) Department of Biochemistry and Molecular Biology, UCL, Gower Street, WC1E 6BT, London, UK.
3) School of Crystallography, Birkbeck, Malet Street, WC1E 7HX London, UK.
*To whom correspondence should be addressed. Email: or
Character Count: 1496
Letters to Editor: New NMR Assignment – Supplementary Material
Resonance assignments of the complex between TraN and the C-terminal domain of TraO from the conjugative plasmid pKM101.
Richard Harris1,2, Richard Bayliss1,3, Gabriel Waksman1,2,3, and Paul C. Driscoll1,2
1 Institute of Structural Molecular Biology, UCL/Birkbeck, Malet Street, WC1E 7HX, London, UK; 2 Department of Biochemistry and Molecular Biology, UCL, Gower Street, WC1E 6BT, London, UK; 3 School of Crystallography, Birkbeck, Malet Street, WC1E 7HX London, UK
Corresponding Author Addresses:
Prof. Paul C .Driscoll
UCL Department of Biochemistry and Molecular Biology
Institute of Structural Molecular Biology
Darwin Building, Gower Street
London, WC1E 6BT
Tel: +44 207 679 7035
Fax: +44 207 679 7193
Email:
Prof. Gabriel Waksman
School of Crystallography, Birkbeck College,
UCL Department of Biochemistry and Molecular Biology
Institute of Structural Molecular Biology
Malet Street
London, WC1E 7HX
Tel: +44 20 7631 6833
Fax: +44 20 7631 6803
Email: or
Key words: TraN, TraO, VirB7, VirB9, pKM101, heteronuclear NMR, resonance assignments
Biological Context
Type IV secretion systems (T4SS) are sophisticated machines that deliver macromolecules across the cell envelope of Gram-negative bacteria. Substrates range from proteins and protein-DNA complexes to peptidoglycans. Several bacterial pathogens such as Helicobacter pylori, Brucella suis and Agrobacterium tumefaciens utilize T4SS to secrete virulence factors directly responsible for disease. In addition, conjugation, the process by which bacteria exchange genetic material, is a T4SS-mediated process (Christie et al., 2005).
The T4SS in E. coli is composed of 12 proteins termed “VirB1-11” and VirD4, which locate either in whole or in part in the cytosol (VirB11, VirD4), the inner-membrane (VirB2, VirB4, VirB6, VirB8, VirB10, and VirD4), the periplasm (VirB1, VirB2, VirB5, VirB7, VirB8, VirB9, and VirB10), the outer-membrane (VirB3, VirB7, VirB9, VirB2, and VirB5) or extracellularly (VirB2, VirB5). The core of the machinery (the core-complex) consists of VirB4, VirB6, VirB7, VirB8, VirB9, and VirB10. The structural biology of T4SS has greatly progressed in the last few years (Yeo and Waksman, 2004). Crystal structures of full-length or fragments of the two ATPases VirD4 and VirB11 have been determined, as well as those of VirB8, VirB10, and VirB5.
VirB7 proteins are small lipidated molecules that locate to the outer membrane, presumably through the lipid moiety. VirB7 is known to interact with and stabilize the core-complex proteins (VirB4, VirB6-10) and also forms a subcomplex with the outer-membrane and extracellularly-located components VirB2 and VirB5 (Christie et al., 2005). VirB9 is an essential component of the core-complex and is known to interact with VirB8 and VirB10 within the core-complex, although the interaction between VirB9 and VirB10 appears to be energy-dependent and reliant on VirB11 and VirD4 ATPase activity (Christie et al., 2005). VirB9 from the Ti plasmid (VirB9Ti) of A. tumefaciens consists of three domains, each of approximately 80-100 residues. The N- and C-terminal domains are conserved among VirB9 proteins and required for both pilus biogenesis and channel activity (Jakubowski et al., 2005). The central domain is poorly conserved and may form the part inserting in the outer-membrane. The C-terminal domain is also the site of disulfide bond formation with VirB7Ti. Recently, we have shown that the C-terminal domain of VirB9, in both the T4SSs of the conjugative plasmid pKM101 and the one encoded by the VirB cluster of B. suis, form a stable complex with its respective VirB7 counterpart.
Here we report the NMR resonance assignments for both VirB7 and the C-terminal domain of VirB9 in the VirB7/VirB9 complex as a first step in determining the three-dimensional solution structure of this complex. The VirB7 and VirB9 homologues under investigation here are the TraN (17-48) and TraO (177-294) proteins of the pKM101 plasmid conjugation T4SS system.
Methods and Results
Sample Preparation. Uniformly 15N- and 13C/15N-labelled recombinant hexahistidine-tagged TraO (177-294) and GST-tagged TraN (17-48) were over-expressed in E. coli BL21 (DE3) CodonPlus RPIL (Stratagene) cells grown on M9 minimal medium with 1.0 g/l (15NH4)2SO4 and 2 g/l 13C6-glucose. TraO protein was first purified by immobilized nickel ion affinity chromatography. TraN lysate was applied to a glutathione sepharose affinity column, washed and then a molar excess of TraO protein was applied. The His6- and GST-tags were subsequently cleaved on-column using tobacco etch virus protease, followed by further purification by immobilized nickel ion affinity chromatography and size exclusion chromatography. For NMR studies, samples of ca. 1.0 mM TraN/TraO in 20mM sodium phosphate buffer (pH 6.7), 100 mM sodium chloride, 0.1mM EDTA, 0.2% sodium azide were prepared.
NMR Spectroscopy. NMR spectra were acquired at either 298 K on Varian UNITYplus and INOVA spectrometers (operating at nominal 1H frequencies of 500 MHz and 600/800 MHz, respectively) equipped with a triple resonance (1H, 13C, 15N) probe including Z-axis pulse field gradients. Sequence-specific resonance assignments were obtained by combining the data from the following 3D gradient sensitivity-enhanced triple resonance experiments: HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB (Yamazaki et al., 1994), CBCA(CO)NH (Muhandiram and Kay, 1994), HA(CA)NH and HA(CACO)NH. Side chain resonance assignments were obtained from 15N-edited TOCSY-HSQC (50ms) and 15N NOESY-HSQC (120ms) experiments in H2O/2H2O (9:1), and 3D HCCH-TOCSY (16ms mixing time) and 13C NOESY-HSQC (120ms) experiments recorded on samples in 2H2O. All spectra were processed using NMRpipe/NMRDraw (Delaglio et al., 1995) and analyzed using ANSIG v3.3 (Kraulis, 1989). Chemical shifts were indirectly referenced to DSS.
Extent of Assignments and Data Deposition
Analysis of the triple resonance experiments allowed identification and sequential assignments for all TraN and 108 out of the 111 TraO (118 less 7 prolines) backbone 15N and amide proton resonances. Definitive assignments have not been obtained for residues G178 and A225-S226. Side chain resonance assignments are 100% complete for TraN and over 97% complete for TraO, with 115 out of 118 residues side chain proton resonances totally assigned. Figure 1 shows assigned 2D 1H-15N HSQC spectra of (a) 13C/15N labelled TraN (VirB7) in complex with unlabelled TraO (VirB9) and (b) 13C/15N labelled TraO (VirB9) in complex with unlabelled TraN (VirB7), both spectra recorded at a 1H frequency of 600 MHz. Backbone chemical shift analysis, using the Chemical Shift Index, (Wishart and Sykes, 1994) suggests that TraO contains 9 b-strands. The full analysis of the three-dimensional solution structure of the complex between TraN/TraO is in progress. The chemical shifts for resonances of TraN and TraO in the TraN/TraO complex have been deposited in the BioMagResBank (accession number BMRB-6936).
Acknowledgements
We are grateful to Dr G. Kelly for providing invaluable assistance with the 800 MHz spectrometer at the MRC National NMR Centre at the National Institute for Medical Research, Mill Hill, London.
References
Christie, P. J., Atmakuri, K., Krishnamoorthy, V., Jakubowski, S., and Cascales, E. (2005) Annu. Rev. Microbiol., 59, 451-485
Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfiefer, J., and Bax, A. (1995) J. Biomol. NMR, 6, 277-293
Jakubowski, S. J., Cascales, E., Krishnamoorthy, V., and Christie, P. J. (2005) J. Bacteriol., 187, 3486-3495
Kraulis, P.J. (1989) J. Magn. Reson., 24, 627-633
Muhandiram, D.R. and Kay L.E. (1994) J. Magn. Reson., B103, 203-216
Wishart, D.S. and Sykes, B.D. (1994) J. Biomol. NMR, 4, 171-180.
Yamazaki, T., Lee, W., Arrowsmith, C.H., Muhandiram, D.R., and Kay L.E. (1994) J. Am. Chem. Soc., 116, 11655-11666
Yeo, H. J., and Waksman, G. (2004) J. Bacteriol., 186, 1919-1926
Figure Legends
Figure 1. Assigned 2D 1H-15N HSQC spectra of (a) 13C/15N-labelled TraN (VirB7) in complex with unlabelled TraO (VirB9) and (b) unlabelled TraN (VirB7) in complex with 13C/15N-labelled TraO (VirB9) recorded on a 600 MHz Varian Inova spectrometer at 298K.