Email updates

Keep up to date with the latest news and content from Retrovirology and BioMed Central.

This article is part of the supplement: AIDS Vaccine 2012

Open Access Poster presentation

NMR spectroscopy of HIV-1 gp120 outer domain

M Sastry1*, L Xu1, S Bhattacharya2, GJ Nabel1, CA Bewley3 and PD Kwong1

  • * Corresponding author: M Sastry

Author Affiliations

1 Vaccine Research Center, NIAID/NIH, Bethesda, MD, USA

2 New York Structural Biology Center, New York, NY, USA

3 NIDDK, National Institutes of Health, Bethesda, MD, USA

For all author emails, please log on.

Retrovirology 2012, 9(Suppl 2):P20  doi:10.1186/1742-4690-9-S2-P20


The electronic version of this article is the complete one and can be found online at: http://www.retrovirology.com/content/9/S2/P20


Published:13 September 2012

© 2012 Sastry et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background

The outer domain (OD) of HIV-1 gp120 has been proposed as a minimal immunogen to elicit broadly neutralizing antibodies. However, OD is heavily glycosylated, contains many flexible regions, and immunization with a number of different OD variants has thus far failed to elicit neutralizing antibodies. An understanding of the conformational space sampled by the OD in its unliganded state, however, may assist in the use of OD as an immunogen.

Methods

We developed a method to isotopically enrich glycoproteins using a mammalian expression system that exploits the high level of protein expression obtained from an adenoviral vector, and employed heteronuclear NMR spectroscopy to obtain structural and dynamic information of unliganded OD. Multidimensional NMR experiments were recorded on uniformly labeled 15N/13C OD as well as on samples selectively enriched in 15N-labeled Gly, Ile, Leu and Val. Experiments for backbone assignments were also recorded on an OD sample enriched in 15N/13C for Ile, Leu and Val.

Results

We successfully produced isotopically labeled OD samples, suitable for NMR analysis. We also identified Gly, Ser, Val, Leu and Ile residues using samples selectively enriched in 15N for Gly, Val, Leu and Ile. Standard triple resonance NMR experiments on the isotopically labeled OD were combined with backbone experiments recorded on a second sample – that was selectively enriched in 15N/13C for Ile, Leu and Val – to assign HN, C’, Calpha and N backbone resonances in about 80 of the 220 residues of OD.

Conclusion

We succeeded in assigning ~1/3 of the backbone for unliganded OD with triple resonance experiments. Extension of these assignments with NOESY experiments is now proceeding. Our results indicate that a solution structure of the highly glycosylated HIV-1 gp120 OD is feasible.