Significance: A major roadblock in the development of an HIV vaccine is the need to develop vaccine regimens that will induce antibodies that bind to conserved regions of the HIV envelope and neutralize many different virus quasispecies. One such envelope target is at the region closest to the membrane, the gp41 membrane proximal external region (MPER). Previous work has demonstrated that antibodies that target this region bind both to the gp41 polypeptide and to the adjacent viral membrane. However, what has been missing is a view of what the MPER neutralizing epitopes may look like in the context of a trimeric orientation with lipids. We have constructed an MPER trimer associated with lipids and solved the trimer structure by NMR spectroscopy.

We report the design and structure determination of a new antigenic membrane-bound MPER trimer for examining immunogenic responses to the HIV-1 viral coat protein gp41. This new design and structure, called gp41-M-MAT, provides important structural information that can further illuminate HIV vaccine development efforts. Our structure is also an important addition to the relatively small number of multimeric membrane-associated structures determined using solution state NMR.

Here is the structure:
PDB Deposition 2m7w:
NMR Solution Structure of gp41-M-MAT, a membrane associated MPER trimer from HIV-1 gp41,
by J. Martin, P. Reardon, H. Sage, D. Moses, A. Munir, B. Haynes, L. Spicer, and B. R. Donald*
Large image

Here is our paper on this structure:
Structure of an HIV-1 Neutralizing Antibody Target: A Lipid Bound gp41 Envelope Membrane Proximal Region Trimer.
by P. Reardon, H. Sage, D. Moses, J. Martin, B. R. Donald, A. Munir, B. Haynes, and L. Spicer.
PNAS 2014; 111(4):1391-6.
[Read the open-access PDF (PNAS website )]

Abstract: The membrane proximal external region (MPER) of HIV-1 gp41 is involved in viral-host cell membrane fusion. It contains short amino acid sequences that are binding sites for the HIV-1 broadly neutralizing antibodies 2F5, 4E10 and 10E8, making these binding sites important targets for HIV-1 vaccine development. We report a high-resolution structure of a designed MPER trimer assembled on a detergent micelle. The NMR solution structure of this trimeric domain, designated gp41-M-MAT, shows the three MPER peptides each adopt symmetric alpha-helical conformations exposing the amino acid side chains of the antibody binding sites. The helices are closely associated at their N-termini, bend between the 2F5 and 4E10 epitopes and gradually separate toward the C-termini where they associate with the membrane. Mabs 2F5 and 4E10 bind gp41-M-MAT with nanomolar affinities, consistent with the substantial exposure of their respective epitopes in the trimer structure. The traditional structure determination of gp41-M-MAT using the Xplor-NIH protocol was validated by independently determining the structure using the DISCO sparse-data protocol, which exploits geometric arrangement algorithms that guarantee to compute all structures and assignments that satisfy the data.

DISCO is described here:

• J. Martin, A. Yan, C. Bailey-Kellogg, P. Zhou and B. R. Donald*. A graphical method for analyzing distance restraints using residual dipolar couplings for structure determination of symmetric protein homo-oligomers. Protein Science 20(6):970-985 (2011) Mar 16. doi: 10.1002/pro.620. [Epub ahead of print] PMID: 21413097
  • [PDF]

Broader Impacts: The abstract above discusses the significance of our work for HIV biology and vaccine design. This work is also of central interest to us, because gp41-M-MAT is an unusual and important designed protein, and our laboratory is very focused on protein design. In addition, there are implications more broadly for structural biology and also for NMR methodology. Specifically, as you may know, the majority of proteins in modern cells are complexes comprised of symmetric homo-oligomers. However, comparatively few of these have been structurally characterized (i.e., few have had their 3D structures determined from experimental measurements). Similarly, it is difficult to determine the structures of membrane-bound and membrane-associated proteins (even though such proteins are common and are very important). We have spent nearly 10 years developing a robust methodology for using NMR and novel computational algorithms to reliably determine the structure of these (relatively) large, symmetric, membrane-bound protein complexes, and our PNAS paper represents the culmination and application of those advanced computational techniques, (whose software we have released open-source). For background, see the DISCO paper in Protein Science, above, and also my textbook (MIT Press, 2011).

Acknowledgements: I would like to acknowledge my Ph.D. students Jeff Martin and Tony Yan, my former postdoc Chris Bailey-Kellogg, and my collaborators Pat Reardon, Harvey Sage, S. Moses Dennison, S. Munir Alam, Bart Haynes, Pei Zhou, and Len Spicer, and thank them for working with us on these projects. This work was funded by a Collaboration for AIDS Vaccine Discovery grant from the Bill and Melinda Gates Foundation to BH and LS and by the National Institutes of Health via two grants to BRD: GM-78031 and GM-65982.

Department of Biochemistry
Department of Computer Science
Human Vaccine Institute

Duke University and
Duke University Medical Center.



Notes: MPER - Membrane-Proximal External Region; gp41 - Glycoprotein 41; HIV-1 - Human Immunodefficiency Virus 1; Mab - Monoclonal antibody.