Proc Natl Acad Sci U S A. 2014 Jan 28;111(4):1391-6.

Structure of an HIV-1-neutralizing antibody target, the lipid-bound gp41 envelope membrane proximal region trimer.

Reardon PN, Sage H, Dennison SM, Martin JW, Donald BR, Alam SM, Haynes BF, Spicer LD.

Department of Biochemistry, Duke Human Vaccine Institute, Duke Department of Medicine, and Department of Radiology, Duke University School of Medicine, Durham, NC 27710.



The membrane proximal external region (MPER) of HIV-1 glycoprotein (gp) 41 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 that the three MPER peptides each adopt symmetric α-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. The 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.

PMID: 24474763



A major obstacle in the development of an HIV vaccine is the lack of vaccine regimens that induce neutralizing antibodies that bind conserved regions of the HIV envelope. The principle objective of this project was to design, biosynthetically synthesize and characterize a realistic representation for one of these envelope targets, a prefusion intermediate HIV-1 gp41, membrane proximal external region (MPER) trimer assembled on a phospholipid membrane surface. Earlier work showed that broadly neutralizing antibodies that target this region bind both to the polypeptide and the adjacent viral membrane. This effort was motivated to assist with HIV-1 vaccine design by creating a molecular model that reasonably represents the structure of the envelope protein when associated with a membrane. Macromolecular constructs evolved from this model may stimulate the immune system to respond and produce the desired neutralizing antibodies. An important element of the design for the macromolecular complex was to ensure solvent exposure of the two amino acid binding sequences (epitopes) recognized by two major HIV-1 broadly neutralizing antibodies, 2F5 and 4E10. The 28 amino acid MPER monomer sequence was biosynthetically produced in E. coli as a chimeric polypeptide with a foldon trimerization domain at the N terminus to assure trimer formation during folding in the presence of detergent micelles. The trimer assembly spontaneously associated with the micelle at near neutral pH producing a complex which avidly bound both 2F5 and 4E10 antibodies. The MPER trimer was successfully transferred to liposomes, and the trimer/liposome, membrane-protein complex exhibited similar high affinity with both antibodies. These findings provided evidence that the design for the envelope protein target was successful.

The structure of the trimer/micelle complex was characterized by NMR using conventional methods and validated by a novel, independent computational method. Results indicated the MPER trimer forms a tripod like structure, of three identical, loosely associated, non-cononical helices having fast dynamical motions. The core epitopes for both 2F5 and 4E10 antibody recognition and their amino acid side chains are solvent exposed accounting for the strong binding affinities of both neutralizing antibodies. An interesting feature of the trimer assembly associated with the micelle surface is that the three-helix structure forms a left handed helical bundle as it enters the phospholipid membrane. Previous computational models for the three transmembrane helicies in the gp41 trimer immediately C terminal to the MPER domain indicate that the three helicies do not interact substantially via inter-strand hydrogen bonds and are not organized into a tight bundle when they exhibit a left handed-twist. This trimer organization within the viral membrane may provide insight into how the gp41 structurally enables membrane fusion between the virus and the host cell leading to transmission of the disease.

ACKNOWLEDGMENTS. NMR experiments were conducted at the Duke NMR center and the David H. Murdoch Research Institute. We are grateful to Dr. Ron Venters for assistance with NMR data collection and interpretation. We thank Dr. Steven Harrison for comments during the preparation of this manuscript. We also thank Dr. Pei Zhou for helpful discussions about RDC analysis, Kara Anasti for assistance in performing the SPR experiments, Dr. Hua-Xin Liao for helpful discussions, and Dr. Mark Conners for providing 10E8 Mab. We gratefully acknowledge Dr. Marian Miller for original artistic contributions. This work was funded by a Collaboration for AIDS Vaccine Discovery grant from the Bill and Melinda Gates Foundation (to B.F.H. and L.D.S.) and by National Institutes of Health Grants GM-78031 and GM-65982 (to B.R.D.).

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Additional-original-figure-of-structureFig 1. HIV-1 gp41 MPER trimer structure as determined when associated with a micelle. This represents a symmetrical trimer sitting on the membrane surface at the base of the tripod. Two strands are represented in ribbon format showing the amino acid side chains in stick form with the third strand in the background represented as a space-filling model. Note the left handed twist exhibited by the three helicies.


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