Structural characterization of HIV gp41 with the membrane-proximal external region

Abstract

Human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein (gp120/gp41) plays a critical role in virus infection and pathogenesis. Three of the six monoclonal antibodies considered to have broadly neutralizing activities (2F5, 4E10, and Z13e1) bind to the membrane-proximal external region (MPER) of gp41. This makes the MPER a desirable template for developing immunogens that can elicit antibodies with properties similar to these monoclonal antibodies, with a long term goal of developing antigens that could serve as novel HIV vaccines. In order to provide a structural basis for rational antigen design, an MPER construct, HR1-54Q, was generated for x-ray crystallographic and x-ray footprinting studies to provide both high resolution atomic coordinates and verification of the solution state of the antigen, respectively. The crystal structure of HR1-54Q reveals a trimeric, coiled-coil six-helical bundle, which probably represents a postfusion form of gp41. The MPER portion extends from HR2 in continuation of a slightly bent long helix and is relatively flexible. The structures observed for the 2F5 and 4E10 epitopes agree well with existing structural data, and enzyme-linked immunosorbent assays indicate that the antigen binds well to antibodies that recognize the above epitopes. Hydroxyl radical-mediated protein footprinting of the antigen in solution reveals specifically protected and accessible regions consistent with the predictions based on the trimeric structure from the crystallographic data. Overall, the HR1-54Q antigen, as characterized by crystallography and footprinting, represents a postfusion, trimeric form of HIV gp41, and its structure provides a rational basis for gp41 antigen design suitable for HIV vaccine development.

FIGURE 1.

A, sequence of the HR1-54Q construct. Green bars mark the processing tags and GGGGS linker; purple and blue bars indicate the gp41 HR1 and HR2+MPER sequences, respectively. B, ELISAs of HR1-54Q with relevant antibodies: 2F5 (red circles), 4E10 (purple squares), Z13e1 (green triangles), and 98-6 (pink diamonds). 98-6 is the antibody that binds to the six-helix bundle.

FIGURE 2.

Structure of HR1-54Q and comparison with other core and MPER structures. A, trimeric structures of HR1-54Q (left), GCN4-gp41 fusion (middle; Protein Data Bank entry 1ENV), and gp41 N36/C34 core (right; Protein Data Bank entry 1AIK). HR1-54Q includes gp41 sequence 547–575 and 630–683 (the last eight residues not shown in the structure). GCN4-gp41 fusion includes gp41 sequences 541–581 and 624–665. N36/C34 core includes gp41 sequences 546–581 and 628–661. B, monomer of HR1-54Q (left), C54 dimer (middle; Protein Data Bank entry 3GWO), and gp41 MPER in docecylphosphocholine micelles (right; Protein Data Bank entry 1JAU).

FIGURE 3.

Structure comparison of HR1-54Q with 2F5 and 4E10 epitope peptides. The MPER from HR1-54Q is shown in blue. The binding epitope from the 2F5 complex is shown in yellow (Protein Data Bank entry 3D0V), and the binding epitope from the 4E10 complex is shown in red (Protein Data Bank entry 1TZG).

FIGURE 4.

Footprinting data mapped onto the HR1-54Q structure. Blue, modified probe residues. Red residues are unmodified probes, yellow shows probe residues for which modification was not verifiable, green indicates residues that are not easily modified, and gray residues are those for which no mass spectrometry signal was identified. A, top view of trimeric (left) and monomeric (right) HR1-54Q. B, side view of trimeric (left) and monomeric (right) HR1-54Q.