Structural basis of local, pH-dependent conformational changes in glycoprotein B from herpes simplex virus type 1

Abstract

Herpesviruses enter cells by membrane fusion either at the plasma membrane or in endosomes, depending on the cell type. Glycoprotein B (gB) is a conserved component of the multiprotein herpesvirus fusion machinery and functions as a fusion protein, with two internal fusion loops, FL1 and FL2. We determined the crystal structures of the ectodomains of two FL1 mutants of herpes simplex virus type 1 (HSV-1) gB to clarify whether their fusion-null phenotypes were due to global or local effects of the mutations on the structure of the gB ectodomain. Each mutant has a single point mutation of a hydrophobic residue in FL1 that eliminates the hydrophobic side chain. We found that neither mutation affected the conformation of FL1, although one mutation slightly altered the conformation of FL2, and we conclude that the fusion-null phenotype is due to the absence of a hydrophobic side chain at the mutated position. Because the ectodomains of the wild-type and the mutant forms of gB crystallized at both low and neutral pH, we were able to determine the effect of pH on gB conformation at the atomic level. For viruses that enter cells by endocytosis, the low pH of the endosome effects major conformational changes in their fusion proteins, thereby promoting fusion of the viral envelope with the endosomal membrane. We show here that upon exposure of gB to low pH, FL2 undergoes a major relocation, probably driven by protonation of a key histidine residue. Relocation of FL2, as well as additional small conformational changes in the gB ectodomain, helps explain previously noted changes in its antigenic and biochemical properties. However, no global pH-dependent changes in gB structure were detected in either the wild-type or the mutant forms of gB. Thus, low pH causes local conformational changes in gB that are very different from the large-scale fusogenic conformational changes in other viral fusion proteins. We propose that these conformational changes, albeit modest, play an important functional role during endocytic entry of HSV.

FIG. 1.

(A) W174R-acidic structure (chain A). A single protomer is colored by domain with domain I, pale blue; domain II, pale green; domain III, yellow; domain IV, orange; and domain V, red. Domains are labeled. New regions, unresolved in the previously determined wt-gB-neutral structure (PDB 2GUM), are shown in purple. Fusion loops FL1, residues 175 to 180, and FL2, residues 258 to 264, are shown in cyan and green, respectively. (B) The region of gB containing the now-visible epitope of the neutralizing MAb H1781, residues 454 to 473 (5), is shown in green. The view in panel B is shown in the same orientation as in panel A. (C) Superposition of the four structures determined here—Y179S-basic (chain C), W174R-acidic (chain B), Y179S-acidic (chain A), and wt-gB-acidic (chain A)—onto the previously determined wt-gB-neutral structure (chain A). The same chains are shown in all subsequent structure figures. Single protomers are shown for clarity. All superpositions were performed with Coot (12) using the entire wt-gB-neutral structure as reference molecule. For any pairwise superposition, the root mean square deviation (RMSD) over all Cα atoms is less than 1.25 Å (see Table S2 in the supplemental material). Relative to the colored protomer in panel A, the protomers in panel C are rotated 40° to the viewer's left around the vertical axis.

FIG. 2.

Low-pH-dependent conformational change in fusion loop FL2. (A) Overlay of the fusion loop regions, in side view. For clarity, only single protomers are shown. The inward and the outward conformations of the FL2 are labeled. The color scheme is the same as in Fig. 1C. The curved arrow indicates low-pH-induced movement of FL2. W174 and Y179 on FL1 are indicated by red asterisks. Residues 261 to 262 are missing in the wt-gB-acidic structure, and the flanking residues 260 and 263 are connected with a black dotted line. (B) Surface view of the fusion loop region. The wt-gB-neutral structure, with residues 258 to 264 and the nitrogen of Y265 omitted, is shown as a white surface. FL2 in each of the five structures is shown as a Cα trace with H263 shown as sticks. All superpositions were performed on residues 185 to 250 using Coot (12) and wt-gB-neutral trimer (chain A) as a reference molecule.

FIG. 3.

Interactions stabilizing the conformation of FL2. (A to E) FL2, residues 258 to 264, is shown as a Cα trace with important side chains shown as sticks. The color scheme is the same as in Fig. 1C and 2. wt-gB-acidic (C) is missing residues 261 to 262; their presumable Cα trace is shown as a dotted yellow line, based on their positions in Y179S-acidic. The gB trimer is shown as a white surface, with residues 258 to 264 and the nitrogen of Y265 omitted. wt-gB-neutral (A) and Y179S-basic (B) structures have FL2 in the inward conformation, whereas wt-gB-acidic (C), Y179S-acidic (D), and W174R-acidic (E) structures have FL2 in the outward conformation. Salt bridges are shown with black dotted lines. The locations of residues Y265 and W174, which form van der Waals interactions with the H263 side chain in the inward conformation of FL2, are labeled in panels A and B. All structures were superposed as described in Fig. 2, and the views in panels A to E are identical. (F) FL2 has distinct conformations in the Y179S-acidic and W174R-acidic structures. Side chains of H263, W174, and R174 are shown as sticks. A red dotted line shows the distance between the closest atoms of R174 from W174R-acidic and H263 from Y179S-acidic.

FIG. 4.

Low-pH conformational changes affect MAb epitopes. Side-by-side surface views of domains I and V of the wt-gB-neutral (A) and W714R-acidic structures (B) (bottom panels) and an enlarged view of the H126 epitope in these structures (top panels). Regions are colored by epitope, and FL2 is colored orange. The side chain of H308 is shown as yellow sticks. The nearby Y303 side chain is shown in purple. Y303N is the MAb resistance mutant (mar) for the H126 neutralizing antibody. Views in panel A and B are shown in the same orientation, obtained by superposing the entire W174R-acidic trimer onto the wt-gB-neutral trimer.

FIG. 5.

Effect of low pH on the overall structure of the gB ectodomain. (A) Electron micrographs of wt gB730 at pH 7.6 (top) and pH 3.0 (bottom). The distinct “crown” and “base” ends are labeled with red arrows in two representative particles, as well as on the surface representation of wt-gB-neutral structure. (B) An overlay of the size exclusion chromatograms for wt gB730 at pH 7.6 (blue) or 3.0 (red) shows that the protein elutes in the same volume at either pH. (C and D) Effect of pH on the electrophoretic mobility of gB730. Purified protein was incubated at the indicated pH and analyzed by SDS-PAGE (0.1% SDS). Samples labeled with “N” were neutralized to pH 7.5 prior to loading. Identical protein amounts were loaded into each lane. ***, Trimer; *, monomer. (C) Coomassie blue-stained gel. (D) Western blot using trimer-specific MAb DL16.