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. 2014 Oct 9;10(10):e1004377.
doi: 10.1371/journal.ppat.1004377. eCollection 2014 Oct.

Structural basis for the recognition of human cytomegalovirus glycoprotein B by a neutralizing human antibody

Nadja Spindler  1 , Uschi Diestel  2 , Joachim D Stump  3 , Anna-Katharina Wiegers  1 , Thomas H Winkler  4 , Heinrich Sticht  3 , Michael Mach  1 , Yves A Muller  2
Affiliations

Structural basis for the recognition of human cytomegalovirus glycoprotein B by a neutralizing human antibody

Nadja Spindler et al. PLoS Pathog. .

Abstract

Human cytomegalovirus (HCMV) infections are life-threating to people with a compromised or immature immune system. Upon adhesion, fusion of the virus envelope with the host cell is initiated. In this step, the viral glycoprotein gB is considered to represent the major fusogen. Here, we present for the first time structural data on the binding of an anti-herpes virus antibody and describe the atomic interactions between the antigenic domain Dom-II of HCMV gB and the Fab fragment of the human antibody SM5-1. The crystal structure shows that SM5-1 binds Dom-II almost exclusively via only two CDRs, namely light chain CDR L1 and a 22-residue-long heavy chain CDR H3. Two contiguous segments of Dom-II are _targeted by SM5-1, and the combining site includes a hydrophobic pocket on the Dom-II surface that is only partially filled by CDR H3 residues. SM5-1 belongs to a series of sequence-homologous anti-HCMV gB monoclonal antibodies that were isolated from the same donor at a single time point and that represent different maturation states. Analysis of amino acid substitutions in these antibodies in combination with molecular dynamics simulations show that key contributors to the picomolar affinity of SM5-1 do not directly interact with the antigen but significantly reduce the flexibility of CDR H3 in the bound and unbound state of SM5-1 through intramolecular side chain interactions. Thus, these residues most likely alleviate unfavorable binding entropies associated with extra-long CDR H3s, and this might represent a common strategy during antibody maturation. Models of entire HCMV gB in different conformational states hint that SM5-1 neutralizes HCMV either by blocking the pre- to postfusion transition of gB or by precluding the interaction with additional effectors such as the gH/gL complex.

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Conflict of interest statement

I have read the journal's policy and the authors of this manuscript have the following competing interests: NS, HS, THW and MM are inventors on a patent application on the antibody described. This does not alter our adherence to all PLOS policies on sharing data and materials.

Figures

Figure 1
Figure 1. HCMV gB Dom-II recognition by antibody SM5-1.
(A) Trimeric model of HCMV glycoprotein B (gB) derived from the crystal structure of the homologous HSV-1 gB protein (PDB ID 2gum, [18]). Of the different gB domains, Dom-II is highlighted in green. N- (Thr-112) and C-terminal (Ser-438) amino acids of the Dom-II expression construct are labelled. Dashed lines represent regions that were not resolved in the crystal structure of HSV-1 gB and are therefore excluded from the model. (B) Cartoon representation of the Dom-II-SM5-1 Fab complex determined at an atomic resolution of 2.1 Å. Dom-II is colored in green, SM5-1 light and heavy chains are colored in red and blue, respectively. The CDR loops of the heavy chain are shown in orange and those of the light chain in yellow. Two segments of gB (residues 112–132 and 344–438) were fused together by an artificial linker segment in order to obtain a sequence-contiguous Dom-II protein . (C) Top view of the antigen-binding site with the structure of Dom-II omitted in the presentation. Colors are as in (B). CDR loops are labeled L1 to L3 and H1 to H3. (D) 2mFo-DFc map contoured at 1σ and displayed within 2.5 Å around any CDR H3 atoms. Contiguous electron density is observed for the entire H3 loop in the antigen-bound structure. (E) Surface representation of the antigen-binding site of SM5-1 oriented as in (C). Shown in white is the surface area of SM5-1 that becomes buried upon antigen binding.
Figure 2
Figure 2. Crystal structures of unbound Dom-II and SM5-1.
(A) Cartoon representation of unbound engineered Dom-II solved at 1.8 Å resolution. No electron density is observed for the artificially introduced linker amino acids (grey dashed line). (B) Cartoon representation of unbound Fab SM5-1 solved at 1.9 Å resolution. The heavy and light chains are colored in blue and red, respectively. The CDR loops are depicted either in orange (H1 to H3) or yellow (L1 to L3). In the free SM5-1 structure, the polypeptide chain between residues H3 107 and 111 as well as heavy chain residues 145 to 152 could not be traced. (C) Superposition of Dom-II in the unbound (in green) and Fab SM5-1-bound state (in black). (D) Superposition of Fab SM5-1 in the unbound state (in color) and in the Dom-II-bound state (in black). The variable domains were superimposed, and the superposition shows that the differences between both structures are limited to CDR H3 and changes in the elbow angle (indicated by an arrow). (E) Close-up showing residual positive mFo-DFc difference electron density in the unbound Dom-II crystal structure. The density is contoured at a 3σ level and extends across a crystallographic two-fold axis (black oval).
Figure 3
Figure 3. Contacts between Dom-II and SM5-1 together with an alignment of the CDRs of related antibodies.
Black lines indicate intermolecular distances smaller than 3.5 Å. The Dom-II sequence is reported in its entirety, while for SM5-1 only the amino acid sequences of the CDRs and of frame work residue Lys67 are displayed. Asterisks indicate mutations in engineered Dom-II in the complex. The CDR sequences of the highly related antibodies are provided and listed either above or below the SM5-1 sequence. Amino acids that differ in any of the sequences are highlighted in red. SM5-1 residues that were substituted in silico for the molecular dynamics simulations are underlined.
Figure 4
Figure 4. Details of the Dom-II-SM5-1 Fab recombining site.
(A) Side chain-specific interactions between CDR H3 and residues 359-EAED-362 of Dom-II. Hydrogen bond distances are reported in Å. (B) Focus on the interaction between SM5-1 and Dom-II residues 379-KQEVN-383. (C, D) Alternative representation of the interactions formed between SM5-1 Fab and Dom-II segment 359-EAED-362 (C) and 379-KQEVN-383 (D). Hydrogen bonds within a distance of <3.5 Å are depicted in black, >3.5 Å in grey. (E) Residue Leu109 from CDR H3 shields a hydrophobic pocket in Dom-II. Side chains lining the hydrophobic cavity (dotted circle) are shown as sticks models. Grey dots highlight the segments 359-EAED-362 and 379-KQEVN-383 of Dom-II.
Figure 5
Figure 5. Neutralization capacity of SM5-1 and of partially germline-reverted SM5-1germ.
Viruses were preincubated with the antibody for 1 h and fibroblasts were infected with the virus-antibody mixture. Medium was changed 4 h after infection. 24 h post infection luciferase activity was measured in relative light units (RLU) as described previously and percent neutralization was calculated . Shown is one data set from two independent experiments.
Figure 6
Figure 6. Conformational flexibility of SM5-1 and SM5-1*.
(A) Plot of the root mean square fluctuations (RMSF) per residue indicating the enhanced flexibility of residues 102–112 (CDR H3) in SM5-1* (red line) compared to SM5-1 (black line). Sequence positions that differ between SM5-1 and SM5-1* are marked by a yellow asterisk. (B, C) Overlay of 6 structures collected every 20 ns over the simulation time for SM5-1 (B) and SM5-1* (C). Note that the CDR H3 loop in SM5-1* exhibits a higher flexibility and deviates further from the Dom-II-bound conformation which served as starting structure. The six residues that are different between SM5-1 and SM5-1* are shown in stick presentation and a pink arrow points towards the CDR H3 loop.
Figure 7
Figure 7. SM5-1 Fab is able to bind to both a prefusion and a postfusion model of entire trimeric HCMV gB.
(A) Prefusion conformation of trimeric HCMV gB modelled according to the crystal structure of VSV-G (PDB ID 2j6j, [19], [26]). (B and C) Two orthogonal views of SM5-1 Fab bound to the prefusion conformation of gB. (D) Postfusion conformation of HCMV gB modelled according to the crystal structure of HSV-1 gB (PDB ID 2gum, [18]). (E and F) SM5-1 bound to the postfusion conformation of gB. The arrow linking panel A to C indicates the conformational transition from a prefusion to a postfusion model of HCMV gB. The arrows linking panel A and B as well as D and E represent SM5-1 binding. Trimeric gB is shown in a surface representation and colored white, grey and black with gB Dom-II highlighted in green. SM5-1 Fab is shown in a cartoon representation.
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YAM and HS acknowledge support from Deutsche Forschungsgemeinschaft CRC-796: Reprogramming host cells by microbial effectors (www.dfg.de)and MM from Deutsche Forschungsgemeinschaft (DFG) grant: MA 929/11-1 (www.dfg.de). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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