Communication
Crystal Structure of a Bivalent Antibody Fab Fragment

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Highlights

  • The Fab of human anti-HCV antibody HC84.26.5D forms bivalent dimers in solution.

  • Crystal structure revealed that Fab HC84.26.5D is a domain-swapped dimer.

  • Dimerization is mediated by deletion of one residue in the H chain elbow region.

  • Domain-swapped Fab dimers are useful as fiducial markers for cryoEM.

Abstract

We determined the crystal structure to 1.8 Å resolution of the Fab fragment of an affinity-matured human monoclonal antibody (HC84.26.5D) that recognizes the E2 envelope glycoprotein of hepatitis C virus (HCV). Unlike conventional Fabs, which are monovalent monomers, Fab HC84.26.5D assembles into a bivalent domain-swapped dimer in which the two VL/VH modules are separated by ~25 Å. In solution, Fab HC84.26.5D exists predominantly as a dimer (~80%) in equilibrium with the monomeric form of the Fab (~20%). Dimerization is mediated entirely by deletion of a single residue, VHSer113 (Kabat numbering), in the elbow region linking the VH and CH1 domains. In agreement with the crystal structure, dimeric Fab HC84.26.5D is able to bind two HCV E2 molecules in solution. This is only the second example of a domain-swapped Fab dimer from among >3000 Fab crystal structures determined to date. Moreover, the architecture of the doughnut-shaped Fab HC84.26.5D dimer is completely different from that of the previously reported Fab 2G12 dimer. We demonstrate that the highly identifiable shape of dimeric Fab HC84.26.5D makes it useful as a fiducial marker for single-particle cryoEM analysis of HCV E2. Bivalent domain-swapped Fab dimers engineered on the basis of HC84.26.5D may also serve as a means of doubling the effective size of conventional Fab–protein complexes for cryoEM.

Introduction

HC84.26 is a broadly neutralizing human monoclonal antibody (HMAb) isolated from a blood donor infected with hepatitis C virus (HCV).1 It recognizes a conformational epitope on the E2 envelope glycoprotein of HCV, designated as antigenic domain D. Affinity maturation of HC84.26 was carried out by yeast surface display of single-chain VL/VH Fv fragments to isolate variants with improved binding and neutralization activities against HCV isolates that were poorly neutralized by wild-type HC84.26.2 One of these affinity-matured variants, HC84.26.5D (KD = 6 nM), showed increased neutralization of diverse HCV isolates in vitro and was able to prevent acute HCV infection in mice. Compared to wild-type HC84.26, affinity-matured HC84.26.5D contains 22 mutations in the light chain variable (VL) region and 5 in the heavy chain variable (VH) region, in addition to a deletion of residue VHSer113 (Kabat numbering) (Figure 1).

We previously determined the crystal structure of HC84.26.5D, expressed as a single-chain Fv (Fv HC84.26.5D), bound to a 13-mer peptide representing a portion of the conformational domain D epitope recognized by this in vitro affinity-matured HMAb.2 Here we report the structure of wild-type HC84.26, expressed as a chimeric Fab fragment (Fab HC84.26chimera) in which human VL and VH regions were linked to mouse light chain constant (CL) and heavy chain constant (CH1) regions, respectively. We also report the structure of affinity-matured HC84.26.5D, expressed as an Fab (Fab HC84.26.5D) in which human VL and VH1 regions were linked to human CL and CH1 regions, respectively. Surprisingly, whereas Fab HC84.26chimera exists as a conventional Fab monomer, two Fab HC84.26.5D molecules assemble into an interlocked domain-swapped dimer that retains the ability to bind HCV E2. To date, the only other antibody shown to be domain-exchanged from among >3000 Fab structures in the Protein Data Bank (PDB) is 2G12, which recognizes oligosaccharide moieties of HIV gp120.3 However, the Fab HC84.26.5D dimer is geometrically completely distinct from the Fab 2G12 dimer. We demonstrate that domain swapping is mediated by a single amino acid deletion in the elbow region between VH and CH1 domains, and that such domain-swapped Fab dimers can be used as fiducial markers in cryo-electron microscopy (cryoEM).

Section snippets

Oligomeric state of Fab HC84.26.5D

Fab HC84.26chimera was produced by in vitro folding from E. coli inclusion bodies, while Fab HC84.26.5D was expressed by secretion from transfected Expi293 mammalian cells (Materials and Methods). We examined the oligomeric state of these proteins by size exclusion chromatography (Figure 2(a)). Fab HC84.26chimera ran as a single peak of 47 kDa, as expected for a monomer. By contrast, Fab HC84.26.5D eluted as two separate peaks: a major peak (~80%) of 118 kDa and minor peak (~20%) of 45 kDa.

Conclusions

According to the original definition of three-dimensional domain swapping,11 both monomeric and oligomeric structures must be observed for an identical protein, with both states found either in crystals or one in a crystal and the other in solution. In the case of Fab HC84.26.5D, the dimeric form is observed in the crystal, while in solution monomeric and dimeric forms exist in equilibrium. Mechanisms to explain domain swapping generally focus on the region that links the exchanging domains

Protein expression and purification

To produce recombinant affinity-matured Fab HC84.26.5D, codon-optimized genes encoding the VH and CH1 domains of the H chain and the VL and CL domains of the L chain were synthesized chemically (GenScript) and cloned into the mammalian expression vector pcDNA3.4Topo. Both chains included an N-terminal immunoglobulin κ signal sequence for secretion. A streptavidin tag (WSHPQFEK) was attached to the C-terminus of the CH1 domain for affinity purification. Fab HC84.26.5D was produced by

Acknowledgements

This work was supported was supported by National Institutes of Health Grants AI132213 (to S.K.H.F, T.R.F. and R.A.M.) and AI123862 (to S.K.H.F.). We thank Alexander V. Kolesnikov and Brian G. Pierce for valuable discussions. We also thank Sneha Rangarajan for assistance in refinements. Results in this report are based on work performed at both Structural Biology Center and SER-CAT beamlines at the Advanced Photon Source of Argonne National Laboratory, operated by UChicago Argonne, LLC, for the

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