title: Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus.
abstract: Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM.
  
  
  OBJECTIVE
  Using cryo-electron microscopy and new direct electron detector technology, we have solved the 4 Å resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified by using a 12 Å structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time.
authors.affiliation: Department of Medicine, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA.
authors.forename: Lindsey J
authors.initials: LJ
authors.lastname: Organtini
authors.affiliation: Department of Medicine, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA.
authors.forename: Hyunwook
authors.initials: H
authors.lastname: Lee
authors.affiliation: Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.
authors.forename: Sho
authors.initials: S
authors.lastname: Iketani
authors.affiliation: Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Huang
authors.affiliation: Department of Medicine, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Ashley
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Makhov
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
authors.forename: James F
authors.initials: JF
authors.lastname: Conway
authors.affiliation: Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.
authors.forename: Colin R
authors.identifier: http://orcid.org/0000-0002-1836-6655
authors.initials: CR
authors.lastname: Parrish
authors.affiliation: Department of Medicine, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA shafenstein@hmc.psu.edu.
authors.forename: Susan
authors.initials: S
authors.lastname: Hafenstein
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D057134:Antibodies, Neutralizing / Q000737:chemistry* / Q000276:immunology*; D000914:Antibodies, Viral / Q000737:chemistry* / Q000276:immunology*; D002213:Capsid / Q000276:immunology; D036022:Capsid Proteins / Q000276:immunology; D004285:Dogs; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology*; D009500:Neutralization Tests / Q000379:methods; D017993:Parvovirus, Canine / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D036022:Capsid Proteins; D007140:Immunoglobulin Fab Fragments; D057127:Single-Chain Antibodies