Crystal structures of mono- and bi-specific diabodies and reduction of their structural flexibility by introduction of disulfide bridges at the Fv interface

• DOI: 10.1038/srep34515
• Publication Date: 2016-09-29T09:40:10Z
• Authors: Jin Hong Kim; Dong Hyun Song; Suk-Jun Youn; Ji Won Kim; Geunyoung Cho; Sun Chang Kim; Hayyoung Lee; Mi Sun Jin; Jie-Oh Lee
• Keywords: Protein Engineering

Abstract Building a sophisticated protein nano-assembly requires method for linking components in predictable and stable structure. Diabodies are engineered antibody fragments that composed of two Fv domains connected by short peptide linkers. They attractive candidates mediators assembling nano-structures because they can simultaneously bind to different proteins rigid enough be crystallized. However, comparison previous crystal structures demonstrates there is substantial structural diversity the interface region diabodies and, therefore, reliable prediction its structure not trivial. Here, we present ten mono- bi-specific diabodies. We found changing an arginine residue threonine greatly reduced also one bispecific underwent unexpected process chain swapping yielding non-functional monospecific diabody. In order further reduce flexibility prevent shuffling, introduced disulfide bridges regions. The disulfide-bridged have may applications crystallizing proteins, analyzing cryo-electron microscopic images building nano-assemblies.