Crystal structures of mono- and bi-specific diabodies and reduction of their structural flexibility by introduction of disulfide bridges at the Fv interface.
Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Diabodies are engineered antibody fragments that are composed of two Fv domains connected by short peptide linkers. They are attractive candidates for mediators in assembling protein nano-structures because they can simultaneously bind to two different proteins and are rigid enough to be crystallized. However, comparison of previous crystal structures demonstrates that there is substantial structural diversity in the Fv interface region of diabodies and, therefore, reliable prediction of its structure is not trivial. Here, we present the crystal structures of ten mono- and bi-specific diabodies. We found that changing an arginine residue in the Fv interface to threonine greatly reduced the structural diversity of diabodies. We also found that one of the bispecific diabodies underwent an unexpected process of chain swapping yielding a non-functional monospecific diabody. In order to further reduce structural flexibility and prevent chain shuffling, we introduced disulfide bridges in the Fv interface regions. The disulfide-bridged diabodies have rigid and predictable structures and may have applications in crystallizing proteins, analyzing cryo-electron microscopic images and building protein nano-assemblies.
Graduate School of Nanoscience and Technology, KAIST, Daejeon, Korea.
Jin Hong
JH
Kim
Agency for Defense Development, Daejeon, Korea.
Dong Hyun
DH
Song
Department of Chemistry, KAIST, Daejeon, Korea.
Suk-Jun
SJ
Youn
Department of Chemistry, KAIST, Daejeon, Korea.
Ji Won
JW
Kim
Department of Chemistry, KAIST, Daejeon, Korea.
Geunyoung
G
Cho
Department of Biological Sciences, KAIST, Daejeon, Korea.
Sun Chang
SC
Kim
Institute of Biotechnology, Chungnam National University, Daejeon, Korea.
Hayyoung
H
Lee
School of Life Sciences, GIST, Gwangju, Korea.
Mi Sun
MS
Jin
Graduate School of Nanoscience and Technology, KAIST, Daejeon, Korea.|Department of Chemistry, KAIST, Daejeon, Korea.
Jie-Oh
JO
Lee
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