TY - JOUR
T1 - Targeted protein engineering provides insights into binding mechanism and affinities of bacterial collagen adhesins
AU - Ross, Caná L.
AU - Liang, Xiaowen
AU - Liu, Qing
AU - Murray, Barbara E.
AU - Höök, Magnus
AU - Ganesh, Vannakambadi K.
N1 - Copyright:
Copyright 2013 Elsevier B.V., All rights reserved.
PY - 2012/10/5
Y1 - 2012/10/5
N2 - The collagen-binding bacterial proteins, Ace and Cna, are well characterized on the biochemical and structural level. Despite overall structural similarity, recombinant forms of the Ace and Cna ligand-binding domains exhibit significantly different affinities and binding kinetics for collagen type I (CI) in vitro. In this study, we sought to understand, in submolecular detail, the bases for these differences. Using a structure-based approach, we engineered Cna and Ace variants by altering specific structural elements within the ligand-binding domains. Surface plasmon resonance-based binding analysis demonstrated that mutations that are predicted to alter the orientation of the Ace and Cna N1 and N2 subdomains significantly affect the interaction between the MSCRAMM (microbial surface components recognizing adhesive matrix molecule) and CI in vitro, including affinity, association/dissociation rates and binding ratio. Moreover, we utilized this information to engineer an Ace variant with an 11,000-fold higher CI affinity than the parent protein. Finally, we noted that several engineered proteins that exhibited a weak interaction with CI recognized more sites on CI, suggesting an inverse correlation between affinity and specificity.
AB - The collagen-binding bacterial proteins, Ace and Cna, are well characterized on the biochemical and structural level. Despite overall structural similarity, recombinant forms of the Ace and Cna ligand-binding domains exhibit significantly different affinities and binding kinetics for collagen type I (CI) in vitro. In this study, we sought to understand, in submolecular detail, the bases for these differences. Using a structure-based approach, we engineered Cna and Ace variants by altering specific structural elements within the ligand-binding domains. Surface plasmon resonance-based binding analysis demonstrated that mutations that are predicted to alter the orientation of the Ace and Cna N1 and N2 subdomains significantly affect the interaction between the MSCRAMM (microbial surface components recognizing adhesive matrix molecule) and CI in vitro, including affinity, association/dissociation rates and binding ratio. Moreover, we utilized this information to engineer an Ace variant with an 11,000-fold higher CI affinity than the parent protein. Finally, we noted that several engineered proteins that exhibited a weak interaction with CI recognized more sites on CI, suggesting an inverse correlation between affinity and specificity.
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U2 - 10.1074/jbc.M112.371054
DO - 10.1074/jbc.M112.371054
M3 - Article
C2 - 22865854
AN - SCOPUS:84867261136
VL - 287
SP - 34856
EP - 34865
JO - The Journal of biological chemistry
JF - The Journal of biological chemistry
SN - 0021-9258
IS - 41
ER -