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Targeted protein engineering provides insights into binding mechanism and affinities of bacterial collagen adhesins.
Ross, Caná L; Liang, Xiaowen; Liu, Qing; Murray, Barbara E; Höök, Magnus; Ganesh, Vannakambadi K.
Afiliação
  • Ross CL; Center for Inflammatory and Infectious Disease, Institute for Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas 77030, USA.
J Biol Chem ; 287(41): 34856-65, 2012 Oct 05.
Article em En | MEDLINE | ID: mdl-22865854
ABSTRACT
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 N(1) and N(2) 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.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Staphylococcus aureus / Proteínas de Bactérias / Engenharia de Proteínas / Proteínas de Transporte / Colágeno / Enterococcus faecalis / Adesinas Bacterianas Idioma: En Revista: J Biol Chem Ano de publicação: 2012 Tipo de documento: Article País de afiliação: Estados Unidos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Staphylococcus aureus / Proteínas de Bactérias / Engenharia de Proteínas / Proteínas de Transporte / Colágeno / Enterococcus faecalis / Adesinas Bacterianas Idioma: En Revista: J Biol Chem Ano de publicação: 2012 Tipo de documento: Article País de afiliação: Estados Unidos