title: Targeted protein engineering provides insights into binding mechanism and affinities of bacterial collagen adhesins.
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.
authors.affiliation: Center for Inflammatory and Infectious Disease, Institute for Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas 77030, USA.
authors.forename: Caná L
authors.initials: CL
authors.lastname: Ross
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Liang
authors.forename: Qing
authors.initials: Q
authors.lastname: Liu
authors.forename: Barbara E
authors.initials: BE
authors.lastname: Murray
authors.forename: Magnus
authors.initials: M
authors.lastname: Höök
authors.forename: Vannakambadi K
authors.initials: VK
authors.lastname: Ganesh
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry* / Q000235:genetics; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D003094:Collagen / Q000737:chemistry* / Q000235:genetics; D013293:Enterococcus faecalis / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013211:Staphylococcus aureus / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: C121675:Ace protein, Enterococcus; D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D002352:Carrier Proteins; C048165:adhesin, Staphylococcus aureus; D003094:Collagen