Abstract
Owing to the thermal instability and low affinity of BlaR-CTD to some β-lactams, the receptor assay based on BlaR-CTD is limited in the detection of abundant variety of drugs and the result is often unstable. In this study, the three-dimensional structure of BlaR-CTD from Bacillus licheniformis ATCC14580 was constructed by homologous modeling based on the crystal structure of BlaR-CTD from B. licheniformis 749/I, and the binding sites of this protein to 40 β-lactams were also obtained by molecular docking. To improve the stability and affinity of the protein, 23 mutant proteins were designed based on docking and homologous alignment results as well as by inserting disulfide bond and building the salt bridge. The mutation was rationality evaluated by SIFT and PloyPhen2 software. The heterologous expressed and purified mutant proteins were then subjected to the activity and stability assay. It was shown that among all mutant proteins, I188K/S19C/G24C, A138E/R50C/Q147C and S190Y/E183C/I188K respectively exhibited a higher affinity to 33, 22 and 21 β-lactams than the wild-type protein, while I188K/S19C/G24C exhibited the best stability. This may due to that the conformation of the active site in mutant protein I188K/S19C/G24C changed, and the random coli in the surface of protein activity increased. Our study suggests a possible structure-function relationship on the stability and affinity of BlaR-CTD, which provides new insights into protein rational design study and lays a solid foundation for establishing the receptor-based screening assay for the detection of β-lactam residues.
Highlights
The penicillin binding protein (PBP) BlaR is a signal transduction membrane protein which induces the synthesis of β-lactamase
Powell et al mutated the amino acid (AA) around the active site of the PBP2 from Neisseria gonorrhoeae, and the results showed that the acylation efficiency for penicillin G was reduced [26]
Evaluation of the affinity of BlaR-CTD mutant proteins The recombinant BlaR-CTD protein originating from B. licheniformis ATCC 14580 and heterologously expressed in E. coli contained a total of 288 residues, corresponding to a theoretical molecular mass of 32,427 Da, since it contained an N-terminal extension sequence originating from pET28 vector (Additional file 1: Figure S3, amino acid residue numbers from − 34 to − 1)
Summary
The penicillin binding protein (PBP) BlaR is a signal transduction membrane protein which induces the synthesis of β-lactamase. The C-terminal domain of BlaR (BlaR-CTD) protein is located in the extracellular region which acts as a drug binding site [19]. BlaR-CTD is firstly the sensor domain of a penicillin receptor that is acylated by penicillin. This protein can identify and bind to a variety of β-lactams [8, 12]. In the active site of the protein, STYK, serine was a key amino acid (AA) that can participate in the binding of β-lactams [31]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
