BLIP-II Employs Differential Hotspot Residues To Bind Structurally Similar Staphylococcus aureus PBP2a and Class A β-Lactamases.
The interaction of β-lactamase inhibitory protein II (BLIP-II) with β-lactamases serves as a model system to investigate the principles underlying protein-protein interactions. Previous studies have focused on identifying the determinants of binding affinity and specificity between BLIP-II and class A β-lactamases. However, interactions between BLIP-II and other bacterial proteins have yet to be explored. Here, we provide evidence that BLIP-II binds penicillin binding protein 2a (PBP2a) from methicillin-resistant Staphylococcus aureus (MRSA) with a KDin the low micromolar range. In comparison to the binding constants for the potent interaction between BLIP-II and TEM-1 β-lactamase (KD= 0.5 pM), the on-rate for BLIP-II binding PBP2a is 44 000 times slower and the off-rate is 170 times faster. Therefore, a slow association rate is a limiting factor for the potency of the interaction between BLIP-II and PBP2a. Results from alanine scanning mutagenesis of the predicted interface residues of BLIP-II indicate that charged residues on the periphery of the BLIP-II interface play a critical role for binding PBP2a, in contrast to previous findings that aromatic residues at the center of the BLIP-II interface are critical for the interaction with β-lactamases. Interestingly, many of the alanine mutants at the BLIP-II interface increase konfor binding PBP2a, consistent with the association rate being a limiting factor for affinity. In summary, the results of the study reveal that BLIP-II binds PBP2a, although weakly compared to binding of β-lactamases, and provides insights into the different binding strategies used for these targets.
Department of Biochemistry and Molecular Biology, ‡Department of Pharmacology, Baylor College of Medicine , Houston, Texas 77030, United States.
Carolyn J
CJ
Adamski
Department of Biochemistry and Molecular Biology, ‡Department of Pharmacology, Baylor College of Medicine , Houston, Texas 77030, United States.
Timothy
0000-0002-5267-0001
T
Palzkill
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D046915:Penicillin-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D000072417:Protein Domains; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism*
D001426:Bacterial Proteins; D046915:Penicillin-Binding Proteins; D001618:beta-Lactamases