BLIP-II Employs Differential Hotspot Residues To Bind Structurally Similar Staphylococcus aureus PBP2a and Class A β-Lactamases

Biochemistry. 2017 Feb 28;56(8):1075-1084. doi: 10.1021/acs.biochem.6b00978. Epub 2017 Feb 16.


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 KD in 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 kon for 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Kinetics
  • Models, Molecular
  • Penicillin-Binding Proteins / chemistry*
  • Penicillin-Binding Proteins / metabolism*
  • Protein Binding
  • Protein Domains
  • Staphylococcus aureus / enzymology*
  • Substrate Specificity
  • beta-Lactamases / chemistry*
  • beta-Lactamases / metabolism*


  • Bacterial Proteins
  • Penicillin-Binding Proteins
  • beta-Lactamases