Role of β-lactamase residues in a common interface for binding the structurally unrelated inhibitory proteins BLIP and BLIP-II

Abstract

The β-lactamase inhibitory proteins (BLIPs) are a model system for examining molecular recognition in protein-protein interactions. BLIP and BLIP-II are structurally unrelated proteins that bind and inhibit TEM-1 β-lactamase. Both BLIPs share a common binding interface on TEM-1 and make contacts with many of the same TEM-1 surface residues. BLIP-II, however, binds TEM-1 over 150-fold tighter than BLIP despite the fact that it has fewer contact residues and a smaller binding interface. The role of eleven TEM-1 amino acid residues that contact both BLIP and BLIP-II was examined by alanine mutagenesis and determination of the association (k on) and dissociation (k off) rate constants for binding each partner. The substitutions had little impact on association rates and resulted in a wide range of dissociation rates as previously observed for substitutions on the BLIP side of the interface. The substitutions also had less effect on binding affinity for BLIP than BLIP-II. This is consistent with the high affinity and small binding interface of the TEM-1-BLIP-II complex, which predicts per residue contributions should be higher for TEM-1 binding to BLIP-II versus BLIP. Two TEM-1 residues (E104 and M129) were found to be hotspots for binding BLIP while five (L102, Y105, P107, K111, and M129) are hotspots for binding BLIP-II with only M129 as a common hotspot for both. Thus, although the same TEM-1 surface binds to both BLIP and BLIP-II, the distribution of binding energy on the surface is different for the two target proteins, that is, different binding strategies are employed.

Keywords: antibiotic resistance; beta-lactamase; binding kinetics; molecular recognition; protein-protein interactions.

Figure 1

Structural comparison of BLIP-TEM-1 (PDB 1JTG) (A) and BLIP-II-TEM-1 (PDB 1JTD) (B). The protruding loop-helix region (red) and the catalytic serine 70 (blue space fill) of TEM-1 (gray) is shown as the primary binding region for both BLIP and BLIP-II (orange).

Figure 2

Kinetic characterization of TEM-1 alanine mutants association with BLIPs. A: Determination of BLIP K_i values for TEM-1 WT and E104A mutant β-lactamase for binding BLIP. B: Enzymatic activity-based measurements of dissociation between BLIP-II and TEM-1 (wild type, N100A, and K111A alanine variants). Data were fitted to first order kinetics to determine dissociation rate constants. C: Representative time-course of the stopped-flow tryptophan fluorescence to determine the association rate constants for BLIP-II/TEM-1 β-lactamase.

Figure 3

Comparison of the ΔΔG values of the TEM-1 alanine variants for binding BLIP (black) and BLIP-II (red) for association constants (A), dissociation rate constants (B) and relative binding energy change (C). Relative free energy changes are defined as the difference in energy between wild-type TEM-1 and the alanine mutant (ΔΔG = −RTln(K_d,wt/K_d,mut)). The top control line is set at 1.35 kcal/mol and the bottom control line is set at −1.35 kcal/mol, which indicates a 10-fold change.

Figure 4

TEM-1 β-lactamase binding hotpots. The TEM-1 β-lactamase structure is shown in gray (PDB ID 1XPB). The catalytic serine 70 residues is colored dark blue. A residue is defined as a hotspot if the alanine substitution reduces binding by >10-fold (Table IV). TEM-1 positions Q99, N100, E110, V216, and M272 are not hotpots for binding either BLIP or BLIP-II and are colored white. TEM-1 residue E104 is a hotspot for binding BLIP but not BLIP-II and is colored light blue. Positions L102, Y105, P107, and K111 are hotspots for binding BLIP-II but not BLIP and are colored orange. TEM-1 residue M129 is a hotspot for binding both BLIP and BLIP-II and is colored red.

Figure 5

Comparison of the ΔΔG values of the TEM-1 alanine variants for binding BLIP (A) and BLIP-II (B) as determined experimentally, and predicted by BeAtMuSiC and Robetta. For experimental data, relative free energy changes are defined as the difference in energy between wild-type TEM-1 and the alanine mutant (ΔΔG = −RTln(K_d,wt/K_d,mut)). The top control line is set at 1.35 kcal/mol and the bottom control line is set at −1.35 kcal/mol, which indicates a 10-fold change or hotspot for binding. BeAtMuSiC results are shown in green, Robetta results are shown in blue and the experimental results are shown in black.