Structural insight into the kinetics and DeltaCp of interactions between TEM-1 beta-lactamase and beta-lactamase inhibitory protein (BLIP)

Abstract

In a previous study, we examined thermodynamic parameters for 20 alanine mutants in beta-lactamase inhibitory protein (BLIP) for binding to TEM-1 beta-lactamase. Here we have determined the structures of two thermodynamically distinctive complexes of BLIP mutants with TEM-1 beta-lactamase. The complex BLIP Y51A-TEM-1 is a tight binding complex with the most negative binding heat capacity change (DeltaG = approximately -13 kcal mol(-1) and DeltaCp = approximately -0.8 kcal mol(-1) K(-1)) among all of the mutants, whereas BLIP W150A-TEM-1 is a weak complex with one of the least negative binding heat capacity changes (DeltaG = approximately -8.5 kcal mol(-1) and DeltaCp = approximately -0.27 kcal mol(-1) K(-1)). We previously determined that BLIP Tyr51 is a canonical and Trp150 an anti-canonical TEM-1-contact residue, where canonical refers to the alanine substitution resulting in a matched change in the hydrophobicity of binding free energy. Structure determination indicates a rearrangement of the interactions between Asp49 of the W150A BLIP mutant and the catalytic pocket of TEM-1. The Asp49 of W150A moves more than 4 angstroms to form two new hydrogen bonds while losing four original hydrogen bonds. This explains the anti-canonical nature of the Trp150 to alanine substitution, and also reveals a strong long distance coupling between Trp150 and Asp49 of BLIP, because these two residues are more than 25 angstroms apart. Kinetic measurements indicate that the mutations influence the dissociation rate but not the association rate. Further analysis of the structures indicates that an increased number of interface-trapped water molecules correlate with poor interface packing in a mutant. It appears that the increase of interface-trapped water molecules is inversely correlated with negative binding heat capacity changes.

FIGURE 1.

The structure of the BLIP-TEM-1 β-lactamase complex indicating the location of several interesting residues and their binding thermodynamics. A, structural representation of BLIP-TEM-1 complex. TEM-1 is represented as a schematic and colored in cyan. BLIP is represented in a white surface model, and the TEM-1 contact residues are colored in yellow. BLIP Trp¹⁵⁰ is represented as a CDW model colored in red, and Tyr⁵¹ is represented as a CDW model colored in green. Asp⁴⁹ and Trp¹¹² are represented in bond models colored red. B, plots of different forms of free energy versus temperature for the formation of four different BLIP mutant-TEM-1 β-lactamase complexes. The thin curves represent the binding free energy, ΔG⁰s, as function of temperature, and are calculated from Equation 1 using the experimentally determined ΔH, ΔS, and ΔCp values. × indicates the specific experimental measurements (16). The hypothetical hydrophobic component “ΔG⁰_hydrs” is calculated using the Equation 2 with the experimentally determined ΔCp and is shown as thick dark curves. The nonhydrophobic component, “ΔG⁰_nonhydrs,” is calculated from Equation 3 in the text and represented as linear dashed lines.

FIGURE 2.

Crystallographic analysis of BLIP mutant-TEM-1 complexes. A, selection of four regions of the electron density maps centered on the mutation sites shows the maps match the mutated residues. B, three-dimensional alignment of α-carbon chains of BLIP mutants from three complex structures. Regions that exhibit large conformational changes are circled. C, comparison of the structures around the Gln⁹⁹ of TEM-1 β-lactamase. The wild type complex is colored red. The Y51A complex is colored blue, showing the Gln⁹⁹ side chain is positioned differently from that in wild type despite the fact that both complexes have the Gln⁹⁹ contact with the Trp¹⁵⁰ of BLIP, with different hydrogen bond configuration between the Gln⁹⁹ and His¹⁴⁸ of the BLIP. The green represents the W150A mutant complex, showing the Gln⁹⁹ residue undergoes a rather large conformational change but retains the original hydrogen bond between TEM-1 Gln⁹⁹ and His¹⁴⁸ of BLIP. D, close-up view of the Asp⁴⁹ region of the BLIPs and the catalytic pocket of TEM-1. It shows the large movement of Asp⁴⁹ in the W150A complex (green). The hydrogen bonds are completely different between Asp⁴⁹ of the BLIP W150A mutant and the catalytic pocket of the TEM-1.

FIGURE 3.

Analysis of interface-trapped water molecules. A, locations of all the identified water molecules in the crystal structures of Y51A-TEM-1 and of W150A-TEM-1 complexes. The complexes were superposed using SUPERPOSE in the CCP4 package. TEM-1 is represented as orange tubes, and the BLIP mutant is in green schematic representation. Green CPK balls are the identified water molecules from the AB complex of W150A-TEM-1 crystal structure, and green dotted circles represent water molecules from the CD complex. Red CPK balls are the identified water molecules from the AB complex of Y51A-TEM-1 crystal structure, and red dotted circles are the water molecules from the CD complex of Y51A-TEM-1 crystal. B, locations of the identified interfacial water molecules on the surface of the BLIP mutants. The BLIP mutants are represented as molecular surfaces. The interfacial water molecules are represented as red balls (for the AB complex in the asymmetric unit) or as dotted spheres (for the CD complex in the asymmetric unit). The W150A-TEM-1 complex is at left, the wild type complex is at center, and to the right is the Y51A-TEM-1 complex. C, histogram of the identified electron density peaks within the BLIP mutant-TEM-1 interfaces. The electron density peaks with B values less than 50 Ų are assigned as water.

FIGURE 4.

Plot of the number of the selected intermolecular atom pairs located within various distance ranges from the TEM-1 surface versus the distance. Selection criterion is the shortest intermolecular atom pairs for each BLIP atom. The solid thick line with the square symbols indicates the intermolecular atom pair distribution of the Y51A-TEM-1 complex that is 20% denser than W150A-TEM-1 complex (thin line with circle symbols) at ∼3.6 Å.

FIGURE 5.

Intrinsic fluorescence signals from the proteins and complexes. Time course traces of the intrinsic fluorescence signals that accompany the association of the BLIP mutants and TEM-1 β-lactamase from stopped-flow spectrometry measurements are shown in the right panels (signals are shown as percent of the average of the equilibrium fluorescence signals). Fluorescence emission spectra are shown in the left panels with the signals normalized to the maximum of the TEM-1 fluorescence peaks. Spectra are labeled with each individual protein name for the corresponding spectra; “complex” is for the spectra of complex solution, and the “sum” labels the arithmetic sum of the two individual protein spectra. At top is the association of the W150A BLIP mutant and TEM-1 fitted with second order kinetics (red line) with a k_on of 2.9 × 10⁵ m^-1 s^-1. The middle graph is from the association of the Y51A BLIP mutant and TEM-1 fitted with second order kinetics (red line) with a k_on value of 3.9 × 10⁵ m^-1 s^-1. At bottom is the association of the W150A BLIP mutant and TEM-1 fitted with second order kinetics (red line) with k_on value of 2.0 × 10⁵ m^-1 s^-1.

FIGURE 6.

Measurements of dissociation rates of the BLIP mutant-TEM-1 β-lactamase complexes. A, plot of the time course of cephalosporin C hydrolysis by TEM-1 after a 7.3-fold dilution of 10 μm of preformed W150A-TEM-1 complex (circles). The curve is a fit of the on- and off-rates simultaneously with an on-rate of 8.4 ± 4 × 10⁵ m^-1 s^-1 and an off-rate of 0.19 ± 0.05 s^-1. B, plot of the time course of intrinsic fluorescence after a 37-fold dilution of 3 μm of preformed W150A-TEM-1 complex (cross). The curve is a fit of the on- and off-rates simultaneously with an on-rate of 3.4 ± 2 × 10⁵ m^-1 s^-1 and an off-rate of 0.05 ± 0.03 s^-1. C, plot of the time course of cephalosporin C hydrolysis activity that is reflective of the fractional amount of TEM-1 after dilution and mixing of the preformed Y51A-TEM-1 complex into 10 μm of inactive S70A TEM-1 mutant. The curve is a first order kinetics fit with k_off = 0.00009/s. D, plot of the time course of cephalosporin C hydrolysis by TEM-1 after dilution and mixing of the preformed wild type complex into 10 μm of inactive S70A TEM-1 mutant. The curve is a first order kinetics with k_off = 0.00013/s.