Thermodynamic Investigation of the Role of Contact Residues of β-Lactamase-inhibitory Protein for Binding to TEM-1 β-Lactamase

Zhen Zhang,Dar-Chone Chow,Jihong Wang,Timothy Palzkill

doi:10.1074/jbc.m611548200

Zhen Zhang, Dar-Chone Chow + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m611548200

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Abstract

We have determined the thermodynamics of binding for the interaction between TEM-1 beta-lactamase and a set of alanine substituted contact residue mutants ofbeta-lactamase-inhibitory protein (BLIP) using isothermal titration calorimetry. The binding enthalpies for these interactions are highly temperature dependent, with negative binding heat capacity changes ranging from -800 to -271 cal mol(-1) K(-1). The isoenthalpic temperatures (at which the binding enthalpy is zero) of these interactions range from 5 to 38 degrees C. The changes in isoenthalpic temperature were used as an indicator of the changes in enthalpy and entropy driving forces, which in turn are related to hydrophobic and hydrophilic interactions. A contact residue of BLIP is categorized as a canonical residue if its alanine substitution mutant exhibits a change of isoenthalpic temperature matching the change of hydrophobicity because of the mutation. A contact position exhibiting a change in isoenthalpic temperature that does not match the change in hydrophobicity is categorized as an anti-canonical residue. Our experimental results reveal that the majority of residues where alanine substitution results in a loss of affinity are canonical (7 of 10), and about half of the residues where alanine substitutions have a minor effect are canonical. The interactions between TEM-1beta-lactamase and BLIP canonical contact residues contribute directly to binding free energy, suggesting potential anchoring sites for binding partners. The anti-canonical behavior of certain residues may be the result of mutation-induced modifications such as structural rearrangements affecting contact residue configurations. Structural inspection of BLIP suggests that the Lys(74) side chain electrostatically holds BLIP loop 2 in position to bind to TEM-1 beta-lactamase, explaining a large loss of entropy-driven binding energy of the K74A mutant and the resulting anti-canonical behavior. The anti-canonical behavior of the W150A mutant may also be due to structural rearrangements. Finally, the affinity enhancing effect of the contact residue mutant Y50A may be due to energetic coupling interactions between Asp(49) and His(41).

Highlights

A protein-protein binding interaction involves a relatively planar interface with a large contact interface area and a large number of contact residues (Ͼ10) [2,3,4]
The analysis of thermodynamic and structural information could provide detailed information of the role of interface residues for binding interactions, and this information could be used in the future design of tight binding peptides or small molecules that could serve as inhibitors of ␤-lactamase
A solu- the displacement ITC method is applicable in this system, the tion of known concentration of a complex of TEM-1 ␤-lacta- displacement ITC measurements were performed with several mase and a low affinity BLIP mutant was placed in the sample combinations of high and low affinity mutants

Summary

EXPERIMENTAL PROCEDURES

Materials—Talon resin was purchased from Clontech (Mountain View, CA). Ion exchange media and columns (Mono Q 5/50 GL, HighTrap Q, Q Sepharose Fast Flow) and sizing columns (Superdex 75 10/300, Superdex 75 prep grade) were purchased from Amersham Biosciences. The bound BLIP mutant proteins were eluted from the Talon cobalt resins using 150 mM imidazole in Tris-buffered saline. Typical titration experiments were carried out by titrating 40 ␮M TEM-1 ␤-lactamase into 4 ␮M BLIP mutant in PBS (50 mM phosphate, pH 7.0, 150 mM NaCl). To avoid inconsistencies in different protein preparations, we pooled enough TEM-1 ␤-lactamase (ϳ60 mg) to titrate many BLIP mutants and enough wild type BLIP (ϳ7 mg) to use as the reference for every mutant titration. The proton linkage effect was tested by measuring the binding enthalpies of some BLIP mutants and TEM-1 ␤-lactamase in Tris buffer. ITC experiments of each BLIP mutant binding to TEM-1 ␤-lactamase were done in no less than three different temperatures within the range of 6 to 30 °C. The following equations were used to calculate K and ⌬H of the tight mutant provided the displacement ITC were competitive [21, 22]:

Kapparent ϭ ϩ

RESULTS

DISCUSSION

High affinity mutants