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Thermodynamic investigation of the role of contact residues of beta-lactamase-inhibitory protein for binding to TEM-1 beta-lactamase.

Wang J, Zhang Z, Palzkill T, Chow DC

Department of Chemistry, University of Houston, Houston, Texas 77204-5003, USA.

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).

Published 11 June 2007 in J Biol Chem, 282(24): 17676-84.
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