Serendipitous Discovery of an Allosteric Zinc Binding Site in an Engineered Protein
A heterotropic allosteric effect involves an effector molecule that is distinct from the substrate or ligand of the protein. How heterotropic allostery originates is an unanswered question. The Ostermeier lab has previously created several heterotropic allosteric enzymes by recombining the genes for TEMI beta-lactamase (BLA) and maltose binding protein (MBP) to create beta-lactamases that are positively or negatively regulated by maltose. One of these engineered enzymes, RG13, was constructed by random insertion of the BLA gene into the gene encoding MBP. In this work, we show that RG 13 has ∼106 M -1 affinity for Zn2+, a property that neither of its parental proteins possesses. Furthermore, Zn2+ is a negative effector that noncompetitively switches off beta-lactam hydrolysis activity. Mutagenesis experiments indicate that the Zn2+-binding site does not involve a histidine or a cysteine, which is atypical of natural Zn 2+-binding sites. These studies also implicate helices 1 and 12 of the BLA domain in allosteric signal propagation. These results support a model for the evolution of heterotropic allostery in which effector affinity and allosteric signaling emerge simultaneously.
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