Substitutions at Histidine 74 and Aspartate 278 Alter Ligand Binding and Allostery in Lactose Repressor Protein

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• 作者：Jennifer K. Barry and Kathleen S. Matthews
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：March 23, 1999
• 年：1999
• 卷：38
• 期：12
• 页码：3579 - 3590
• 全文大小：171K
• 年卷期：v.38,no.12(March 23, 1999)
• ISSN：1520-4995

文摘

In the inducer-bound structure of the lac repressor protein, the side chains of H74 and D278are positioned to form an ion pair between monomers that appears to be disrupted upon operator binding(Lewis, M., Chang, G., Horton, N. C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G.,and Lu, P. (1996) Science 271, 1247-1254). A series of single substitutions at H74 and D278 and adouble mutant, H74D-D278H, were generated to determine the influence of this interaction on ligandbinding and allostery in lac repressor. Introduction of apolar amino acids at H74 resulted in distinct effectson ligand binding. Alanine and leucine substitutions decreased operator binding, while tryptophan andphenylalanine increased affinity for operator DNA. Introduction of a negatively charged residue at position74 in H74D had minimal effects, and "inverting" the side chains in H74D/D278H did not significantlyalter inducer or operator binding at neutral pH. In contrast, all substitutions of D278 increased affinity foroperator DNA and diminished inducer binding. These observations can be interpreted in the context ofthe Monod-Wyman-Changeux model. If a salt bridge were essential for stabilizing or destabilizing theinducer-bound conformation, a mutation at either residue that interrupts this interaction should have asimilar effect on allostery. Because the type and degree of alteration in ligand binding properties dependedon the nature of the substitution at these residues, the individual roles played by H74 and D278 in lacrepressor allostery appear more important than their direct contact across the monomer-monomer interface.