Modulation of effector affinity by hinge region mutations also modulates switching activity in an engineered allosteric TEM1 beta-lactamase switch.
RG13 is an engineered allosteric beta-lactamase (BLA) for which maltose is a positive effector. RG13 is a hybrid protein between TEM1 BLA and maltose-binding protein (MBP). Maltose binding to MBP is known to convert the open form of the protein to the closed form through conformational changes about the hinge region. We have constructed and genetically selected several variants of RG13 modified in the hinge region of the MBP domain and explored their effect on beta-lactam hydrolysis, maltose affinity and maltose-induced switching. Hinge mutations that increased maltose affinity the most (and thus presumably close the apo-MBP domain the most) also abrogated switching the most. We provide evidence for a model of RG13 switching in which there exists a threshold conformation between the open to closed form of the MBP domain that divides states that catalyze beta-lactam hydrolysis with different relative rates of acylation and deacylation.
Kim
Jin Ryoun
JR
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA.
Ostermeier
Marc
M
D000215:Acylation; D000494:Allosteric Regulation / Q000235:genetics* / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002511:Cephalosporins / Q000378:metabolism; D019020:Directed Molecular Evolution; D006868:Hydrolysis; D019811:Hydroxylamine / Q000494:pharmacology; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D002511:Cephalosporins; D058619:Maltose-Binding Proteins; D019811:Hydroxylamine; D008320:Maltose; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; C021720:nitrocefin