Ligand binding and allostery can emerge simultaneously.
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. We have previously created several heterotropic allosteric enzymes by recombining the genes for TEM1 beta-lactamase (BLA) and maltose binding protein (MBP) to create BLAs that are positively or negatively regulated by maltose. We show here that one of these engineered enzymes has approximately 10(6) M(-1) affinity for Zn(2+), a property that neither of the parental proteins possesses. Furthermore, Zn(2+) is a negative effector that noncompetitively switches off beta-lactam hydrolysis activity. Mutagenesis experiments indicate that the Zn(2+)-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.
Liang
Jing
J
Program in Molecular and Computational Biophysics, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Kim
Jin Ryoun
JR
Boock
Jason T
JT
Mansell
Thomas J
TJ
Ostermeier
Marc
M
D000494:Allosteric Regulation; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019143:Evolution, Molecular; D008024:Ligands; D058619:Maltose-Binding Proteins; D008957:Models, Genetic; D016296:Mutagenesis; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D008024:Ligands; D058619:Maltose-Binding Proteins; D011506:Proteins; D001618:beta-Lactamases; D015032:Zinc