Engineered disulfide linking the hinge regions within lactose repressor dimer increases operator affinity, decreases sequence selectivity, and alters allostery.
The hinge domain encompasses amino acids 51-60 of lactose repressor (LacI) and plays an important role in its regulatory interaction with operator DNA. This segment makes both hinge-DNA and hinge-hinge' contacts that are critical to DNA binding. Furthermore, this small region serves as a central element in communicating the allosteric response to inducer. Introducing a disulfide bond between partner hinges within a dimer via the mutation V52C results in a protein that has increased affinity for O(1) operator DNA compared to wild-type LacI and abolishes allosteric response to inducer [Falcon, C. M., Swint-Kruse, L., and Matthews, K. S. (1997) J. Biol. Chem. 272, 26818]. We have established that this high affinity is maintained for the disulfide-linked protein even when symmetry and half-site spacing within the operator region are altered, whereas binding by the reduced protein, as for wild-type LacI, is severely diminished by these alterations. Interestingly, the allosteric response to inducer for V52C-oxidized remains intact for a small group of operator variants. Temperature studies demonstrate that the presence of the disulfide alters the thermodynamics of the protein-DNA interaction, with a DeltaC(p) of significantly smaller magnitude compared to wild-type LacI. The results presented here establish the hinge region as an important element not only for LacI high-affinity operator binding but also for the essential communication between ligand binding domains. Moreover, the results confirm that DNA sequence/conformation can profoundly influence allostery for this prototypic regulatory protein.
Falcon
C M
CM
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251, USA.
Matthews
K S
KS
D000494:Allosteric Regulation / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D056968:Lac Repressors; D016297:Mutagenesis, Site-Directed; D009875:Operator Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013696:Temperature; D014633:Valine / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004220:Disulfides; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012097:Repressor Proteins; D014633:Valine; D003545:Cysteine