title: Integrated insights from simulation, experiment, and mutational analysis yield new details of LacI function.
abstract: Protein structural change underlies many signal transduction processes. Although end-state structures are known for various allosteric proteins, intermediates are difficult to observe. Recently, targeted molecular dynamics simulation (TMD) was used to examine the conformational transition and predict relevant intermediates for wild-type lactose repressor (LacI). A catalog of involved residues suggests that the transition of this homodimer is asymmetric and that K84 is a prominent participant in the dynamic N-subdomain interface. Previous experiments indicated that hydrophobic substitutions at position 84 engender slowed, biphasic inducer binding kinetics, which might reflect the same phenomena observed in TMD. Here, we report biochemical confirmation that DNA and inducer binding remain allosterically linked in K84A and K84L, albeit with a differential smaller than that found in wild-type LacI. Other features of these mutant proteins are consistent with an allosteric conformational shift that approximates that of the wild type. As a consequence, these repressors can be utilized to explore an unanswered question about LacI function: How many inducers (one or two per dimer) are required to diminish operator affinity? The biphasic natures of the K84L and K84A inducer association rates allow direct correlation between the two distinct inducer binding events and operator release. Indeed, the kinetics of operator release for the K84A and K84L closely parallel those for the second inducer binding event. Together with implications from previous equilibrium results for wild-type and mutant proteins, these kinetic data demonstrate that binding of two inducers per dimeric DNA binding unit is required to release the operator in these variant LacI proteins.
authors.affiliation: Department of Biochemistry and Molecular Biology, MS 3030, The University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
authors.forename: Liskin
authors.initials: L
authors.lastname: Swint-Kruse
authors.forename: Hongli
authors.initials: H
authors.lastname: Zhan
authors.forename: Kathleen Shive
authors.initials: KS
authors.lastname: Matthews
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D007763:Lac Operon / Q000235:genetics; D056968:Lac Repressors; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D056968:Lac Repressors; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside