title: Chemical shift imprint of intersubunit communication in a symmetric homodimer.
abstract: Allosteric communication is critical for protein function and cellular homeostasis, and it can be exploited as a strategy for drug design. However, unlike many protein-ligand interactions, the structural basis for the long-range communication that underlies allostery is not well understood. This lack of understanding is most evident in the case of classical allostery, in which a binding event in one protomer is sensed by a second symmetric protomer. A primary reason why study of interdomain signaling is challenging in oligomeric proteins is the difficulty in characterizing intermediate, singly bound species. Here, we use an NMR approach to isolate and characterize a singly ligated state ("lig1") of a homodimeric enzyme that is otherwise obscured by rapid exchange with apo and saturated forms. Mixed labeled dimers were prepared that simultaneously permit full population of the lig1 state and isotopic labeling of either protomer. Direct visualization of peaks from lig1 yielded site-specific ligand-state multiplets that provide a convenient format for assessing mechanisms of intersubunit communication from a variety of NMR measurements. We demonstrate this approach on thymidylate synthase from Escherichia coli, a homodimeric enzyme known to be half-the-sites reactive. Resolving the dUMP1 state shows that active site communication occurs not upon the first dUMP binding, but upon the second. Surprisingly, for many sites, dUMP1 peaks are found beyond the limits set by apo and dUMP2 peaks, indicating that binding the first dUMP pushes the enzyme ensemble to further conformational extremes than the apo or saturated forms. The approach used here should be generally applicable to homodimers.
authors.affiliation: Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599;
authors.forename: Bradley T
authors.initials: BT
authors.lastname: Falk
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Sapienza
authors.affiliation: Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 drewlee@unc.edu.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Lee
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003856:Deoxyuracil Nucleotides / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009587:Nitrogen Isotopes; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D013816:Thermodynamics; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D003856:Deoxyuracil Nucleotides; D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D021122:Protein Subunits; D011994:Recombinant Proteins; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase
keywords: NMR; allostery; homodimer; subunit communication; thymidylate synthase