title: The structure of the catechin-binding site of human sulfotransferase 1A1.
abstract: We are just beginning to understand the allosteric regulation of the human cytosolic sulfotransferase (SULTs) family-13 disease-relevant enzymes that regulate the activities of hundreds, if not thousands, of signaling small molecules. SULT1A1, the predominant isoform in adult liver, harbors two noninteracting allosteric sites, each of which binds a different molecular family: the catechins (naturally occurring flavonols) and nonsteroidal antiinflammatory drugs (NSAIDs). Here, we present the structure of an SULT allosteric binding site-the catechin-binding site of SULT1A1 bound to epigallocatechin gallate (EGCG). The allosteric pocket resides in a dynamic region of the protein that enables EGCG to control opening and closure of the enzyme's active-site cap. Furthermore, the structure offers a molecular explanation for the isozyme specificity of EGCG, which is corroborated experimentally. The binding-site structure was obtained without X-ray crystallography or multidimensional NMR. Instead, a SULT1A1 apoprotein structure was used to guide positioning of a small number of spin-labeled single-Cys mutants that coat the entire enzyme surface with a paramagnetic field of sufficient strength to determine its contribution to the bound ligand's transverse (T2) relaxation from its 1D solution spectrum. EGCG protons were mapped to the protein surface by triangulation using the T2values to calculate their distances to a trio of spin-labeled Cys mutants. The final structure was obtained using distance-constrained molecular dynamics docking. This approach, which is readily extensible to other systems, is applicable over a wide range of ligand affinities, requires little protein, avoids the need for isotopically labeled protein, and has no protein molecular weight limitations.
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461-1926.
authors.forename: Ian
authors.initials: I
authors.lastname: Cook
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461-1926.
authors.forename: Ting
authors.initials: T
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461-1926.
authors.forename: Mark
authors.initials: M
authors.lastname: Girvin
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461-1926; tom.leyh@einstein.yu.edu.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Leyh
mesh_terms: D000495:Allosteric Site; D015239:Arylsulfotransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002392:Catechin / Q000031:analogs & derivatives / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013113:Spin Labels; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D013113:Spin Labels; D002392:Catechin; C045651:epigallocatechin gallate; D015239:Arylsulfotransferase; C117544:SULT1A1 protein, human
keywords: NMR; allostery; catechin; structure; sulfotransferase