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Title: Structural studies of glycogen branching enzyme (GLGB) & CRFE2alpha : urucortin complexes
Keywords: Protein crystallography,Mycobacterum tuberculosis, Glycogen branching enzyme (GlgB), hCRFR2-extra cellular domain (ECD), Urocortins (Ucn), Selectivity
Issue Date: 27-Dec-2010
Citation: KUNTAL PAL (2010-12-27). Structural studies of glycogen branching enzyme (GLGB) & CRFE2alpha : urucortin complexes. ScholarBank@NUS Repository.
Abstract: The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an a-1,4-glucan branching enzyme (MtbGlgB, EC, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1?4 bond and making a new 1?6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33? resolution. MtbGlgBWT contains four domains: N1 ?-sandwich, N2 ?-sandwich, a central (?/a)8 domain that houses the catalytic site, and a C-terminal ?-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) Mtb?108GlgB protein. The N1 ?-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 ?-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and Mtb?108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1?4 bond breakage) and isomerization (1?6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and Mtb?108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (EC?112GlgB). The mammalian corticotropin releasing factor (CRF)/urocortin (Ucn) peptide hormones include four structurally similar peptides, CRF, Ucn1, Ucn2, and Ucn3, that regulate stress responses, metabolism, and cardiovascular function by activating either of two related class B G protein-coupled receptors, CRFR1 and CRFR2. CRF and Ucn1 activate both receptors, whereas Ucn2 and Ucn3 are CRFR2-selective. The molecular basis for selectivity is unclear. Here, we show that the purified N-terminal extracellular domains (ECDs) of human CRFR1 and the CRFR2alpha isoform are sufficient to discriminate the peptides, and we present three crystal structures of the CRFR2alpha ECD bound to each of the Ucn peptides. The CRFR2alpha ECD forms the same fold observed for the CRFR1 and mouse CRFR2beta ECDs, but contains a unique N-terminal alpha-helix formed by its pseudo signal peptide. The CRFR2alpha ECD peptide-binding site architecture is similar to that of CRFR1 and binding of the alpha-helical Ucn peptides closely resembles CRF binding to CRFR1. Comparing the electrostatic surface potentials of the ECDs suggests a charge compatibility mechanism for ligand discrimination involving a single amino acid difference in the receptors (CRFR1 Glu104/CRFR2alpha Pro100) at a site proximate to peptide residue 35 (Arg in CRF/Ucn1, Ala in Ucn2/3). CRFR1 Glu104 acts as a selectivity filter preventing Ucn2/3 binding because the nonpolar Ala35 is incompatible with the negatively charged Glu104. The structures explain the mechanisms of ligand recognition and discrimination and provide a molecular template for the rational design of therapeutic agents selectively targeting these receptors.
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