Nature reviews

Nature reviews. In an unusual proteolytic maturation process(9C11), any of six centrally located 20C26 amino acid sequence repeats called HCF-1PRO repeats (Fig. 1A) are cleaved by OGT in the presence of UDP-GlcNAc(12), providing a link between cell-cycle progression and nutrient levels. The HCF-1PRO repeats contain two essential regions for proteolysis: a threonine-rich region proposed to be an OGT-binding site and the cleavage site, which contains a conserved Cys-Glu-Thr (CET) sequence (10, 11). Open in a separate window Fig. 1 Effect of mutations in OGT and HCF-1 constructs on cleavage and glycosylation(A) to an HCF-1PRO repeat mutant containing mutations in the threonine-rich region (T17C22A). WT (lane 2) and mutant (lanes 1, 3 and 4) GSTCHCF-1rep1 substrates were tested for OGT binding in the presence of UDP-GlcNAc using an OGT-directed pull-down assay. Anti-GST and anti-T7 antibodies were used to detect GSTCHCF-1rep1 (upper panel) and OGT (lower panel), respectively, by western blotting. Because intact UDP-GlcNAc is required for OGT-catalyzed cleavage of HCF-1PRO-repeats, we thought the UDP-5SGlcNAc analogue might stabilize density for a full repeat. Indeed, we obtained a structure of OGT with UDP-5SGlcNAc and a 26 amino acid peptide corresponding to HCF-1PRO rep2, but with an E10Q substitution. In this structure the C-terminal threonine-rich region binds to the TPR domain as described above, and the N-terminal cleavage region is now visible (Fig. 4A) and forms an extensive binding interface with UDP-5SGlcNAc (Fig. 4B). A structure containing UDP-GlcNAc and a wild-type repeat confirms the binding mode of the E10Q peptide (fig. S9). Remarkably, the cleavage region binds in a mode almost identical to that of a glycosylation-competent peptide substrate(14), and residue 10 aligns perfectly with the glycosyl acceptor amino acid (Fig. 4C). The structures suggested an E10S mutation, which prevents cleavage (Fig. 1D), would be glycosylated at residue 10. Indeed, unlike HCF3R-EAA, the E10S analog (HCF3R-SAA) was glycosylated efficiently (Fig. 4D and fig. S10). Therefore, the identity of the amino acid at position 10 of an HCF-1PRO repeat glutamate or serine can dictate whether OGT cleaves or glycosylates the substrate. Open in Buparvaquone a separate windowpane Fig. 4 HCF-1 cleavage takes place in the glycosyltransferase active site of OGT(A) Overall structure of the OGT:UDP-5SGlcNAc:HCF-1-E10Q1C26 complex. The HCF-1 peptide is definitely demonstrated as spheres in cyan with the UDP-5SGlcNAc in yellow. (B) Close-up look at of the two substrate analogs shown in yellow in the OGT active site. The entire cleavage region can be seen and the C-E10Q-T residues are annotated. The anomeric carbon of UDP-5SGlcNAc is definitely indicated (C1). (C) Overlay of the substrate analogs Buparvaquone from your OGT:UDP-5SGlcNAc:HCF-1 peptide complex (yellow) and the previously reported SEMA3E OGT:UDP-5SGlcNAc:CKIIA complex (cyan). CKII is definitely a well-characterized OGT glycosylation substrate. The E10Q sidechain of the HCF-1 peptide is definitely shown as transparent Buparvaquone just after the -carbon. (D) Mutating E10 to S in an HCF-1PRO repeat converts a cleavage substrate (HCF3R-EAA) into a glycosylation substrate (HCF-SAA), which is definitely defective in cleavage. (Remaining panel) Cleavage products of HCF3R-EAA and HCF3R-SAA were separated by SDS-PAGE and stained with Coomassie Blue. (Right panel) Glycosylation of wild-type and mutant HCF3R substrates was carried out with 14C-UDP-GlcNAc and analyzed by PAGE. Full gels are demonstrated fig. S10. Earlier hypotheses suggested that OGT consists of a dedicated protease active site or functions as a co-protease to template HCF-1 autocatalysis(12). Instead, OGT promotes cleavage of the HCF-1PRO repeat using the same catalytic region as for glycosylation. The threonine-rich region of the HCF-1PRO repeat binds in the channel formed from the TPR website of OGT, stabilized from the contacts explained above. The cleavage region threads into the active site and binds over UDP-GlcNAc in the same conformation that a glycosylation substrate would, with the glutamate part chain positioned near the anomeric carbon of the sugars. Since a pyroglutamate product is definitely created, and spontaneous cyclization of N-terminal glutamates is definitely kinetically Buparvaquone very sluggish(22), the glutamate part chain is likely activated by formation of an ester species as part of the cleavage mechanism. We speculate the glutamate part chain traps a transient oxocarbenium ion created within the active site, producing a glutamyl ester that can undergo intramolecular assault, leading ultimately to formation of the N-terminal pyroglutamate. We note that pyroglutamates are proposed species in additional biological phenomena(23). Possible mechanisms for cleavage proceeding from a glutamyl ester are suggested.