6tyy

Hedgehog autoprocessing mutant D46H

spinqualitylabelsall models 6tyy, resolution 1.36Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 6tyy is a 1 chain structure with sequence from Drosophila melanogaster. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.36Å Ligands: CSD Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function HH_DROME Intercellular signal essential for a variety of patterning events during development. Establishes the anterior-posterior axis of the embryonic segments and patterns the larval imaginal disks. Binds to the patched (ptc) receptor, which functions in association with smoothened (smo), to activate the transcription of target genes wingless (wg), decapentaplegic (dpp) and ptc. In the absence of hh, ptc represses the constitutive signaling activity of smo through fused (fu). The hedgehog protein N-product constitutes the active species in both local and long-range signaling, whereas the C-terminal product has no signaling activity. It acts as a morphogen, and diffuses long distances despite its lipidation. Heparan sulfate proteoglycans of the extracellular matrix play an essential role in diffusion. Lipophorin is required for diffusion, probably by acting as vehicle for its movement, explaining how it can spread over long distances despite its lipidation. The hedgehog protein C-product, which mediates the autocatalytic activity, has no signaling activity. Publication Abstract from PubMed Hedgehog (Hh) autoprocessing converts Hh precursor protein to cholesterylated Hh ligand for downstream signaling. A conserved active-site aspartate residue, D46, plays a key catalytic role in Hh autoprocessing by serving as a general base to activate substrate cholesterol. Here we report that a charge-altering Asp-to-His mutant (D46H) expands native cholesterylation activity and retains active-site conformation. Native activity toward cholesterol was established for D46H in vitro using a continuous FRET-based autoprocessing assay and in cellulo with stable expression in human 293T cells. The catalytic efficiency of cholesterylation with D46H is similar to that with wild type (WT), with kmax/KM = 2.1 x 10(3) and 3.7 x 10(3) M(-1) s(-1), respectively, and an identical pKa = 5.8 is obtained for both residues by NMR. To our knowledge this is the first example where a general base substitution of an Asp for His preserves both the structure and activity as a general base. Surprisingly, D46H exhibits increased catalytic efficiency toward non-native substrates, especially coprostanol (>200-fold) and epicoprostanol (>300-fold). Expanded substrate tolerance is likely due to stabilization by H46 of the negatively charged tetrahedral intermediate using electrostatic interactions, which are less constrained by geometry than H-bond stabilization by D46. In addition to providing fundamental insights into Hh autoprocessing, our findings have important implications for protein engineering and enzyme design. General Base Swap Preserves Activity and Expands Substrate Tolerance in Hedgehog Autoprocessing.,Zhao J, Ciulla DA, Xie J, Wagner AG, Castillo DA, Zwarycz AS, Lin Z, Beadle S, Giner JL, Li Z, Li H, Banavali N, Callahan BP, Wang C J Am Chem Soc. 2019 Nov 7. doi: 10.1021/jacs.9b08914. PMID:31682419 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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