title: A Single Aspartate Coordinates Two Catalytic Steps in Hedgehog Autoprocessing.
abstract: Hedgehog (Hh) signaling is driven by the cholesterol-modified Hh ligand, generated by autoprocessing of Hh precursor protein. Two steps in Hh autoprocessing, N-S acyl shift and transesterification, must be coupled for efficient Hh cholesteroylation and downstream signal transduction. In the present study, we show that a conserved aspartate residue, D46 of the Hh autoprocessing domain, coordinates these two catalytic steps. Mutagenesis demonstrated that D46 suppresses non-native Hh precursor autoprocessing and is indispensable for transesterification with cholesterol. NMR measurements indicated that D46 has a pKa of 5.6, ∼2 units above the expected pKa of aspartate, due to a hydrogen-bond between protonated D46 and a catalytic cysteine residue. However, the deprotonated form of D46 side chain is also essential, because a D46N mutation cannot mediate cholesteroylation. On the basis of these data, we propose that the proton shuttling of D46 side chain mechanistically couples the two steps of Hh cholesteroylation.
authors.forename: Jian
authors.initials: J
authors.lastname: Xie
authors.affiliation: Department of Chemistry, Binghamton University, State University of New York , 4400 Vestal Parkway East, Binghamton, New York 13902, United States.
authors.forename: Timothy
authors.initials: T
authors.lastname: Owen
authors.forename: Ke
authors.initials: K
authors.lastname: Xia
authors.affiliation: Department of Chemistry, Binghamton University, State University of New York , 4400 Vestal Parkway East, Binghamton, New York 13902, United States.
authors.forename: Brian
authors.initials: B
authors.lastname: Callahan
authors.forename: Chunyu
authors.initials: C
authors.lastname: Wang
mesh_terms: D000818:Animals; D001224:Aspartic Acid; D055162:Biocatalysis; D017124:Conserved Sequence; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism*; D004951:Esterification; D053823:Hedgehog Proteins / Q000737:chemistry* / Q000378:metabolism*; D000072417:Protein Domains; D011499:Protein Processing, Post-Translational; D011522:Protons; D015398:Signal Transduction
chemical_list: D029721:Drosophila Proteins; D053823:Hedgehog Proteins; D011522:Protons; D001224:Aspartic Acid