Abstract
ATP phosphoribosyl transferase (ATP-PRT) joins ATP and 5-phosphoribosyl-1-pyrophosphate (PRPP) in a highly regulated reaction that initiates histidine biosynthesis. The unusual hetero-octameric version of ATP-PRT includes four HisG(S) catalytic subunits based on the periplasmic binding protein fold and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. Here, we present the first structure of a PRPP-bound ATP-PRT at 2.9 A and provide a structural model for allosteric activation based on comparisons with other inhibited and activated ATP-PRTs from both the hetero-octameric and hexameric families. The activated state of the octameric enzyme is characterized by an interstitial phosphate ion in the HisZ-HisG interface and new contacts between the HisZ motif 2 loop and the HisG(S) dimer interface. These contacts restructure the interface to recruit conserved residues to the active site, where they activate pyrophosphate to promote catalysis. Additionally, mutational analysis identifies the histidine binding sites within a region highly conserved between HisZ and the functional HisRS. Through the oligomerization and functional re-assignment of protein domains associated with aminoacylation and phosphate binding, the HisZ-HisG octameric ATP-PRT acquired the ability to initiate the synthesis of a key metabolic intermediate in an allosterically regulated fashion.
Highlights
Phosphoribosyl transferases (PRTs)5 catalyze the attack of a nitrogenous and/or aromatic base on 5-phosphoribosyl-1-pyrophosphate (PRPP), and thereby participate in essential reactions in the biosynthesis of nucleotides and the amino acids tryptophan and histidine [1, 2]
Hetero-octameric ATP phosphoribosyl transferase (ATP-PRT) possess a second subunit type, HisZ, which is related to the catalytic domain of functional histidyl-tRNA synthetases (HisRSs), but is inactive on its own [21,22,23]
Wilson plot a N1-methyl-ATP and PRPP substrates were co-crystallized with the HisZG ATP-PRTase. b ATP was present in the mother liquor during crystallization. c The numbers in parentheses are the statistics for the highest resolution shell. d Rmerge ϭIϪI/͚I, whereIis the average intensity from multiple observations of symmetry-related reflections. e Riso ϭFPH Ϫ FP/͚ ͉FP, where FP is the observed structure factor amplitude for the reference data set (Se-Peak), and FPH is the observed structure factor amplitude for the heavy atom derivative. f Figure of merit, before and after density modification. g Rwork and Rfree ϭhkl ʈFo ϪFcʈ/͚hklFo, where Fo and Fc are the observed and calculated structure factor amplitudes
Summary
Construction of Mutant Proteins—Mutant versions (E130A and Y268F/Y269F) of the HisZ-HisG ATP-PRT were derived from a Lactococcus lactis pQE30 expression construct [21] by use of the QuikChange® procedure (Stratagene). The PO4-bound dataset served as the reference for refinement, and was collected at 1.2124 Å on beamline X26C from a tungstatederivatized crystal that diffracted to 2.9 Å. An additional 3.2-Å dataset was collected from a crystal grown in the presence of N-1-ATP and PRPP. Structure Determination and Refinement—Twenty of the 48 possible selenium sites in the asymmetric unit were initially determined with SHELXD [28]. These sites were refined with SOLVE [29], allowing identification of 24 additional selenium atoms. The PRPP bound model was refined using rigid body refinement followed by simulated annealing in CNS [32]. Reactions included ATP-PRT at a concentration of 100 nM, and were initiated with PRPP. The baseline absorbance was established by setting A290 to zero for the reaction
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