Trapping and structure determination of an intermediate in the allosteric transition of aspartate transcarbamoylase

Abstract

X-ray crystallography and small-angle X-ray scattering (SAXS) in solution have been used to show that a mutant aspartate transcarbamoylase exists in an intermediate quaternary structure between the canonical T and R structures. Additionally, the SAXS data indicate a pH-dependent structural alteration consistent with either a pH-induced conformational change or a pH-induced alteration in the T to R equilibrium. These data indicate that this mutant is not a model for the R state, as has been proposed, but rather represents the enzyme trapped along the path of the allosteric transition between the T and R states.A lthough many aspects of allosteric regulation and cooperativity have been established for Escherichia coli aspartate transcarbamoylase (ATCase), the enzyme that catalyzes the committed step in pyrimidine nucleotide biosynthesis, many of the details of how the active sites change from low-activity, lowaffinity to high-activity, high-affinity during the T to R transition have not been delineated (1). The major limitation has been that stabilization of the enzyme in the R state requires the presence of active site ligands, thus making interpretation of the structural rearrangements upon the T to R transition, in the absence of ligands, difficult. Newell and Schachman (2) have concluded, from sedimentation velocity experiments, that the quaternary structures of ATCase with the catalytic chain mutation K164E and the catalytic chain double-mutation K164E and E239K exist in the R state, irrespective of the presence of active site ligands. The enzymatic properties of the double mutant (K164E/E239K ATCase) include a lack of homotropic cooperativity and an inability to be activated by ATP or inhibited by CTP: properties coincident of an enzyme that cannot transition between the two allosteric states (2). More recently, Velyvis et al. (3) demonstrated that the partially labeled K164E/E239K ATCase exhibited certain chemical shifts in solution NMR studies that they conclude are characteristic of the R state of the enzyme. These same chemical shifts were also observed when the wild-type enzyme was bound with the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) (3), which is known to stabilize fully the R state of the enzyme (4). Thus, a crystal structure of K164E/ E239K ATCase should provide us a means to obtain the structural details of an R-state active site in the absence of ligands. However, here we report the X-ray crystal structure and the solution small-angle X-ray scattering (SAXS) data for the K164E/ E239K ATCase, which clearly demonstrate that this doublemutant enzyme is not in the R-quaternary structure. Instead this mutant enzyme is in a unique intermediate state on the path between the T and R structural states. Results and DiscussionCrystal Structure of K164E/E239K ATCase Is Different from the WildType R State Structure. The K164E/E239K ATCase was purified and subsequently crystallized in the absence of ligands as described in Methods. The crystals were determined to be in the P2 1 2 1 2 1 space gro...

Applications of small-angle X-ray scattering to biomacromolecular solutions

“…In the present review we largely mentioned the SAXS applications, which emerged from the collaborative projects at the EMBL biological SAXS beamlines, largely to illustrate the capacities of the novel data analysis methods. The number of biological SAXS applications by numerous groups worldwide is rapidly growing and especially exciting results are obtained in hybrid modeling projects, where the method is used together with other structural and computational techniques (Bach et al, 2012, Clerici et al, 2009, Guo et al, 2012, Hilge et al, 2009, Jehle et al, 2010. In this respect, standardization of the reporting of the SAXS data and its treatment is of high importance for adequate assessment by the reviewers and readers.…”

Structural Characterisation of Non-Deamidated Acidic Variants of Erwinia chrysanthemi L-asparaginase Using Small-Angle X-ray Scattering and Ion-Mobility Mass Spectrometry

“…However, these differences are subtle indicating the solution structure is not in a different global arrangement compared with the crystal structure. Small differences observed for proteins by SAXS observed in the Kratky or P(r) treatments of the data have been shown to have significance (37)(38)(39). It is worth noting that the crystal structure 1O7J represents ErA without bound substrate and waters.…”

New regulatory mechanism‐based inhibitors of aspartate transcarbamoylase for potential anticancer drug development

“…However, in all ATCase with regulatory subunit (ecATCase‐holo), most structures adopt R167 ‘in’ state no matter domain closure occurs or not. There are only four ecATCase‐holo structures that adopt R167 ‘out’ state, two of which (PDB ID: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=9ATC and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4E2F) have mutations destabilizing R state of ecATCase‐holo and the other two (PDB ID: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=1R0C and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2AIR) bind with substrate analogs or products in an unusual way . A structure alignment between R167 ‘in’ and ‘out’ state of ecATCase‐holo lacking domain closure is shown (Fig.…”

“…However, in all ATCase with regulatory subunit (ecATCaseholo), most structures adopt R167 'in' state no matter domain closure occurs or not. There are only four ecATCase-holo structures that adopt R167 'out' state, two of which (PDB ID: 9ATC and 4E2F) have mutations destabilizing R state of ecATCase-holo [23][24][25] and the other two (PDB ID: 1R0C and 2AIR) bind with substrate analogs or products in an unusual way [22,26]. A structure alignment between R167 'in' and 'out' state of ecATCase-holo lacking domain closure is shown (Fig.…”