We describe the first structure determination of a type II citrate synthase, an enzyme uniquely found in Gram-negative bacteria. Such enzymes are hexameric and are strongly and specifically inhibited by NADH through an allosteric mechanism. This is in contrast to the widespread dimeric type I citrate synthases found in other organisms, which do not show allosteric properties. Our structure of the hexameric type II citrate synthase from Escherichia coli is composed of three identical dimer units arranged about a central 3-fold axis. The interactions that lead to hexamer formation are concentrated in a relatively small region composed of helix F, FG and IJ helical turns, and a seven-residue loop between helices J and K. This latter loop is present only in type II citrate synthase sequences. Running through the middle of the hexamer complex, and along the 3-fold axis relating dimer units, is a remarkable pore lined with 18 cationic residues and an associated hydrogen-bonded network. Also unexpected was the observation of a novel N-terminal domain, formed by the collective interactions of the first 52 residues from the two subunits of each dimer. The domain formed is rich in beta-sheet structure and has no counterpart in previous structural studies of type I citrate synthases. This domain is located well away from the dimer-dimer contacts that form the hexamer, and it is not involved in hexamer formation. Another surprising observation from the structure of type II E. coli citrate synthase is the unusual polypeptide chain folding found at the putative acetylcoenzyme A binding site. Key parts of this region, including His264 and a portion of polypeptide chain known from type I structures to form an adenine binding loop (residues 299-303), are shifted by as much as 10 A from where they must be for substrate binding and catalysis to occur. Furthermore, the adjacent polypeptide chain composed of residues 267-297 is extremely mobile in our structure. Thus, acetylcoenzyme A binding to type II E. coli citrate synthase would require substantial structural shifts and a concerted refolding of the polypeptide chain to form an appropriate binding subsite. We propose that this essential rearrangement of the acetylcoenzyme A binding part of the active site is also a major feature of allostery in type II citrate synthases. Overall, this study suggests that the evolutionary development of hexameric association, the elaboration of a novel N-terminal domain, introduction of a NADH binding site, and the need to refold a key substrate binding site are all elements that have been developed to allow for the allosteric control of catalysis in the type II citrate synthases.
Study of the hexameric and allosterically regulated citrate synthases (type II CS) provides a rare opportunity to gain not only an understanding of a novel allosteric mechanism but also insight into how such properties can evolve from an unregulated structural platform (the dimeric type I CS). To address both of these issues, we have determined the structure of the complex of NADH (a negative allosteric effector) with the F383A variant of type II Escherichia coli CS. This variant was chosen because its kinetics indicate it is primarily in the T or inactive allosteric conformation, the state that strongly binds to NADH. Our structural analyses show that the six NADH binding sites in the hexameric CS complex are located at the interfaces between dimer units such that most of each site is formed by one subunit, but a number of key residues are drawn from the adjacent dimer. This arrangement of interactions serves to explain why NADH allosteric regulation is a feature only of hexameric type II CS. Surprisingly, in both the wild-type enzyme and the NADH complex, the two subunits of each dimer within the hexameric conformation are similar but not identical in structure, and therefore, while the general characteristics of NADH binding interactions are similar in each subunit, the details of these are somewhat different between subunits. Detailed examination of the observed NADH binding sites indicates that both direct charged interactions and the overall cationic nature of the sites are likely responsible for the ability of these sites to discriminate between NADH and NAD(+). A particularly novel characteristic of the complex is the horseshoe conformation assumed by NADH, which is strikingly different from the extended conformation found in its complexes with most proteins. Sequence homology studies suggest that this approach to binding NADH may arise out of the evolutionary need to add an allosteric regulatory function to the base CS structure. Comparisons of the amino acid sequences of known type II CS enzymes, from different Gram-negative bacteria taxonomic groups, show that the NADH-binding residues identified in our structure are strongly conserved, while hexameric CS molecules that are insensitive to NADH have undergone key changes in the sequence of this part of the protein.
The citrate synthase of Escherichia coli is an example of a Type II citrate synthase, a hexamer that is subject to allosteric inhibition by NADH. In previous crystallographic work, we defined the NADH binding sites, identifying nine amino acids whose side chains were proposed to make hydrogen bonds with the NADH molecule. Here, we describe the functional properties of nine sequence variants, in which these have been replaced by nonbonding residues. All of the variants show some changes in NADH binding and inhibition and small but significant changes in kinetic parameters for catalysis. In three cases, Y145A, R163L, and K167A, NADH inhibition has become extremely weak. We have used nanospray/time-of-flight mass spectrometry, under nondenaturing conditions, to show that two of these, R163L and K167A, do not form hexamers in response to NADH binding, unlike the wild type enzyme. One variant, R109L, shows tighter NADH binding. We have crystallized this variant and determined its structure, with and without bound NADH. Unexpectedly, the greatest structural changes in the R109L variant are in two regions outside the NADH binding site, both of which, in wild type citrate synthase, have unusually high mobilities as measured by crystallographic thermal factors. In the R109L variant, both regions (residues 260 -311 and 316 -342) are much less mobile and have rearranged significantly. We argue that these two regions are elements in the path of communication between the NADH binding sites and the active sites and are centrally involved in the regulatory conformational change in E. coli citrate synthase.The Type II citrate synthases (CS) 1 of Gram-negative bacteria, such as Escherichia coli, are hexamers of identical subunits, which are strongly and specifically inhibited by the biological reducing agent, NADH, binding at a location distinct from the active site (1, 2). These properties distinguish them clearly from the Type I citrate synthases found in Gram-positive bacteria, archaea, and eukaryotes, which are homodimers without regulatory properties. Type I and Type II CS subunits all have the same overall fold and very similar active sites, so that, as a first approximation, a Type II hexamer may be regarded as a trimer of Type I dimers (3). From this we have argued that NADH inhibition in the Type II enzymes is an evolutionary add-on; Type I dimers were altered by a series of mutations to generate NADH sites and new contact surfaces so that hexamers could form. Our previous structural work on E. coli CS has shown that the six NADH binding sites are remote from the active sites and are located near the dimerdimer interfaces, with some residues contributed to each site by two subunits on either side of the interface (4). This finding explained why NADH binding induces a dimer to hexamer shift in the dimer-hexamer equilibrium shown by E. coli CS at moderate concentrations (1-20 M) (5).With the structure of the CS-NADH complex in hand, we can describe in detail the interactions between the protein and the nucleotide (4). The...
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