1NXG

The F383A variant of type II Citrate Synthase complexed with NADH

Summary for 1NXG

• Entry DOI: 10.2210/pdb1nxg/pdb
• Related: 1K3P 1NXE
• Descriptor: Citrate synthase, SULFATE ION, 1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE, ... (4 entities in total)
• Functional Keywords: nadh, citrate synthase, f383a, allosteric, transferase
• Biological source: Escherichia coli
• Total number of polymer chains: 2
• Total formula weight: 97812.94

Authors

Maurus, R.,Nguyen, N.T.,Stokell, D.J.,Ayed, A.,Hultin, P.G.,Duckworth, H.W.,Brayer, G.D. (deposition date: 2003-02-10, release date: 2003-04-08, Last modification date: 2024-02-14)

Primary citation

Maurus, R.,Nguyen, N.T.,Stokell, D.J.,Ayed, A.,Hultin, P.G.,Duckworth, H.W.,Brayer, G.D.
Insights into the evolution of allosteric properties. The NADH binding site of hexameric type II citrate synthases.
Biochemistry, 42:5555-5565, 2003

PubMed Abstract: 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.

PubMed: 12741811
DOI: 10.1021/bi020622s

Experimental method

X-RAY DIFFRACTION (2.5 Å)