Structure of Lactate Dehydrogenase at 2.8 Å Resolution

Adams, Margaret; Ford, Geoffrey C.; Koekoek, Roelof; Lentz, P. J.; McPherson, Alexander; Rossmann, Michael G.; Smiley, Ira E.; Schevitz, Richard W.; Wonacott, A.

doi:10.1038/2271098a0

Cited by 231 publications

(66 citation statements)

References 31 publications

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“…Although one of the substrates of DT is NAD, the structure of dimeric DT complexed with the inhibitor ApUp (DT-ApUp) is distinct from the Rossmann NAD-binding fold found in the dehydrogenases (Adams et al, 1970). Figure 7A shows the position of the ApUp molecule in the context of the C a coordinates of the C domain (see also Kinemage 2).…”

Section: Dinucleotide-binding Foldmentioning

confidence: 99%

Refined structure of dimeric diphtheria toxin at 2.0 Å resolution

1994

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The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections ( F > l o ( F ) ) , yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3'-5' uridine 3' monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp.The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an "open" structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by "domain swapping" to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.Keywords: ADP-ribosyltransferase; diphtheria; membrane insertion Diphtheria toxin (DT) is secreted from toxic strains of the bacterium Corynebacterium diphtheriae. Toxigenic conversion of C. diphtheriae occurs by lysogenization with corynephage 0 (Freeman, 1951), which carries the DT gene encoding a 535-residue protein (M, 58,342) (Uchida et al., 1971; Greenfield et al., 1983). DT kills eukaryotic cells by inactivating an essential component of the protein translation machinery, elongation factor 2 (EF-2) (Collier, 1975).DT is produced as a protomer of 2 fragments connected by a loop containing a proteolysis site and a disulfide bond. Limited proteolysis with trypsin and reduction of the disulfide separates the 2 fragments, which are denoted fragment A and fragment B (Drazin et al., 1971). Fragment A consists of an independent folding domain, the catalytic (C) domain (residues 1-190) (Choe et al., 1992). Fragment B consists of 2 folding domains, the transmembrane (T) domain (residues 191-378) and receptor-binding (R) domain (residues 379-535) (Choe et al., 1992).

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Section: Dinucleotide-binding Foldmentioning

confidence: 99%

Refined structure of dimeric diphtheria toxin at 2.0 Å resolution

1994

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“…NAD(P) exerts its functions by association with proteins. The protein fold that binds NAD(P) was discovered by Rossmann when the crystal structure of lactate dehydrogenase was determined (11). The Rossmann fold is the most common fold, based on its predicted occurrence from the genes known today (12).…”

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confidence: 99%

Restructuring of the dinucleotide-binding fold in an NADP(H) sensor protein

Zheng¹,

Dai²,

Zhao³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Rossmann fold ͉ signal transduction N AD and its phosphorylated form, NADP, is a universal cofactor in a large number of redox reactions carried out by a variety of enzymes, especially dehydrogenases. It is widely recognized that NAD(P) is an essential molecule in energy metabolism in all organisms. More recently, mounting evidence suggests that, in addition to its central role in energy metabolism, NAD(P) is also involved in signaling pathways that regulate fundamental processes of cellular functions, including gene transcription and apoptosis (1,2). This realization generated a link between the energy metabolism and the regulated networks of biological processes.One of the NAD signaling mechanisms is through gene silencer proteins known as sirtuins, such as Sir2 in yeast, a NAD-dependent histone deacetylase. Increased activities of Sir2 induced by an increase in NAD production extended lifespan (3). It was shown that a product of NAD-dependent deacetylation by Sir2, nicotinamide, strongly inhibits the activity of Sir2 (4, 5). Similar mechanisms are present in other eukaryotes, including humans (6). NAD is also directly related to transcription regulation. PolyADP-ribose polymerases (PARPs) can modify the acceptor proteins by synthesizing a polyADP-ribose molecule using NAD ϩ as the substrate. The transcription factors that can be modified by PARPs include p53, YY1, NF-B, and TATA-binding protein (1). In other cases, transcription regulation is not carried out by any enzymatic reaction. For instance, the C-terminal-binding protein CtBP is a corepressor that has an increased affinity to its partners, such as adenovirus E1A or cellular repressor ZEB, when NADH binds to CtBP (7). Crystal structures showed that NADH binding to CtBP induced a conformational switch that stabilizes the dimerization of CtBP, which in turn promotes its binding to the repressors (8). In the case of the negative transcriptional regulator NmrA, NAD ϩ binding to NmrA controls the rate of nuclear entry of the GATA transcription-activating protein AreA (9, 10). NmrA has a similar structure to short-chain dehydrogenase/reductase (SDR) family proteins but no enzyme activities, because of the lack of conserved active-site residues (9). NAD(P) exerts its functions by association with proteins. The protein fold that binds NAD(P) was discovered by Rossmann when the crystal structure of lactate dehydrogenase was determined (11). The Rossmann fold is the most common fold, based on its predicted occurrence from the genes known today (12). This motif consists of six ␤-strands connected by ␣-helices with the NAD(P) molecule bound at the top [supporting information (SI) Fig. 5]. The Rossmann fold has always been present as a rigid-body domain in all other known crystal structures until the structure of HSCARG was determined. We found that the common ␣E that connects ␤5 to ␤6 in the Rossmann fold is deformed as an extended loop when NADP is not bound with HSCARG. This allows the formation of an asymmetric dimer between a subunit with NADP bound and an emp...

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“…The complicated geometrical situation a t the active site of the enzyme as concluded from this work may be compared with that determined for the enzymatically simpler lactate dehydrogenase by X-ray crystallography, which resulted in a system of holes and clefts [32].…”

Section: And-imentioning

confidence: 99%

Spin Labeling Studies of d‐Glyceraldehyde‐3‐phosphate Dehydrogenase

Balthasar

1971

European Journal of Biochemistry

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~-Glyceraldehyde-3-phosphate dehydrogenase has been reacted with spin labels, which were designed to elucidate the geometry of the active site of the enzyme. Three series of spin labels were used, which represented either substrate and coenzyme analogs or irreversible inhibitors, the separation of the active site directing part from the nitroxide radical being readily adjusted through the length of the intervening methylene chain.The effect of this variation on the electron paramagnetic resonance spectrum of the labeled protein as well as the effect of coenzyme binding on these spectra have been investigated. All spectra are complex and represent the superposition of a t least three different types of spectra. These three types of spectra are correlated to nitroxide radicals in three different types of motion: (a) very fast rotation in a fluid region, (b) medium fast rotation of a hindered radical and (c) slow rotation of an almost immobilized radical. The spin labels differ only in the relative population in the three states related to the three types of motion. Also addition of the coenzyme NAD only alters this relative population. A spin labeled coenzyme analog, which has the nitroxide in the region of the sugar-nicotinamide bond, is bound to the protein and displays an electron paramagnetic resonance spectrum typical for an immobilized radical. The geometry of the active of ~-glyceraldehyde-3-phosphate dehydrogenase is complex and cannot be described as a simple cleft, but instead as a system of narrow and wider spaces, which is changed by the binding of the coenzyme.The spin labeling technique has been introduced by McConnell [l]. It has been applied to a wide spectrum of problems and has been especially successful in biochemical and biological applications. Reviews on spin labels have appeared [Z, 31. Spin labels have been used in biochemistry to detect conformational changes of haemoglobin [3,4], aspartate transcarbamylase [5] as well as to study the activity [6] and the geometry of the active site [7] of ol-chymotrypsin. Spin labels were used in this work in an attempt to determine conformational and geometrical properties of the tetrameric enzyme D-glyceraldehyde-3-phosphate dehydrogenase. ~-Glyceraldehyde

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Structure of Lactate Dehydrogenase at 2.8 Å Resolution

Cited by 231 publications

References 31 publications

Refined structure of dimeric diphtheria toxin at 2.0 Å resolution

Refined structure of dimeric diphtheria toxin at 2.0 Å resolution

Restructuring of the dinucleotide-binding fold in an NADP(H) sensor protein

Spin Labeling Studies of d‐Glyceraldehyde‐3‐phosphate Dehydrogenase

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