The crystal structure of Fe₄S₄ quinolinate synthase unravels an enzymatic dehydration mechanism that uses tyrosine and a hydrolase-type triad.

International audienceQuinolinate synthase (NadA) is a Fe4S4 cluster-containing dehydrating enzyme involved in the synthesis of quinolinic acid (QA), the universal precursor of the essential nicotinamide adenine dinucleotide (NAD) coenzyme. A previously determined apo NadA crystal structure revealed the binding of one substrate analog, providing partial mechanistic information. Here, we report on the holo X-ray structure of NadA. The presence of the Fe4S4 cluster generates an internal tunnel and a cavity in which we have docked the last precursor to be dehydrated to form QA. We find that the only suitably placed residue to initiate this process is the conserved Tyr21. Furthermore, Tyr21 is close to a conserved Thr-His-Glu triad reminiscent of those found in proteases and other hydrolases. Our mutagenesis data show that all of these residues are essential for activity and strongly suggest that Tyr21 deprotonation, to form the reactive nucleophilic phenoxide anion, is mediated by the triad. NadA displays a dehydration mechanism significantly different from the one found in archetypical dehydratases such as aconitase, which use a serine residue deprotonated by an oxyanion hole. The X-ray structure of NadA will help us unveil its catalytic mechanism, the last step in the understanding of NAD biosynthesis

Crystal Structure of the Transcription Regulator RsrR Reveals a [2Fe-2S] Cluster Coordinated by Cys, Glu and His Residues

The recently discovered Rrf2 family transcriptional regulator RsrR coordinates a [2Fe-2S] cluster. Remarkably, binding of the protein to RsrR-regulated promoter DNA sequences is switched on and off through the facile cycling of the [2Fe-2S] cluster be-tween +2 and +1 states. Here, we report high resolution crystal structures of the RsrR dimer, revealing that the [2Fe-2S] cluster is asymmetrically coordinated across the RsrR monomer-monomer interface by two Cys residues from one subunit and His and Glu residues from the other. To our knowledge, this is the first example of a protein bound [Fe-S] cluster with three different amino acid side chains as ligands, and of Glu acting as ligand to a [2Fe-2S] cluster. Analyses of RsrR structures revealed a conformation-al change, centered on Trp9, which results in a significant shift in the DNA-binding helix-turn-helix region

The crystal structure of the global anaerobic transcriptional regulator FNR explains its extremely fine-tuned monomer-dimer equilibrium

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Low-temperature switching by photoinduced protonation in photochromic fluorescent proteins

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Structural basis of X-ray-induced transient photobleaching in a photoactivatable green fluorescent protein.

International audienceWe have observed the photoactivatable fluorescent protein IrisFP in a transient dark state with near-atomic resolution. This dark state is assigned to a radical species that either relaxes to the ground state or evolves into a permanently bleached chromophore. We took advantage of X-rays to populate the radical, which presumably forms under illumination with visible light by an electron-transfer reaction in the triplet state. The combined X-ray diffraction and in crystallo UV-vis absorption, fluorescence, and Raman data reveal that radical formation in IrisFP involves pronounced but reversible distortion of the chromophore, suggesting a transient loss of pi conjugation. These results reveal that the methylene bridge of the chromophore is the Achilles' heel of fluorescent proteins and help unravel the mechanisms of blinking and photobleaching in FPs, which are of importance in the rational design of photostable variants

Crystal structure of human iron regulatory protein 1 as cytosolic aconitase.

International audienceIron regulatory proteins (IRPs) control the translation of proteins involved in iron uptake, storage and utilization by binding to specific noncoding sequences of the corresponding mRNAs known as iron-responsive elements (IREs). This strong interaction assures proper iron homeostasis in animal cells under iron shortage. Conversely, under iron-replete conditions, IRP1 binds a [4Fe-4S] cluster and functions as cytosolic aconitase. Regulation of the balance between the two IRP1 activities is complex, and it does not depend only on iron availability. Here, we report the crystal structure of human IRP1 in its aconitase form. Comparison with known structures of homologous enzymes reveals well-conserved folds and active site environments with significantly different surface shapes and charge distributions. The specific features of human IRP1 allow us to propose a tentative model of an IRP1-IRE complex that agrees with a range of previously obtained data

Structural basis of bacteriophage T5 infection trigger and E. coli cell wall perforation

Abstract The vast majority of bacteriophages (phages) - bacterial viruses - present a tail that allows host recognition, cell wall perforation and safe channelling of the viral DNA from the capsid to the cytoplasm of the infected bacterium. The majority of tailed phages bears a long flexible tail ( Siphoviridae ) at the distal end of which a tip complex, often called baseplate, harbours one or more Receptor Binding Protein·s (RBPs). Interaction between the RBPs and the host surface triggers cell wall perforation and DNA ejection, but little is known on these mechanisms for Siphoviridae . Here, we present the structure of siphophage T5 tip at high resolution, determined by electron cryo-microscopy, allowing to trace most of its constituting proteins, including 35 C-terminal residues of the Tape Measure Protein. We also present the structure of T5 tip after interaction with its E. coli receptor FhuA reconstituted into nanodisc. It brings out the dramatic conformational changes underwent by T5 tip upon infection, i . e . bending of the central fibre on the side, opening of the tail tube and its anchoring to the membrane, and formation of a transmembrane channel. These new structures shed light on the mechanisms of host recognition and activation of the viral entry for Siphoviridae

Crystal Structures of Quinolinate Synthase in Complex with a Substrate Analogue, the Condensation Intermediate, and Substrate-Derived Product.

International audienceThe enzyme NadA catalyzes the synthesis of quinolinic acid (QA), the precursor of the universal nicotinamide adenine dinucleotide (NAD) cofactor. Here, we report the crystal structures of complexes between the Thermotoga maritima (Tm) NadA K219R/Y107F variant and (i) the first intermediate (W) resulting from the condensation of dihydroxyacetone phosphate (DHAP) with iminoaspartate and (ii) the DHAP analogue and triose-phosphate isomerase inhibitor phosphoglycolohydroxamate (PGH). In addition, using the TmNadA K219R/Y21F variant, we have reacted substrates and obtained a crystalline complex between this protein and the QA product. We also show that citrate can bind to both TmNadA K219R and its Y21F variant. The W structure indicates that condensation causes dephosphorylation. We propose that catalysis by the K219R/Y107F variant is arrested at the W intermediate because the mutated protein is unable to catalyze its aldo-keto isomerization and/or cyclization that ultimately lead to QA formation. Intriguingly, PGH binds to NadA with its phosphate group at the site where the carboxylate groups of W also bind. Our results shed significant light on the mechanism of the reaction catalyzed by NadA

Probing the Reactivity of [4Fe-4S] Fumarate and Nitrate Reduction (FNR) Regulator with O2 and NO: Increased O2 Resistance and Relative Specificity for NO of the [4Fe-4S] L28H FNR Cluster

The Escherichia coli fumarate and nitrate reduction (FNR) regulator acts as the cell’s master switch for the transition between anaerobic and aerobic respiration, controlling the expression of >300 genes in response to O2 availability. Oxygen is perceived through a reaction with FNR’s [4Fe-4S] cluster cofactor. In addition to its primary O2 signal, the FNR [4Fe-4S] cluster also reacts with nitric oxide (NO). In response to physiological concentrations of NO, FNR de-represses the transcription of hmp, which encodes a principal NO-detoxifying enzyme, and fails to activate the expression of the nitrate reductase (nar) operon, a significant source of endogenous cellular NO. Here, we show that the L28H variant of FNR, which is much less reactive towards O2 than wild-type FNR, remains highly reactive towards NO. A high resolution structure and molecular dynamics (MD) simulations of the closely related L28H-FNR from Aliivibrio fischeri revealed decreased conformational flexibility of the Cys20-Cys29 cluster-binding loop that is suggested to inhibit outer-sphere O2 reactivity, but only partially impair inner-sphere NO reactivity. Our data provide new insights into the mechanistic basis for how iron–sulfur cluster regulators can distinguish between O2 and NO