Crystal structure of the transcriptional repressor DdrO: insight into the metalloprotease/repressor-controlled radiation response in Deinococcus.

Exposure to harmful conditions such as radiation and desiccation induce oxidative stress and DNA damage. In radiation-resistant Deinococcus bacteria, the radiation/desiccation response is controlled by two proteins: the XRE family transcriptional repressor DdrO and the COG2856 metalloprotease IrrE. The latter cleaves and inactivates DdrO. Here, we report the biochemical characterization and crystal structure of DdrO, which is the first structure of a XRE protein targeted by a COG2856 protein. DdrO is composed of two domains that fold independently and are separated by a flexible linker. The N-terminal domain corresponds to the DNA-binding domain. The C-terminal domain, containing three alpha helices arranged in a novel fold, is required for DdrO dimerization. Cleavage by IrrE occurs in the loop between the last two helices of DdrO and abolishes dimerization and DNA binding. The cleavage site is hidden in the DdrO dimer structure, indicating that IrrE cleaves DdrO monomers or that the interaction with IrrE induces a structural change rendering accessible the cleavage site. Predicted COG2856/XRE regulatory protein pairs are found in many bacteria, and available data suggest two different molecular mechanisms for stress-induced gene expression: COG2856 protein-mediated cleavage or inhibition of oligomerization without cleavage of the XRE repressor.

Structural insight into magnetochrome-mediated magnetite biomineralization.

Magnetotactic bacteria align along the Earth's magnetic field using an organelle called the magnetosome, a biomineralized magnetite (Fe(II)Fe(III)2O4) or greigite (Fe(II)Fe(III)2S4) crystal embedded in a lipid vesicle. Although the need for both iron(II) and iron(III) is clear, little is known about the biological mechanisms controlling their ratio. Here we present the structure of the magnetosome-associated protein MamP and find that it is built on a unique arrangement of a self-plugged PDZ domain fused to two magnetochrome domains, defining a new class of c-type cytochrome exclusively found in magnetotactic bacteria. Mutational analysis, enzyme kinetics, co-crystallization with iron(II) and an in vitro MamP-assisted magnetite production assay establish MamP as an iron oxidase that contributes to the formation of iron(III) ferrihydrite eventually required for magnetite crystal growth in vivo. These results demonstrate the molecular mechanisms of iron management taking place inside the magnetosome and highlight the role of magnetochrome in iron biomineralization.

Rhodobactersphaeroides methionine sulfoxide reductase P reduces R- and S-diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates.

Methionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as R- and S-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown to be absolutely stereospecific for the reduction of S-diastereomer and R-diastereomer, respectively. Recently, a new enzymatic system, MsrQ/MsrP which is conserved in all gram-negative bacteria, was identified as a key actor for the reduction of oxidized periplasmic proteins. The haem-binding membrane protein MsrQ transmits reducing power from the electron transport chains to the molybdoenzyme MsrP, which acts as a protein-MetO reductase. The MsrQ/MsrP function was well established genetically, but the identity and biochemical properties of MsrP substrates remain unknown. In this work, using the purified MsrP enzyme from the photosynthetic bacteria Rhodobacter sphaeroides as a model, we show that it can reduce a broad spectrum of protein substrates. The most efficiently reduced MetO is found in clusters, in amino acid sequences devoid of threonine and proline on the C-terminal side. Moreover, R. sphaeroides MsrP lacks stereospecificity as it can reduce both R- and S-diastereomers of MetO, similarly to its Escherichia coli homolog, and preferentially acts on unfolded oxidized proteins. Overall, these results provide important insights into the function of a bacterial envelop protecting system, which should help understand how bacteria cope in harmful environments.

Structural basis for regulation of the human acetyl-CoA thioesterase 12 and interactions with the steroidogenic acute regulatory protein-related lipid transfer (START) domain.

Acetyl-CoA plays a fundamental role in cell signaling and metabolic pathways, with its cellular levels tightly controlled through reciprocal regulation of enzymes that mediate its synthesis and catabolism. ACOT12, the primary acetyl-CoA thioesterase in the liver of human, mouse, and rat, is responsible for cleavage of the thioester bond within acetyl-CoA, producing acetate and coenzyme A for a range of cellular processes. The enzyme is regulated by ADP and ATP, which is believed to be mediated through the ligand-induced oligomerization of the thioesterase domains, whereby ATP induces active dimers and tetramers, whereas apo- and ADP-bound ACOT12 are monomeric and inactive. Here, using a range of structural and biophysical techniques, it is demonstrated that ACOT12 is a trimer rather than a tetramer and that neither ADP nor ATP exert their regulatory effects by altering the oligomeric status of the enzyme. Rather, the binding site and mechanism of ADP regulation have been determined to occur through two novel regulatory regions, one involving a large loop that links the thioesterase domains (Phe(154)-Thr(178)), defined here as RegLoop1, and a second region involving the C terminus of thioesterase domain 2 (Gln(304)-Gly(326)), designated RegLoop2. Mutagenesis confirmed that Arg(312) and Arg(313) are crucial for this mode of regulation, and novel interactions with the START domain are presented together with insights into domain swapping within eukaryotic thioesterases for substrate recognition. In summary, these experiments provide the first structural insights into the regulation of this enzyme family, revealing an alternate hypothesis likely to be conserved throughout evolution.

The emerging role of acyl-CoA thioesterases and acyltransferases in regulating peroxisomal lipid metabolism.

The importance of peroxisomes in lipid metabolism is now well established and peroxisomes contain approximately 60 enzymes involved in these lipid metabolic pathways. Several acyl-CoA thioesterase enzymes (ACOTs) have been identified in peroxisomes that catalyze the hydrolysis of acyl-CoAs (short-, medium-, long- and very long-chain), bile acid-CoAs, and methyl branched-CoAs, to the free fatty acid and coenzyme A. A number of acyltransferase enzymes, which are structurally and functionally related to ACOTs, have also been identified in peroxisomes, which conjugate (or amidate) bile acid-CoAs and acyl-CoAs to amino acids, resulting in the production of amidated bile acids and fatty acids. The function of ACOTs is to act as auxiliary enzymes in the α- and ß-oxidation of various lipids in peroxisomes. Human peroxisomes contain at least two ACOTs (ACOT4 and ACOT8) whereas mouse peroxisomes contain six ACOTs (ACOT3, 4, 5, 6, 8 and 12). Similarly, human peroxisomes contain one bile acid-CoA:amino acid N-acyltransferase (BAAT), whereas mouse peroxisomes contain three acyltransferases (BAAT and acyl-CoA:amino acid N-acyltransferases 1 and 2: ACNAT1 and ACNAT2). This review will focus on the human and mouse peroxisomal ACOT and acyltransferase enzymes identified to date and discuss their cellular localizations, emerging structural information and functions as auxiliary enzymes in peroxisomal metabolic pathways.

Magnetochrome: a c-type cytochrome domain specific to magnetotatic bacteria.

Magnetotactic bacteria consist of a group of taxonomically, physiologically and morphologically diverse prokaryotes, with the singular ability to align with geomagnetic field lines, a phenomenon referred to as magnetotaxis. This magnetotactic property is due to the presence of iron-rich crystals embedded in lipidic vesicles forming an organelle called the magnetosome. Magnetosomes are composed of single-magnetic-domain nanocrystals of magnetite (Fe(3)O(4)) or greigite (Fe(3)S(4)) embedded in biological membranes, thereby forming a prokaryotic organelle. Four specific steps are described in this organelle formation: (i) membrane specialization, (ii) iron acquisition, (iii) magnetite (or greigite) biocrystallization, and (iv) magnetosome alignment. The formation of these magnetic crystals is a genetically controlled process, which is governed by enzyme-catalysed processes. On the basis of protein sequence analysis of genes known to be involved in magnetosome formation in Magnetospirillum magneticum AMB-1, we have identified a subset of three membrane-associated or periplasmic proteins containing a double cytochrome c signature motif CXXCH: MamE, MamP and MamT. The presence of these proteins suggests the existence of an electron-transport chain inside the magnetosome, contributing to the process of biocrystallization. We have performed heterologous expression in E. coli of the cytochrome c motif-containing domains of MamE, MamP and MamT. Initial biophysical characterization has confirmed that MamE, MamP and MamT are indeed c-type cytochromes. Furthermore, determination of redox potentials for this new family of c-type cytochromes reveals midpoint potentials of -76 and -32 mV for MamP and MamE respectively.

Structural determination of functional domains in early B-cell factor (EBF) family of transcription factors reveals similarities to Rel DNA-binding proteins and a novel dimerization motif.

The early B-cell factor (EBF) transcription factors are central regulators of development in several organs and tissues. This protein family shows low sequence similarity to other protein families, which is why structural information for the functional domains of these proteins is crucial to understand their biochemical features. We have used a modular approach to determine the crystal structures of the structured domains in the EBF family. The DNA binding domain reveals a striking resemblance to the DNA binding domains of the Rel homology superfamily of transcription factors but contains a unique zinc binding structure, termed zinc knuckle. Further the EBF proteins contain an IPT/TIG domain and an atypical helix-loop-helix domain with a novel type of dimerization motif. The data presented here provide insights into unique structural features of the EBF proteins and open possibilities for detailed molecular investigations of this important transcription factor family.

Reduction of Protein Bound Methionine Sulfoxide by a Periplasmic Dimethyl Sulfoxide Reductase.

In proteins, methionine (Met) can be oxidized into Met sulfoxide (MetO). The ubiquitous methionine sulfoxide reductases (Msr) A and B are thiol-oxidoreductases reducing MetO. Reversible Met oxidation has a wide range of consequences, from protection against oxidative stress to fine-tuned regulation of protein functions. Bacteria distinguish themselves by the production of molybdenum-containing enzymes reducing MetO, such as the periplasmic MsrP which protects proteins during acute oxidative stress. The versatile dimethyl sulfoxide (DMSO) reductases were shown to reduce the free amino acid MetO, but their ability to reduce MetO within proteins was never evaluated. Here, using model oxidized proteins and peptides, enzymatic and mass spectrometry approaches, we showed that the Rhodobacter sphaeroides periplasmic DorA-type DMSO reductase reduces protein bound MetO as efficiently as the free amino acid L-MetO and with catalytic values in the range of those described for the canonical Msrs. The identification of this fourth type of enzyme able to reduce MetO in proteins, conserved across proteobacteria and actinobacteria, suggests that organisms employ enzymatic systems yet undiscovered to regulate protein oxidation states.

Reactive Neurogenesis and Down-Regulation of the Potassium-Chloride Cotransporter KCC2 in the Cochlear Nuclei after Cochlear Deafferentation.

While many studies have been devoted to investigating the homeostatic plasticity triggered by cochlear hearing loss, the cellular and molecular mechanisms involved in these central changes remain elusive. In the present study, we investigated the possibility of reactive neurogenesis after unilateral cochlear nerve section in the cochlear nucleus (CN) of cats. We found a strong cell proliferation in all the CN sub-divisions ipsilateral to the lesion. Most of the newly generated cells survive up to 1 month after cochlear deafferentation in all cochlear nuclei (except the dorsal CN) and give rise to a variety of cell types, i.e., microglial cells, astrocytes, and neurons. Interestingly, many of the newborn neurons had an inhibitory (GABAergic) phenotype. This result is intriguing since sensory deafferentation is usually accompanied by enhanced excitation, consistent with a reduction in central inhibition. The membrane potential effect of GABA depends, however, on the intra-cellular chloride concentration, which is maintained at low levels in adults by the potassium chloride co-transporter KCC2. The KCC2 density on the plasma membrane of neurons was then assessed after cochlear deafferentation in the cochlear nuclei ipsilateral and contralateral to the lesion. Cochlear deafferentation is accompanied by a strong down-regulation of KCC2 ipsilateral to the lesion at 3 and 30 days post-lesion. This study suggests that reactive neurogenesis and down-regulation of KCC2 is part of the vast repertoire involved in homeostatic plasticity triggered by hearing loss. These central changes may also play a role in the generation of tinnitus and hyperacusis.

Functional interactions between the MutL and Vsr proteins of Escherichia coli are dependent on the N-terminus of Vsr.

The crystal structure of the Escherichia coli Vsr endonuclease bound to a C(T/G)AGG substrate revealed that the DNA is held by a pincer composed of a trio of aromatic residues which intercalate into the major groove, and an N-terminus alpha helix which lies across the minor groove. We have constructed an N-terminus truncation (Delta14) which removes most of the alpha helix. The mutant is still fairly proficient in mediating very short patch repair. However, its endonuclease activity is considerably reduced and, in contrast to that of the wild type protein, cannot be stimulated by MutL. We had shown previously that excess Vsr in vivo causes mutagenesis, probably by inhibiting the participation of MutL in mismatch repair. The Delta14 mutant has diminished mutagenicity. In contrast, four enzymatically inactive mutants, with intact N-termini, are as mutagenic as the wild type protein. On the basis of these results we suggest that MutL causes a conformational change in the N-terminus of Vsr which enhances Vsr activity, and that this functional interaction between Vsr and MutL decreases the ability of MutL to carry out mismatch repair.

Sulphur shuttling across a chaperone during molybdenum cofactor maturation.

Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP-used as a surrogate of the molybdenum cofactor's nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.

Structure and evolution of the magnetochrome domains: no longer alone.

Magnetotactic bacteria (MTB) can swim along Earth's magnetic field lines, thanks to the alignment of dedicated cytoplasmic organelles. These organelles, termed magnetosomes, are proteolipidic vesicles filled by a 35-120 nm crystal of either magnetite or greigite. The formation and alignment of magnetosomes are mediated by a group of specific genes, the mam genes, encoding the magnetosome-associated proteins. The whole process of magnetosome biogenesis can be divided into four sequential steps; (i) cytoplasmic membrane invagination, (ii) magnetosomes alignment, (iii) iron crystal nucleation and (iv) species-dependent mineral size and shape control. Since both magnetite and greigite are a mix of iron (III) and iron (II), iron redox state management within the magnetosome vesicle is a key issue. Recently, studies have started pointing out the importance of a MTB-specific c-type cytochrome domain found in several magnetosome-associated proteins (MamE, P, T, and X). This magnetochrome (MCR) domain is almost always found in tandem, and this tandem is either found alone (MamT), in combination with a PDZ domain (MamP), a domain of unknown function (MamX) or with a trypsin combined to one or two PDZ domains (MamE). By taking advantage of new genomic data available on MTB and a recent structural study of MamP, which helped define the MCR domain boundaries, we attempt to retrace the evolutionary history within and between the different MCR-containing proteins. We propose that the observed tandem repeat of MCR is the result of a convergent evolution and attempt to explain why this domain is rarely found alone.

High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation.

The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 Å) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBP's concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.

Comparative structural analysis of lipid binding START domains.

BACKGROUND: Steroidogenic acute regulatory (StAR) protein related lipid transfer (START) domains are small globular modules that form a cavity where lipids and lipid hormones bind. These domains can transport ligands to facilitate lipid exchange between biological membranes, and they have been postulated to modulate the activity of other domains of the protein in response to ligand binding. More than a dozen human genes encode START domains, and several of them are implicated in a disease. PRINCIPAL FINDINGS: We report crystal structures of the human STARD1, STARD5, STARD13 and STARD14 lipid transfer domains. These represent four of the six functional classes of START domains. SIGNIFICANCE: Sequence alignments based on these and previously reported crystal structures define the structural determinants of human START domains, both those related to structural framework and those involved in ligand specificity. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.