RESUMO
The crystal structures of two homologous endopeptidases from cyanobacteria Anabaena variabilis and Nostoc punctiforme were determined at 1.05 and 1.60 A resolution, respectively, and contain a bacterial SH3-like domain (SH3b) and a ubiquitous cell-wall-associated NlpC/P60 (or CHAP) cysteine peptidase domain. The NlpC/P60 domain is a primitive, papain-like peptidase in the CA clan of cysteine peptidases with a Cys126/His176/His188 catalytic triad and a conserved catalytic core. We deduced from structure and sequence analysis, and then experimentally, that these two proteins act as gamma-D-glutamyl-L-diamino acid endopeptidases (EC 3.4.22.-). The active site is located near the interface between the SH3b and NlpC/P60 domains, where the SH3b domain may help define substrate specificity, instead of functioning as a targeting domain, so that only muropeptides with an N-terminal L-alanine can bind to the active site.
Assuntos
Endopeptidases/química , Endopeptidases/metabolismo , Peptidoglicano/química , Peptidoglicano/metabolismo , Sequência de Aminoácidos , Anabaena variabilis/química , Anabaena variabilis/enzimologia , Domínio Catalítico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/fisiologia , Endopeptidases/fisiologia , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Nostoc/química , Nostoc/enzimologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Domínios de Homologia de srcRESUMO
SSO2064 is the first structural representative of PF01796 (DUF35), a large prokaryotic family with a wide phylogenetic distribution. The structure reveals a novel two-domain architecture comprising an N-terminal, rubredoxin-like, zinc ribbon and a C-terminal, oligonucleotide/oligosaccharide-binding (OB) fold domain. Additional N-terminal helical segments may be involved in protein-protein interactions. Domain architectures, genomic context analysis and functional evidence from certain bacterial representatives of this family suggest that these proteins form a novel fatty-acid-binding component that is involved in the biosynthesis of lipids and polyketide antibiotics and that they possibly function as acyl-CoA-binding proteins. This structure has led to a re-evaluation of the DUF35 family, which has now been split into two entries in the latest Pfam release (v.24.0).
Assuntos
Acil Coenzima A/química , Proteínas Arqueais/química , Dobramento de Proteína , Sulfolobus solfataricus/química , Zinco/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Cristalografia por Raios X , Genoma Arqueal , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/metabolismoRESUMO
A novel aminoacyl-tRNA synthetase that contains an iron-sulfur cluster in the tRNA anticodon-binding region and efficiently charges tRNA with tryptophan has been found in Thermotoga maritima. The crystal structure of TmTrpRS (tryptophanyl-tRNA synthetase; TrpRS; EC 6.1.1.2) reveals an iron-sulfur [4Fe-4S] cluster bound to the tRNA anticodon-binding (TAB) domain and an L-tryptophan ligand in the active site. None of the other T. maritima aminoacyl-tRNA synthetases (AARSs) contain this [4Fe-4S] cluster-binding motif (C-x22-C-x6-C-x2-C). It is speculated that the iron-sulfur cluster contributes to the stability of TmTrpRS and could play a role in the recognition of the anticodon.
Assuntos
Proteínas Ferro-Enxofre/química , Thermotoga maritima/enzimologia , Triptofano-tRNA Ligase/química , Sequência de Aminoácidos , Animais , Sequência Conservada , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
In the plant pathogen Xanthomonas campestris pv. campestris, the product of the tcmJ gene, XcTcmJ, encodes a protein belonging to the RmlC family of cupins. XcTcmJ was crystallized in a monoclinic space group (C2) in the presence of zinc acetate and the structure was determined to 1.6â Å resolution. Previously, the apo structure has been reported in the absence of any bound metal ion [Chin et al. (2006), Proteins, 65, 1046-1050]. The most significant difference between the apo structure and the structure of XcTcmJ described here is a reorganization of the binding site for zinc acetate, which was most likely acquired from the crystallization solution. This site is located in the conserved metal ion-binding domain at the putative active site of XcTcmJ. In addition, an acetate was also bound within coordination distance of the zinc. In order to accommodate this binding, rearrangement of a conserved histidine ligand is required as well as several nearby residues within and around the putative active site. These observations indicate that binding of zinc serves a functional role in this cupin protein.
Assuntos
Proteínas de Bactérias/química , Domínio Catalítico , Xanthomonas campestris/química , Acetato de Zinco/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência Conservada , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia Estrutural de Proteína , Xanthomonas campestris/metabolismo , Acetato de Zinco/metabolismoRESUMO
The Structural Genomics of Pathogenic Protozoa (SGPP) Consortium aimed to determine crystal structures of proteins from trypanosomatid and malaria parasites in a high throughput manner. The pipeline of target selection, protein production, crystallization, and structure determination, is sketched. Special emphasis is given to a number of technology developments including domain prediction, the use of "co-crystallants," and capillary crystallization. "Fragment cocktail crystallography" for medical structural genomics is also described.
Assuntos
Genômica/métodos , Plasmodium/genética , Proteínas de Protozoários/química , Trypanosomatina/genética , Animais , Cristalização , Cristalografia por Raios X/métodosRESUMO
Structural information on helicase proteins has expanded recently beyond the DNA helicases Rep and PcrA, and the hepatitis C virus RNA helicase to include UvrB, members of the DEA(D/H)-box RNA helicase family, examples of DnaB-related helicases and RuvB. The expanding database of structures has clarified the structural 'theme and variations' that relate the different helicase families. Furthermore, information is emerging on the functions of the conserved helicase motifs and their participation in the mechanisms by which these proteins catalyze the remodeling of DNA and RNA in ATP-dependent activities.
Assuntos
DNA Helicases/química , RNA Helicases/química , Motivos de Aminoácidos , Proteínas de Bactérias/fisiologia , Sequência Conservada , DNA Helicases/fisiologia , DnaB Helicases , Família Multigênica , Conformação Proteica , RNA Helicases/fisiologia , Homologia de Sequência , Relação Estrutura-AtividadeRESUMO
The crystal structure of D-glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH) from the major malaria parasite Plasmodium falciparum is solved at 2.25 A resolution. The structure of PfGAPDH is of interest due to the dependence of the malaria parasite in infected human erythrocytes on the glycolytic pathway for its energy generation. Recent evidence suggests that PfGAPDH may also be required for other critical activities such as apical complex formation. The cofactor NAD(+) is bound to all four subunits of the tetrameric enzyme displaying excellent electron densities. In addition, in all four subunits a completely unexpected large island of extra electron density in the active site is observed, approaching closely the nicotinamide ribose of the NAD(+). This density is most likely the protease inhibitor AEBSF, found in maps from two different crystals. This putative AEBSF molecule is positioned in a crucial location and hence our structure, with expected and unexpected ligands bound, can be of assistance in lead development and design of novel antimalarials.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/química , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Sequência Conservada , Cristalografia por Raios X , Citoplasma/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , NAD/química , NAD/metabolismo , Plasmodium falciparum/genética , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaRESUMO
The Bacillus subtilis RNA helicase YxiN is a modular three-domain protein. The first two domains form a conserved helicase core that couples an ATPase activity to an RNA duplex-destabilization activity, while the third domain recognizes a stem-loop of 23S ribosomal RNA with high affinity and specificity. The structure of the second domain, amino-acid residues 207-368, has been solved to 1.95 A resolution, revealing a parallel alphabeta-fold. The crystallographic asymmetric unit contains two protomers; superposition shows that they differ substantially in two segments of peptide that overlap the conserved helicase sequence motifs V and VI, while the remainder of the domain is isostructural. The conformational variability of these segments suggests that induced fit is intrinsic to the recognition of ligands (ATP and RNA) and the coupling of the ATPase activity to conformational changes.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , RNA Helicases DEAD-box/química , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
We have determined the crystal structures of three homologous proteins from the pathogenic protozoans Leishmania donovani, Leishmania major, and Trypanosoma cruzi. We propose that these proteins represent a new subfamily within the isochorismatase superfamily (CDD classification cd004310). Their overall fold and key active site residues are structurally homologous both to the biochemically well-characterized N-carbamoylsarcosine-amidohydrolase, a cysteine hydrolase, and to the phenazine biosynthesis protein PHZD (isochorismase), an aspartyl hydrolase. All three proteins are annotated as mitochondrial-associated ribonuclease Mar1, based on a previous characterization of the homologous protein from L. tarentolae. This would constitute a new enzymatic activity for this structural superfamily, but this is not strongly supported by the observed structures. In these protozoan proteins, the extended active site is formed by inter-subunit association within a tetramer, which implies a distinct evolutionary history and substrate specificity from the previously characterized members of the isochorismatase superfamily. The characterization of the active site is supported crystallographically by the presence of an unidentified ligand bound at the active site cysteine of the T. cruzi structure.
Assuntos
Hidrolases/química , Modelos Moleculares , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Hidrolases/classificação , Hidrolases/metabolismo , Leishmania donovani/enzimologia , Leishmania major/enzimologia , Dados de Sequência Molecular , Proteínas de Protozoários/classificação , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Trypanosoma cruzi/enzimologiaRESUMO
The history of fragment-based drug discovery, with an emphasis on crystallographic methods, is sketched, illuminating various contributions, including our own, which preceded the industrial development of the method. Subsequently, the creation of the BMSC fragment cocktails library is described. The BMSC collection currently comprises 68 cocktails of 10 compounds that are shape-wise diverse. The utility of these cocktails for initiating lead discovery in structure-based drug design has been explored by soaking numerous protein crystals obtained by our MSGPP (Medical Structural Genomics of Pathogenic Protozoa) consortium. Details of the fragment selection and cocktail design procedures, as well as examples of the successes obtained are given. The BMSC Fragment Cocktail recipes are available free of charge and are in use in over 20 academic labs.
Assuntos
Descoberta de Drogas/métodos , Infecções por Protozoários/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Cristalografia por Raios X , Desenho de Fármacos , Genoma de Protozoário , Humanos , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Ribonuclease E (RNase E) is a multifunctional endoribonuclease that has been evolutionarily conserved in both Gram-positive and Gram-negative bacteria. X-ray crystallography and biochemical studies have concluded that the Escherichia coli RNase E protein functions as a homotetramer formed by Zn linkage of dimers within a region extending from amino acid residues 416 through 529 of the 116-kDa protein. Using fragments of RNase E proteins from E. coli and Haemophilus influenzae, we show here that RNase E derivatives that are as short as 395 amino acid residues and that lack the Zn-link region shown previously to be essential for tetramer formation (i.e. amino acid residues 400-415) are catalytically active enzymes that retain the 5' to 3' scanning ability and cleavage site specificity characteristic of full-length RNase E and that also confer colony forming ability on rne null mutant bacteria. Further truncation leads to loss of these properties. Our results, which identify a minimal catalytically active RNase E sequence, indicate that contrary to current models, a tetrameric quaternary structure is not required for RNase E to carry out its core enzymatic functions.
Assuntos
Endorribonucleases/química , Peptídeos/química , Domínio Catalítico , Cromatografia em Gel , Escherichia coli/metabolismo , Teste de Complementação Genética , Haemophilus influenzae/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Zinco/químicaRESUMO
DEx(D/H) proteins, typically described as RNA helicases, participate in rearrangement of RNA-RNA and possibly RNA-protein complexes in the cell. Aside from the conserved DEx(D/H) core, members of this protein family often contain N- and C-terminal extensions that are responsible for additional functions. The Bacillus subtilis DEx(D/H)-box protein YxiN and its Escherichia coli ortholog DbpA contain an approximately 80 amino acid C-terminal extension that has been proposed to specifically interact with a region of 23 S ribosomal RNA including hairpin 92. In this study, the DEx(D/H)-box core and the C-terminal domain of YxiN were expressed and characterized as separate proteins. The isolated DEx(D/H)-box core, YxCat, had weak, nonspecific RNA binding activity and showed RNA-stimulated ATPase activity with a Km(ATP) that resembled several non-specific DEx(D/H) proteins. The isolated C-terminal domain, YxRBD, bound RNA with the high affinity and specificity seen with full-length YxiN. Thus, YxiN is a modular protein combining the activities of the YxCat and YxRBD domains. Footprinting of YxiN and YxRBD on a 172-nucleotide fragment of 23 S rRNA was used to identify the sites of interaction of the C-terminal and helicase domains with the RNA.