bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystal Structures of Active Fully Assembled Substrate- and Product-Bound Complexes of UDP-N-Acetylmuramic Acid:l-Alanine Ligase (MurC) fromHaemophilus influenzae
Authors: Mol Clifford D. , Brooun Alexei , Dougan Douglas R. , Hilgers Mark T. , Tari Leslie W. , Wijnands Robert A. , Knuth Mark W. , McRee Duncan E. , Swanson Ronald V. 
Published: July 2003

ABSTRACTUDP-N-acetylmuramic acid:l-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures ofHaemophilus influenzaeMurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg2+and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-l-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn2+have been determined to 1.85- and 1.7-Å resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the γ-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates.
Comparative Analysis of the
Escherichia coli
Ketopantoate Hydroxymethyltransferase Crystal Structure Confirms that It Is a Member of the (βα)
8
Phosphoenolpyruvate/Pyruvate Superfamily
Authors: Schmitzberger Florian , Smith Alison G. , Abell Chris , Blundell Tom L. 
Published: July 2003

ABSTRACT

Escherichia coli
ketopantoate hydroxymethyltransferase (KPHMT) catalyzes the first step in the biosynthesis pathway of pantothenate (vitamin B
5
), the transfer of a hydroxymethyl group onto α-ketoisovalerate. Here we describe a detailed comparative analysis of the KPHMT crystal structure and the identification of structural homologues, some of which have remarkable similarities in their active sites, modes of binding to substrates, and mechanisms. We show that KPHMT forms a family within the phosphoenolpyruvate/pyruvate superfamily. Based on the analysis, we propose that in this superfamily there should be a subdivision into two groups. This paper completes our structural analysis of the
E. coli
enzymes in the pantothenate pathway.
Mycobacterium tuberculosisChaperonin 10 Heptamers Self-Associate through Their Biologically Active Loops
Authors: Roberts Michael M. , Coker Alun R. , Fossati Gianluca , Mascagni Paolo , Coates Anthony R. M. , Wood Steve P. 
Published: July 2003

ABSTRACTThe crystal structure ofMycobacterium tuberculosischaperonin 10 (cpn10Mt) has been determined to a resolution of 2.8 Å. Two dome-shaped cpn10Mtheptamers complex through loops at their bases to form a tetradecamer with 72 symmetry and a spherical cage-like structure. The hollow interior enclosed by the tetradecamer is lined with hydrophilic residues and has dimensions of 30 Å perpendicular to and 60 Å along the sevenfold axis. Tetradecameric cpn10Mthas also been observed in solution by dynamic light scattering. Through its base loop sequence cpn10Mtis known to be the agent in the bacterium responsible for bone resorption and for the contribution towards its strong T-cell immunogenicity. Superimposition of the cpn10Mtsequences 26 to 32 and 66 to 72 andE. coliGroES 25 to 31 associated with bone resorption activity shows them to have similar conformations and structural features, suggesting that there may be a common receptor for the bone resorption sequences. The base loops of cpn10s in general also attach to the corresponding chaperonin 60 (cpn60) to enclose unfolded protein and to facilitate its correct folding in vivo. Electron density corresponding to a partially disordered protein subunit appears encapsulated within the interior dome cavity of each heptamer. This suggests that the binding of substrates to cpn10 is possible in the absence of cpn60.
Structural Analysis of the Domain Interface in DrrB, a Response Regulator of the OmpR/PhoB Subfamily
Authors: Robinson Victoria L. , Wu Ti , Stock Ann M. 
Published: July 2003

ABSTRACT

The N-terminal regulatory domains of bacterial response regulator proteins catalyze phosphoryl transfer and function as phosphorylation-dependent regulatory switches to control the output activities of C-terminal effector domains. Structures of numerous isolated regulatory and effector domains have been determined. However, a detailed understanding of regulatory interactions among these domains has been limited by the relative paucity of structural data for intact multidomain response regulator proteins. The first multidomain structures determined, those of transcription factor NarL and methylesterase CheB, both revealed extensive interdomain interfaces. The regulatory domains obstruct access to the functional sites of the effector domains, indicating a regulatory mechanism based on inhibition. In contrast, the recently determined structure of the OmpR/PhoB homologue DrrD revealed no significant interdomain interface, suggesting that the domains are tethered by a flexible linker and lack a fixed orientation relative to each other. To address the generality of this feature, we have determined the 1.8-Å resolution crystal structure of
Thermotoga maritima
DrrB, providing a second structure of a multidomain response regulator of the OmpR/PhoB subfamily. The structure reveals an extensive domain interface of 751 Å
2
and therefore differs greatly from that observed in DrrD. Residues that are crucial players in defining the activation state of the regulatory domain contribute to this interface, implying that conformational changes associated with phosphorylation will influence these intramolecular contacts. The DrrB and DrrD structures are suggestive of different signaling mechanisms, with intramolecular communication between N- and C-terminal domains making substantially different contributions to effector domain regulation in individual members of the OmpR/PhoB family.
Probing the Role of Divalent Metal Ions in a Bacterial Psychrophilic Metalloprotease: Binding Studies of an Enzyme in the Crystalline State by X-Ray Crystallography
Authors: Ravaud Stephanie , Gouet Patrice , Haser Richard , Aghajari Nushin 
Published: July 2003

ABSTRACTThe psychrophilic alkaline metalloprotease (PAP) produced by aPseudomonasbacterium isolated in Antarctica belongs to the clan of metzincins, for which a zinc ion is essential for catalytic activity. Binding studies in the crystalline state have been performed by X-ray crystallography in order to improve the understanding of the role of the zinc and calcium ions bound to this protease. Cocrystallization and soaking experiments with EDTA in a concentration range from 1 to 85 mM have resulted in five three-dimensional structures with a distinct number of metal ions occupying the ion-binding sites. Evolution of the structural changes observed in the vicinity of each cation-binding site has been studied as a function of the concentration of EDTA, as well as of time, in the presence of the chelator. Among others, we have found that the catalytic zinc ion was the first ion to be chelated, ahead of a weakly bound calcium ion (Ca 700) exclusive to the psychrophilic enzyme. Upon removal of the catalytic zinc ion, the side chains of the active-site residues His-173, His-179 and Tyr-209 shifted ∼4, 1.0, and 1.6 Å, respectively. Our studies confirm and also explain the sensitivity of PAP toward moderate EDTA concentrations and propose distinct roles for the calcium ions. A new crystal form of native PAP validates our previous predictions regarding the adaptation of this enzyme to cold environments as well as the proteolytic domain calcium ion being exclusive for PAP independent of crystallization conditions.
The Solution Structure of YbcJ from
Escherichia coli
Reveals a Recently Discovered αL Motif Involved in RNA Binding
Authors: Volpon Laurent , Lievre Carine , Osborne Michael J. , Gandhi Shaifali , Iannuzzi Pietro , Larocque Robert , Cygler Miroslaw , Gehring Kalle , Ekiel Irena 
Published: July 2003

ABSTRACT

The structure of the recombinant
Escherichia coli
protein YbcJ, a representative of a conserved family of bacterial proteins (COG2501), was determined by nuclear magnetic resonance. The fold of YbcJ identified it as a member of the larger family of S4-like RNA binding domains. These domains bind to structured RNA, such as that found in tRNA, rRNA, and a pseudoknot of mRNA. The structure of YbcJ revealed a highly conserved patch of basic residues, comprising amino acids K26, K38, R55, K56, and K59, which likely participate in RNA binding.
Crystal Structures of the Quinone Oxidoreductase from
Thermus thermophilus
HB8 and Its Complex with NADPH: Implication for NADPH and Substrate Recognition
Authors: Shimomura Yoshimitsu , Kakuta Yoshimitsu , Fukuyama Keiichi 
Published: July 2003

ABSTRACT

The crystal structures of the ζ-crystalline-like soluble quinone oxidoreductase from
Thermus thermophilus
HB8 (QOR
Tt
) and of its complex with NADPH have been determined at 2.3- and 2.8-Å resolutions, respectively. QOR
Tt
is composed of two domains, and its overall fold is similar to the folds of
Escherichia coli
quinone oxidoreductase (QOR
Ec
) and horse liver alcohol dehydrogenase. QOR
Tt
forms a homodimer in the crystal by interaction of the βF-strands in domain II, forming a large β-sheet that crosses the dimer interface. High thermostability of QOR
Tt
was evidenced by circular dichroic measurement. NADPH is located between the two domains in the QOR
Tt
-NADPH complex. The disordered segment involved in the coenzyme binding of apo-QOR
Tt
becomes ordered upon NADPH binding. The segment covers an NADPH-binding cleft and may serve as a lid. The 2′-phosphate group of the adenine of NADPH is surrounded by polar and positively charged residues in QOR
Tt
, suggesting that QOR
Tt
binds NADPH more readily than NADH. The putative substrate-binding site of QOR
Tt
, unlike that of QOR
Ec
, is largely blocked by nearby residues, permitting access only to small substrates. This may explain why QOR
Tt
has weak
p
-benzoquinone reduction activity and is inactive with such large substrates of QOR
Ec
as 5-hydroxy-1,4-naphthoquinone and phenanthraquinone.
Crystal Structure of the SarS Protein from
Staphylococcus aureus
Authors: Li Ronggui , Manna Adhar C. , Dai Shaodong , Cheung Ambrose L. , Zhang Gongyi 
Published: July 2003

ABSTRACT

The expression of virulence determinants in
Staphylococcus aureus
is controlled by global regulatory loci (e.g.,
sarA
and
agr
). One of these determinants, protein A (
spa
), is activated by
sarS
, which encodes a 250-residue DNA-binding protein. Genetic analysis indicated that the
agr
locus likely mediates
spa
repression by suppressing the transcription of
sarS
. Contrary to SarA and SarR, which require homodimer formation for proper function, SarS is unusual within the SarA protein family in that it contains two homologous halves, with each half sharing sequence similarity to SarA and SarR. Here we report the 2.2 Å resolution X-ray crystal structure of the SarS protein. SarS has folds similar to those of SarR and, quite plausibly, the native SarA structure. Two typical winged-helix DNA-binding domains are connected by a well-ordered loop. The interactions between the two domains are extensive and conserved. The putative DNA-binding surface is highly positively charged. In contrast, negatively charged patches are located opposite to the DNA-binding surface. Furthermore, sequence alignment and structural comparison revealed that MarR has folds similar to those of SarR and SarS. Members of the MarR protein family have previously been implicated in the negative regulation of an efflux pump involved in multiple antibiotic resistance in many gram-negative species. We propose that MarR also belongs to the winged-helix protein family and has a similar mode of DNA binding as SarR and SarS and possibly the entire SarA protein family member. Based on the structural differences of SarR, SarS, and MarR, we further classified these winged-helix proteins to three subfamilies, SarA, SarS, and MarR. Finally, a possible transcription regulation mechanism is proposed.
A Bacterial TrwC Relaxase Domain Contains a Thermally Stable α-Helical Core
Authors: Arrondo José-Luis R. , Echabe Izaskun , Iloro Ibón , Hernando Miguel-Ángel , de la Cruz Fernando , Goñi Félix M. 
Published: July 2003

ABSTRACT
The TrwC protein is the relaxase-helicase responsible for the initiation and termination reactions of DNA processing during plasmid R388 conjugation. The TrwC-N275 fragment comprises the 275-amino-acid N-terminal domain of the protein that contains the DNA cleavage and strand transfer activities (the relaxase domain). It can be easily purified by keeping a cell lysate at 90°C for 10 min. Infrared spectroscopy shows that this domain has a predominantly α/β structure with some amount of unordered structure. Fast heating and cooling does not change the secondary structure, whereas slow heating produces two bands in the infrared spectrum characteristic of protein aggregation. The denaturation temperature is increased in the protein after the fast-heating thermal shock. Two-dimensional infrared correlation spectroscopy shows that thermal unfolding is a very cooperative two-state process without any appreciable steps prior to aggregation. After aggregation, the α-helix percentage is not altered and α-helix signal does not show in the correlation maps, meaning that the helices are not affected by heating. The results indicate that the domain has an α-helix core resistant to temperature and responsible for folding after fast heating and an outer layer of β-sheet and unordered structure that aggregates under slow heating. The combination of a compact core and a flexible outer layer could be related to the structural requirements of DNA-protein binding.
Mechanisms of Activation of Phosphoenolpyruvate Carboxykinase from
Escherichia coli
by Ca
2+
and of Desensitization by Trypsin
Authors: Sudom Athena , Walters Robert , Pastushok Landon , Goldie Douglas , Prasad Lata , Delbaere Louis T. J. , Goldie Hughes 
Published: July 2003

ABSTRACT

The 1.8-Å resolution structure of the ATP-Mg
2+
-Ca
2+
-pyruvate quinary complex of
Escherichia coli
phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg
2+
-Mn
2+
-pyruvate-PCK, except for the Ca
2+
and Mn
2+
binding sites. Ca
2+
was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca
2+
bound only at the active site, indicating that there is likely no surface allosteric site.
45
Ca
2+
bound to PCK with a
K

d

of 85 μM and
n
of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca
2+
. Separate roles of Mg
2+
, which binds the nucleotide, and Ca
2+
, which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca
2+
-bound structure. Partial trypsin digestion abolishes Ca
2+
activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca
2+
binding site, probably stabilizing the C terminus. Phe409Ala, ΔPhe409, Phe413Ala, Δ397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca
2+
site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes.
Structural Study of Binding of Flagellin by Toll-Like Receptor 5
Authors: Jacchieri Saul G. , Torquato Ricardo , Brentani Ricardo R. 
Published: July 2003

ABSTRACTIn order to predict the binding regions within the complex formed by Toll-like receptor 5 (TLR-5) and flagellin, a complementary hydropathy between the two proteins was sought. A region common to the flagellins ofSalmonella entericaserovar Typhimurium,Pseudomonas aeruginosa, andListeria monocytogeneswas shown to be hydropathically complementary to the 552-to-561 fragment of TLR-5, whose sequence is EILDISRNQL. The hydrophobicity profile of this region is shared with flagellins of 377 bacterial species out of a total of 723 publicly available sequences. A conformational analysis of the predicted binding site of TLR-5, whose structure is still unknown, was carried out with a methodology already applied to similar problems. To sample the conformations available to the peptide chain, a plot of the number of conformations per unit energy interval (density of states) versus energy was built. Following a theoretical argument, conformations belonging to maxima in this plot were selected. The most stable structure obtained in this search, an α-helical conformation, was shown to form the electrostatic interactions Glu552-Gln89, Asp555-Arg92, and Arg558-Glu93 with the predicted binding site of the flagellin ofS.entericaserovar Typhimurium, formed by the 88-to-97 chain fragment (LQRVRELAVQ), which is likewise α helical.
Structural Basis for Thermostability of β-Glycosidase from the Thermophilic Eubacterium
Thermus nonproteolyticus
HG102
Authors: Wang Xinquan , He Xiangyuan , Yang Shoujun , An Xiaomin , Chang Wenrui , Liang Dongcai 
Published: July 2003

ABSTRACT

The three-dimensional structure of a thermostable β-glycosidase (Gly
Tn
) from the thermophilic eubacterium
Thermus nonproteolyticus
HG102 was determined at a resolution of 2.4 Å. The core of the structure adopts the (βα)
8
barrel fold. The sequence alignments and the positions of the two Glu residues in the active center indicate that Gly
Tn
belongs to the glycosyl hydrolases of retaining family 1. We have analyzed the structural features of Gly
Tn
related to the thermostability and compared its structure with those of other mesophilic glycosidases from plants, eubacteria, and hyperthermophilic enzymes from archaea. Several possible features contributing to the thermostability of Gly
Tn
were elucidated.
Mycobacterium tuberculosisChaperonin 10 Is Secreted in the Macrophage Phagosome: Is Secretion Due to Dissociation and Adoption of a Partially Helical Structure at the Membrane?
Authors: Fossati Gianluca , Izzo Gaetano , Rizzi Emanuele , Gancia Emanuela , Modena Daniela , Moras Maria Luisa , Niccolai Neri , Giannozzi Elena , Spiga Ottavia , Bono Letizia , Marone Piero , Leone Eugenio , Mangili Francesca , Harding Stephen , Errington Neil , Walters Christopher , Henderson Brian , Roberts Michael M. , Coates Anthony R. M. , Casetta Bruno , Mascagni Paolo 
Published: July 2003

ABSTRACTTo confirm thatMycobacterium tuberculosischaperonin 10 (Cpn10) is secreted outside the live bacillus, infected macrophages were examined by electron microscopy. This revealed that the mycobacterial protein accumulates both in the wall of the bacterium and in the matrix of the phagosomes in which ingested mycobacteria survive within infected macrophages. To understand the structural implications underlying this secretion, a structural study ofM.tuberculosisCpn10 was performed under conditions that are generally believed to mimic the membrane environment. It was found that in buffer-organic solvent mixtures, the mycobacterial protein forms two main species, namely, a partially helical monomer that prevails in dilute solutions at room temperature and a dimer that folds into a β-sheet-dominated structure and prevails in either concentrated protein solutions at room temperature or in dilute solutions at low temperature. A partially helical monomer was also found and was completely associated with negatively charged detergents in a micelle-bound state. Remarkably, zwitterionic lipids had no effect on the protein structure. By using N- and C-truncated forms of the protein, the C- and N-terminal sequences were identified as possessing an amphiphilic helical character and as selectively associating with acidic detergent micelles. When the study was extended to other chaperonins, it was found that human Cpn10 is also monomeric and partially helical in dilute organic solvent-buffer mixtures. In contrast,Escherichia coliCpn10 is mostly dimeric and predominately β-sheet in both dilute and concentrated solutions. Interestingly, human Cpn10 also crosses biological membranes, whereas theE.colihomologue is strictly cytosolic. These results suggest that dissociation to partially helical monomers and interaction with acidic lipids may be two important steps in the mechanism of secretion ofM.tuberculosisCpn10 to the external environment.
Detecting Protein-Protein Interactions in the Intact Cell of
Bacillus subtilis
(ATCC 6633)
Authors: Winters Michael S. , Day R. A. 
Published: July 2003

ABSTRACT

The salt bridge, paired group-specific reagent cyanogen (ethanedinitrile; C
2
N
2
) converts naturally occurring pairs of functional groups into covalently linked products. Cyanogen readily permeates cell walls and membranes. When the paired groups are shared between associated proteins, isolation of the covalently linked proteins allows their identity to be assigned. Examination of organisms of known genome sequence permits identification of the linked proteins by mass spectrometric techniques applied to peptides derived from them. The cyanogen-linked proteins were isolated by polyacrylamide gel electrophoresis. Digestion of the isolated proteins with proteases of known specificity afforded sets of peptides that could be analyzed by mass spectrometry. These data were compared with those derived theoretically from the Swiss Protein Database by computer-based comparisons (Protein Prospector;
http://prospector.ucsf.edu
). Identification of associated proteins in the ribosome of
Bacillus subtilis
strain ATCC 6633 showed that there is an association homology with the association patterns of the ribosomal proteins of
Haloarcula marismortui
and
Thermus thermophilus
. In addition, other proteins involved in protein biosynthesis were shown to be associated with ribosomal proteins.
Structural Insight into the Antibiotic Action of Telithromycin against Resistant Mutants
Authors: Berisio Rita , Harms Joerg , Schluenzen Frank , Zarivach Raz , Hansen Harly A. S. , Fucini Paola , Yonath Ada 
Published: July 2003

ABSTRACT

The crystal structure of the ketolide telithromycin bound to the
Deinococcus radiodurans
large ribosomal subunit shows that telithromycin blocks the ribosomal exit tunnel and interacts with domains II and V of the 23S RNA. Comparisons to other clinically relevant macrolides provided structural insights into its enhanced activity against macrolide-resistant strains.