bluuebunny/crossref_metadata_2025 huggingface dataset:
Horizontal array beamforming in an azimuthally anisotropic internal wave field
Authors: Finette Steven , Oba Roger
Published: July 2003

A numerical study of beamforming on a horizontal array is performed in a shallow water waveguide where a summer thermocline is perturbed by a time evolving realization of an internal wave field. The components of the internal wave field consist of a horizontally (azimuthally) isotropic, spatially homogeneous contribution, and a horizontally anisotropic, spatially inhomogeneous component. These terms represent a diffuse (“background”) internal wave field and a localized solitary wave packet, respectively. Conventional beamforming is performed as a function of time while the internal wave field evolves throughout a computational volume containing the source–receiver paths. Source–receiver orientation with respect to the azimuthally anisotropic component has a significant effect on the beamformed output. When the source–receiver configuration is oriented approximately parallel to the solitary wave crests, beam wander, fading, beam splitting and coherence length degradation occurs in a time-dependent manner as the solitary wave packet passes through the environment. Both horizontal refraction of energy and a time-dependent modal source excitation distribution are responsible for these beamforming effects. In cases where source–receiver orientation is not approximately parallel to the wave crests, these effects are substantially reduced or eliminated, indicating that an azimuthally selective perturbation of the acoustic field can be attributed to the wave packet. Modal decomposition of the acoustic field and single mode starting fields are used to infer that, for the source–receiver orientation along the wave crests and troughs, acoustic propagation is predominantly adiabatic. A modal phase speed analysis explains several features associated with the beamformed power.
Ecological Consequences of a Century of Warming in Lake Tanganyika
Authors: Verburg Piet , Hecky Robert E. , Kling Hedy
Published: July 2003

Deep tropical lakes are excellent climate monitors because annual mixing is shallow and flushing rates are low, allowing heat to accumulate during climatic warming. We describe effects of warming on Lake Tanganyika: A sharpened density gradient has slowed vertical mixing and reduced primary production. Increased warming rates during the coming century may continue to slow mixing and further reduce productivity in Lake Tanganyika and other deep tropical lakes.
Melanopsin Is Required for Non-Image-Forming Photic Responses in Blind Mice
Authors: Panda Satchidananda , Provencio Ignacio , Tu Daniel C. , Pires Susana S. , Rollag Mark D. , Castrucci Ana Maria , Pletcher Mathew T. , Sato Trey K. , Wiltshire Tim , Andahazy Mary , Kay Steve A. , Van Gelder Russell N. , Hogenesch John B.
Published: July 2003

Although mice lacking rod and cone photoreceptors are blind, they retain many eye-mediated responses to light, possibly through photosensitive retinal ganglion cells. These cells express melanopsin, a photopigment that confers this photosensitivity. Mice lacking melanopsin still retain nonvisual photoreception, suggesting that rods and cones could operate in this capacity. We observed that mice with both outer-retinal degeneration and a deficiency in melanopsin exhibited complete loss of photoentrainment of the circadian oscillator, pupillary light responses, photic suppression of arylalkylamine-
N
-acetyltransferase transcript, and acute suppression of locomotor activity by light. This indicates the importance of both nonvisual and classical visual photoreceptor systems for nonvisual photic responses in mammals.
Separation of Metallic from Semiconducting Single-Walled Carbon Nanotubes
Authors: Krupke Ralph , Hennrich Frank , Löhneysen Hilbert v. , Kappes Manfred M.
Published: July 2003

We have developed a method to separate metallic from semiconducting single-walled carbon nanotubes from suspension using alternating current dielectrophoresis. Our method takes advantage of the difference of the relative dielectric constants of the two species with respect to the solvent, resulting in an opposite movement of metallic and semiconducting tubes along the electric field gradient. Metallic tubes are attracted toward a microelectrode array, leaving semiconducting tubes in the solvent. Proof of the effectiveness of separation is given by a comparative Raman spectroscopy study on the dielectrophoretically deposited tubes and on a reference sample.
Old Dogs and New Tricks, Proteases, Inhibitors, and Cell Migration
Authors: Stefansson Steingrimur , Lawrence Daniel A.
Published: July 2003

A new model for the actions of plasminogen activator inhibitors (PAIs) on cell migration may resolve the conflicting research data on these proteins in metastasis and angiogenesis. Results from two groups reveal a role for PAI-1 in promoting cycles of attachment and detachment of the cell from the extracellular matrix that is independent of its role as an enzymatic inhibitor of urokinase-type plasminogen activator (uPA). Through the formation of a complex of integrins, uPA and its receptor, and the clearance receptors of the low-density lipoprotein family, PAI-1 may promote endocytosis and recycling of these adhesion-controlling proteins, allowing cycling of cellular attachment and detachment.
Crystal Structure of the YchF Protein Reveals Binding Sites for GTP and Nucleic Acid
Authors: Teplyakov Alexey , Obmolova Galina , Chu Seung Y. , Toedt John , Eisenstein Edward , Howard Andrew J. , Gilliland Gary L.
Published: July 2003

The bacterial protein encoded by the geneychFis 1 of 11 universally conserved GTPases and the only one whose function is unknown. The crystal structure determination of YchF was sought to help with the functional assignment of the protein. The YchF protein fromHaemophilus influenzaewas cloned and expressed, and the crystal structure was determined at 2.4 Å resolution. The polypeptide chain is folded into three domains. The N-terminal domain has a mononucleotide binding fold typical for the P-loop NTPases. An 80-residue domain next to it has a pronounced α-helical coiled coil. The C-terminal domain features a six-stranded half-barrel that curves around an α-helix. The crablike three-domain structure of YchF suggests the binding site for a double-stranded nucleic acid in the cleft between the domains. The structure of the putative GTP-binding site is consistent with the postulated guanine specificity of the protein. Fluorescence measurements have demonstrated the ability of YchF to bind a double-stranded nucleic acid and GTP. Taken together with other experimental data and genomic analysis, these results suggest that YchF may be part of a nucleoprotein complex and may function as a GTP-dependent translation factor.
Crystal Structure ofd-Hydantoinase fromBurkholderia pickettiiat a Resolution of 2.7 Angstroms: Insights into the Molecular Basis of Enzyme Thermostability
Authors: Xu Zhen , Liu Yunqing , Yang Yunliu , Jiang Weihong , Arnold Eddy , Ding Jianping
Published: July 2003

d-Hydantoinase (d-HYD) is an industrial enzyme that is widely used in the production ofd-amino acids which are precursors for semisynthesis of antibiotics, peptides, and pesticides. This report describes the crystal structure ofd-hydantoinase fromBurkholderia pickettii(HYDBp) at a 2.7-Å resolution. The structure of HYDBpconsists of a core (α/β)8triose phosphate isomerase barrel fold and a β-sheet domain, and the catalytic active site consists of two metal ions and six highly conserved amino acid residues. Although HYDBpshares only moderate sequence similarity withd-HYDs fromThermussp. (HYDTsp) andBacillus stearothermophilus(HYDBst), whose structures have recently been solved, the overall structure and the structure of the catalytic active site are strikingly similar. Nevertheless, the amino acids that compose the substrate-binding site are less conserved and have different properties, which might dictate the substrate specificity. Structural comparison has revealed insights into the molecular basis of the differential thermostability ofd-HYDs. The more thermostable HYDTspcontains more aromatic residues in the interior of the structure than HYDBpand HYDBst. Changes of large aromatic residues in HYDTspto smaller residues in HYDBpor HYDBstdecrease the hydrophobicity and create cavities inside the structure. HYDTsphas more salt bridges and hydrogen-bonding interactions and less oxidation susceptible Met and Cys residues on the protein surface than HYDBpand HYDBst. Besides, HYDTspalso contains more rigid Pro residues. These factors are likely to make major contributions to the varying thermostability of these enzymes. This information could be exploited in helping to engineer more thermostable mesophilic enzymes.
Dimer-Tetramer Transition between Solution and Crystalline States of Streptavidin and Avidin Mutants
Authors: Pazy Yael , Eisenberg-Domovich Yael , Laitinen Olli H. , Kulomaa Markku S. , Bayer Edward A. , Wilchek Meir , Livnah Oded
Published: July 2003

The biotin-binding tetrameric proteins, streptavidin from
Streptomyces avidinii
and chicken egg white avidin, are excellent models for the study of subunit-subunit interactions of a multimeric protein. Efforts are thus being made to prepare mutated forms of streptavidin and avidin, which would form monomers or dimers, in order to examine their effect on quaternary structure and assembly. In the present communication, we compared the crystal structures of binding site W→K mutations in streptavidin and avidin. In solution, both mutant proteins are known to form dimers, but upon crystallization, both formed tetramers with the same parameters as the native proteins. All of the intersubunit bonds were conserved, except for the hydrophobic interaction between biotin and the tryptophan that was replaced by lysine. In the crystal structure, the binding site of the mutated apo-avidin contains 3 molecules of structured water instead of the 5 contained in the native protein. The lysine side chain extends in a direction opposite that of the native tryptophan, the void being partially filled by an adjacent lysine residue. Nevertheless, the binding-site conformation observed for the mutant tetramer is an artificial consequence of crystal packing that would not be maintained in the solution-phase dimer. It appears that the dimer-tetramer transition may be concentration dependent, and the interaction among subunits obeys the law of mass action.
Crystal Structure of a Putative Methyltransferase from
Mycobacterium tuberculosis
: Misannotation of a Genome Clarified by Protein Structural Analysis
Authors: Johnston Jodie M. , Arcus Vickery L. , Morton Craig J. , Parker Michael W. , Baker Edward N.
Published: July 2003

Bioinformatic analyses of whole genome sequences highlight the problem of identifying the biochemical and cellular functions of many gene products that are at present uncharacterized. The open reading frame Rv3853 from
Mycobacterium tuberculosis
has been annotated as
menG
and assumed to encode an
S
-adenosylmethionine (SAM)-dependent methyltransferase that catalyzes the final step in menaquinone biosynthesis. The Rv3853 gene product has been expressed, refolded, purified, and crystallized in the context of a structural genomics program. Its crystal structure has been determined by isomorphous replacement and refined at 1.9 Å resolution to an
R
factor of 19.0% and
R
free
of 22.0%. The structure strongly suggests that this protein is not a SAM-dependent methyltransferase and that the gene has been misannotated in this and other genomes that contain homologs. The protein forms a tightly associated, disk-like trimer. The monomer fold is unlike that of any known SAM-dependent methyltransferase, most closely resembling the phosphohistidine domains of several phosphotransfer systems. Attempts to bind cofactor and substrate molecules have been unsuccessful, but two adventitiously bound small-molecule ligands, modeled as tartrate and glyoxalate, are present on each monomer. These may point to biologically relevant binding sites but do not suggest a function. In silico screening indicates a range of ligands that could occupy these and other sites. The nature of these ligands, coupled with the location of binding sites on the trimer, suggests that proteins of the Rv3853 family, which are distributed throughout microbial and plant species, may be part of a larger assembly binding to nucleic acids or proteins.
Crystal Structure of the Hyperthermophilic Archaeal DNA-Binding Protein Sso10b2 at a Resolution of 1.85 Angstroms
Authors: Chou Chia-Cheng , Lin Ting-Wan , Chen Chin-Yu , Wang Andrew H.-J.
Published: July 2003

The crystal structure of a small, basic DNA binding protein, Sso10b2, from the thermoacidophilic archaeon
Sulfolobus solfataricus
was determined by the Zn multiwavelength anomalous diffraction method and refined to 1.85 Å resolution. The 89-amino-acid protein adopts a βαβαββ topology. The structure is similar to that of Sso10b1 (also called Alba) from the same organism. However, Sso10b2 contains an arginine-rich loop RDRRR motif, which may play an important role in nucleic acid binding. There are two independent Sso10b2 proteins in the asymmetric unit, and a plausible stable dimer could be deduced from the crystal structure. Topology comparison revealed that Sso10b2 is similar to several RNA-binding proteins, including IF3-C, YhhP, and DNase I. Models of the Sso10b2 dimer bound to either B-DNA or A-DNA have been constructed.
A Novel Adenylate Binding Site Confers Phosphopantetheine Adenylyltransferase Interactions with Coenzyme A
Authors: Izard Tina
Published: July 2003

Phosphopantetheine adenylyltransferase (PPAT) regulates the key penultimate step in the essential coenzyme A (CoA) biosynthetic pathway. PPAT catalyzes the reversible transfer of an adenylyl group from Mg
2+
:ATP to 4′-phosphopantetheine to form 3′-dephospho-CoA (dPCoA) and pyrophosphate. The high-resolution crystal structure of PPAT complexed with CoA has been determined. Remarkably, CoA and the product dPCoA bind to the active site in distinct ways. Although the phosphate moiety within the phosphopantetheine arm overlaps, the pantetheine arm binds to the same pocket in two distinct conformations, and the adenylyl moieties of these two ligands have distinct binding sites. Moreover, the PPAT:CoA crystal structure confirms the asymmetry of binding to the two trimers within the hexameric enzyme. Specifically, the pantetheine arm of CoA bound to one protomer within the asymmetric unit displays the dPCoA-like conformation with the adenylyl moiety disordered, whereas CoA binds the twofold-related protomer in an ordered and unique fashion.
Determinants of the Src Homology Domain 3-Like Fold
Authors: D'Aquino J. Alejandro , Ringe Dagmar
Published: July 2003

In eukaryotes, the Src homology domain 3 (SH3) is a very important motif in signal transduction. SH3 domains recognize poly-proline-rich peptides and are involved in protein-protein interactions. Until now, the existence of SH3 domains has not been demonstrated in prokaryotes. However, the structure of the C-terminal domain of DtxR clearly shows that the fold of this domain is very similar to that of the SH3 domain. In addition, there is evidence that the C-terminal domain of DtxR binds to poly-proline-rich regions. Other bacterial proteins have domains that are structurally similar to the SH3 domain but whose functions are unknown or differ from that of the SH3 domain. The observed similarities between the structures of the C-terminal domain of DtxR and the SH3 domain constitute a perfect system to gain insight into their function and information about their evolution. Our results show that the C-terminal domain of DtxR shares a number of conserved key hydrophobic positions not recognizable from sequence comparison that might be responsible for the integrity of the SH3-like fold. Structural alignment of an ensemble of such domains from unrelated proteins shows a common structural core that seems to be conserved despite the lack of sequence similarity. This core constitutes the minimal requirements of protein architecture for the SH3-like fold.
The Purine Repressor of
Bacillus subtilis
: a Novel Combination of Domains Adapted for Transcription Regulation
Authors: Sinha Sangita C. , Krahn Joseph , Shin Byung Sik , Tomchick Diana R. , Zalkin Howard , Smith Janet L.
Published: July 2003

The purine repressor from
Bacillus subtilis
, PurR, represses transcription from a number of genes with functions in the synthesis, transport, and metabolism of purines. The 2.2-Å crystal structure of PurR reveals a two-domain protein organized as a dimer. The larger C-terminal domain belongs to the PRT structural family, in accord with a sequence motif for binding the inducer phosphoribosylpyrophosphate (PRPP). The PRT domain is fused to a smaller N-terminal domain that belongs to the winged-helix family of DNA binding proteins. A positively charged surface on the winged-helix domain likely binds specific DNA sequences in the recognition site. A second positively charged surface surrounds the PRPP site at the opposite end of the PurR dimer. Conserved amino acids in the sequences of PurR homologs in 21 gram-positive bacteria cluster on the proposed recognition surface of the winged-helix domain and around the PRPP binding site at the opposite end of the molecule, supporting a common function of DNA and PRPP binding for all of the proteins. The structure supports a binding mechanism in which extended regions of DNA interact with extensive protein surface. Unlike most PRT proteins, which are phosphoribosyltransferases (PRTases), PurR lacks catalytic activity. This is explained by a tyrosine side chain that blocks the site for a nucleophile cosubstrate in PRTases. Thus,
B. subtilis
has adapted an enzyme fold to serve as an effector-binding domain and has used it in a novel combination with the DNA-binding winged-helix domain as a repressor of purine genes.
Functional Dissection of the
Bacillus subtilis pur
Operator Site
Authors: Bera Aloke Kumar , Zhu Jianghai , Zalkin Howard , Smith Janet L.
Published: July 2003

Bacillus subtilis
PurR represses transcription of several genes involved in purine synthesis, metabolism, and transport and cofactor synthesis. PurR binds specifically to DNAs containing an inverted repeat of a 14-nucleotide “PurBox” located in the upstream control regions of genes in the PurR regulon. Further biochemical investigation of the interaction of PurR with a series of shortened upstream DNA fragments of the
pur
operon determined the minimum length and specificity elements of the operator. The relative affinities of the two PurBoxes differ significantly, such that upstream PurBox1 (−81 to −68 relative to the transcription start site) is designated “strong” and downstream PurBox2 (−49 to −36) is designated “weak.” Two PurBoxes are required for high-affinity PurR binding, and one of these must be strong. The shortest DNA construct with high affinity for PurR is a 74-bp perfect palindrome in which weak PurBox2 and its flanking sequences are replaced by strong PurBox1 and flanking sequences. Two PurR dimers bind to this symmetric construct. Phosphoribosylpyrophosphate (PRPP), the effector molecule that reduces affinity of PurR for DNA, requires one weak PurBox in the DNA construct to inhibit PurR binding. PRPP binds, as expected, to a PRPP-motif in PurR. A tracks outside the central conserved CGAA sequence of the PurBox may facilitate DNA bending, leading to a proposal for strong and weak designations of PurBoxes in the control regions of other genes regulated by PurR.
Structure of a Coenzyme A Pyrophosphatase from
Deinococcus radiodurans
: a Member of the Nudix Family
Authors: Kang Lin-Woo , Gabelli Sandra B. , Bianchet Mario A. , Xu Wen Lian , Bessman Maurice J. , Amzel L. Mario
Published: July 2003

Gene
Dr1184
from
Deinococcus radiodurans
codes for a Nudix enzyme (DR-CoAse) that hydrolyzes the pyrophosphate moiety of coenzyme A (CoA). Nudix enzymes with the same specificity have been found in yeast, humans, and mice. The three-dimensional structure of DR-CoAse, the first of a Nudix hydrolase with this specificity, reveals that this enzyme contains, in addition to the fold observed in other Nudix enzymes, insertions that are characteristic of a CoA-hydrolyzing Nudix subfamily. The structure of the complex of the enzyme with Mg
2+
, its activating cation, reveals the position of the catalytic site. A helix, part of the N-terminal insertion, partially occludes the binding site and has to change its position to permit substrate binding. Comparison of the structure of DR-CoAse to those of other Nudix enzymes, together with the location in the structure of the sequence characteristic of CoAses, suggests a mode of binding of the substrate to the enzyme that is compatible with all available data.
Structure ofMycobacterium tuberculosisMethionine Sulfoxide Reductase A in Complex with Protein-Bound Methionine
Authors: Taylor Alexander B. , Benglis, David M. , Dhandayuthapani Subramanian , Hart P. John
Published: July 2003

Peptide methionine sulfoxide reductase (MsrA) repairs oxidative damage to methionine residues arising from reactive oxygen species and reactive nitrogen intermediates. MsrA activity is found in a wide variety of organisms, and it is implicated as one of the primary defenses against oxidative stress. Disruption of the gene encoding MsrA in several pathogenic bacteria responsible for infections in humans results in the loss of their ability to colonize host cells. Here, we present the X-ray crystal structure of MsrA from the pathogenic bacteriumMycobacterium tuberculosisrefined to 1.5 Å resolution. In contrast to the three catalytic cysteine residues found in previously characterized MsrA structures,M. tuberculosisMsrA represents a class containing only two functional cysteine residues. The structure reveals a methionine residue of one MsrA molecule bound at the active site of a neighboring molecule in the crystal lattice and thus serves as an excellent model for protein-bound methionine sulfoxide recognition and repair.
X-Ray Crystal Structure of the Multidomain Endoglucanase Cel9G from
Clostridium cellulolyticum
Complexed with Natural and Synthetic Cello-Oligosaccharides
Authors: Mandelman David , Belaich Anne , Belaich J. P. , Aghajari Nushin , Driguez Hugues , Haser Richard
Published: July 2003

Complete cellulose degradation is the first step in the use of biomass as a source of renewable energy. To this end, the engineering of novel cellulase activity, the activity responsible for the hydrolysis of the β-1,4-glycosidic bonds in cellulose, is a topic of great interest. The high-resolution X-ray crystal structure of a multidomain endoglucanase from
Clostridium cellulolyticum
has been determined at a 1.6-Å resolution. The endoglucanase, Cel9G, is comprised of a family 9 catalytic domain attached to a family III
c
cellulose-binding domain. The two domains together form a flat platform onto which crystalline cellulose is suggested to bind and be fed into the active-site cleft for endolytic hydrolysis. To further dissect the structural basis of cellulose binding and hydrolysis, the structures of Cel9G in the presence of cellobiose, cellotriose, and a DP-10 thio-oligosaccharide inhibitor were resolved at resolutions of 1.7, 1.8, and 1.9 Å, respectively.
The 1.3-Angstrom-Resolution Crystal Structure of β-Ketoacyl-Acyl Carrier Protein Synthase II fromStreptococcus pneumoniae
Authors: Price Allen C. , Rock Charles O. , White Stephen W.
Published: July 2003

The β-ketoacyl-acyl carrier protein synthases are members of the thiolase superfamily and are key regulators of bacterial fatty acid synthesis. As essential components of the bacterial lipid metabolic pathway, they are an attractive target for antibacterial drug discovery. We have determined the 1.3 Å resolution crystal structure of the β-ketoacyl-acyl carrier protein synthase II (FabF) from the pathogenic organismStreptococcus pneumoniae. The protein adopts a duplicated βαβαβαββ fold, which is characteristic of the thiolase superfamily. The two-fold pseudosymmetry is broken by the presence of distinct insertions in the two halves of the protein. These insertions have evolved to bind the specific substrates of this particular member of the thiolase superfamily. Docking of the pantetheine moiety of the substrate identifies the loop regions involved in substrate binding and indicates roles for specific, conserved residues in the substrate binding tunnel. The active site triad of this superfamily is present in spFabF as His 303, His 337, and Cys 164. Near the active site is an ion pair, Glu 346 and Lys 332, that is conserved in the condensing enzymes but is unusual in our structure in being stabilized by an Mg2+ion which interacts with Glu 346. The active site histidines interact asymmetrically with Lys 332, whose positive charge is closer to His 303, and we propose a specific role for the lysine in polarizing the imidazole ring of this histidine. This asymmetry suggests that the two histidines have unequal roles in catalysis and provides new insights into the catalytic mechanisms of these enzymes.
The Crystal Structure of Shikimate Dehydrogenase (AroE) Reveals a Unique NADPH Binding Mode
Authors: Ye Sheng , von Delft Frank , Brooun Alexei , Knuth Mark W. , Swanson Ronald V. , McRee Duncan E.
Published: July 2003

Shikimate dehydrogenase catalyzes the NADPH-dependent reversible reduction of 3-dehydroshikimate to shikimate. We report the first X-ray structure of shikimate dehydrogenase from
Haemophilus influenzae
to 2.4-Å resolution and its complex with NADPH to 1.95-Å resolution. The molecule contains two domains, a catalytic domain with a novel open twisted α/β motif and an NADPH binding domain with a typical Rossmann fold. The enzyme contains a unique glycine-rich P-loop with a conserved sequence motif, GAGGXX, that results in NADPH adopting a nonstandard binding mode with the nicotinamide and ribose moieties disordered in the binary complex. A deep pocket with a narrow entrance between the two domains, containing strictly conserved residues primarily contributed by the catalytic domain, is identified as a potential 3-dehydroshikimate binding pocket. The flexibility of the nicotinamide mononucleotide portion of NADPH may be necessary for the substrate 3-dehydroshikimate to enter the pocket and for the release of the product shikimate.
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

UDP-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

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

The 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

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

The 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

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

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

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

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

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

In 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

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

To 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.