bluuebunny/crossref_metadata_2025_split huggingface dataset:
Computational design of a water‐soluble analog of phospholamban
Authors: Slovic Avram M. , Summa Christopher M. , Lear James D. , DeGrado William F. 
Published: January 2003

AbstractMembrane proteins and water‐soluble proteins share a similar core. This similarity suggests that it should be possible to water‐solubilize membrane proteins by mutating only their lipid‐exposed residues. We have developed computational tools to design water‐soluble variants of helical membrane proteins, using the pentameric phospholamban (PLB) as our test case. To water‐solublize PLB, the membrane‐exposed positions were changed to polar or charged amino acids, while the putative core was left unaltered. We generated water‐soluble phospholamban (WSPLB), and compared its properties to its predecessor PLB. In aqueous solution, WSPLB mimics all of the reported properties of PLB including oligomerization state, helical structure, and stabilization upon phosphorylation. We also characterized the truncated mutant WSPLB (21–52) comprising only the former transmembrane segment of PLB. This peptide shows a decreased specificity for forming a pentameric oligomerization state.
Crystal structure of a trimeric form of dephosphocoenzyme A kinase from Escherichia coli
Authors: O'Toole Nicholas , Barbosa João A.R.G. , Li Yunge , Hung Li‐Wei , Matte Allan , Cygler Miroslaw 
Published: January 2003

AbstractCoenzyme A (CoA) is an essential cofactor used in a wide variety of biochemical pathways. The final step in the biosynthesis of CoA is catalyzed by dephosphocoenzyme A kinase (DPCK, E.C. 2.7.1.24). Here we report the crystal structure of DPCK from Escherichia coli at 1.8 Å resolution. This enzyme forms a tightly packed trimer in its crystal state, in contrast to its observed monomeric structure in solution and to the monomeric, homologous DPCK structure from Haemophilus influenzae. We have confirmed the existence of the trimeric form of the enzyme in solution using gel filtration chromatography measurements. Dephospho‐CoA kinase is structurally similar to many nucleoside kinases and other P‐loop‐containing nucleotide triphosphate hydrolases, despite having negligible sequence similarity to these enzymes. Each monomer consists of five parallel β‐strands flanked by α‐helices, with an ATP‐binding site formed by a P‐loop motif. Orthologs of the E. coli DPCK sequence exist in a wide range of organisms, including humans. Multiple alignment of orthologous DPCK sequences reveals a set of highly conserved residues in the vicinity of the nucleotide/CoA binding site.
Trapping of peptide‐based surrogates in an artificially created channel of cytochrome c peroxidase
Authors: Hays Anna‐Maria A. , Gray Harry B. , Goodin David B. 
Published: January 2003

AbstractAs recently described, the deliberate removal of the proposed electron transfer pathway from cytochrome c peroxidase resulted in the formation of an extended ligand‐binding channel. The engineered channel formed a template for the removed peptide segment, suggesting that synthetic surrogates might be introduced to replace the native electron transfer pathway. This approach could be united with the recent development of sensitizer‐linked substrates to initiate and study electron transfer, allowing access to unresolved issues about redox mechanism of the enzyme. Here, we present the design, synthesis, and screening of a peptide library containing natural and unnatural amino acids to identify the structural determinants for binding this channel mutant. Only one peptide, (benzimidazole‐propionic acid)‐Gly‐Ala‐Ala, appeared to interact, and gave evidence for both reversible and kinetically trapped binding, suggesting multiple conformations for the channel protein. Notably, this peptide was the most analogous to the removed electron transfer sequence, supporting the use of a cavity‐template strategy for design of specific sensitizer‐linked substrates as replacements for the native electron transfer pathway.
Structural variation and inhibitor binding in polypeptide deformylase from four different bacterial species
Authors: Smith Kathrine J. , Petit Chantal M. , Aubart Kelly , Smyth Martin , McManus Edward , Jones Jo , Fosberry Andrew , Lewis Ceri , Lonetto Michael , Christensen Siegfried B. 
Published: January 2003

AbstractPolypeptide deformylase (PDF) catalyzes the deformylation of polypeptide chains in bacteria. It is essential for bacterial cell viability and is a potential antibacterial drug target. Here, we report the crystal structures of polypeptide deformylase from four different species of bacteria: Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, and Escherichia coli. Comparison of these four structures reveals significant overall differences between the two Gram‐negative species (E. coli and H. influenzae) and the two Gram‐positive species (S. pneumoniae and S. aureus). Despite these differences and low overall sequence identity, the S1′ pocket of PDF is well conserved among the four enzymes studied. We also describe the binding of nonpeptidic inhibitor molecules SB‐485345, SB‐543668, and SB‐505684 to both S. pneumoniae and E. coli PDF. Comparison of these structures shows similar binding interactions with both Gram‐negative and Gram‐positive species. Understanding the similarities and subtle differences in active site structure between species will help to design broad‐spectrum polypeptide deformylase inhibitor molecules.
Vibrio cholerae cytolysin is composed of an α‐hemolysin‐like core
Authors: Olson Rich , Gouaux Eric 
Published: January 2003

AbstractThe enteric pathogen Vibrio cholerae secretes a water‐soluble 80‐kD cytolysin, Vibrio cholerae cytolysin (VCC) that assembles into pentameric channels following proteolytic activation by exogenous proteases. Until now, VCC has been placed in a unique class of pore‐forming toxins, distinct from paradigms such as Staphyloccal α‐hemolysin. However, as reported here, amino acid sequence analysis and three‐dimensional structure modeling indicate that the core component of the VCC toxin is related in sequence and structure to a family of hemolysins from Staphylococcus aureus that include leukocidin F and α‐hemolysin. Furthermore, our analysis has identified the channel‐forming region of VCC and a potential lipid head‐group binding site, and suggests a conserved mechanism of assembly and lysis. An additional domain in the VCC toxin is related to plant lectins, conferring additional target cell specificity to the toxin.
Expression, solubilization, and biochemical characterization of the tight junction transmembrane protein claudin‐4
Authors: Mitic Laura L. , Unger Vinzenz M. , Anderson James Melvin 
Published: January 2003

AbstractThe tight junction tetraspan protein claudin‐4 creates a charge‐selective pore in the paracellular pathway across epithelia. The structure of the pore is unknown, but is presumed to result from transcellular adhesive contacts between claudin's extracellular loops. Here we report the expression of claudin‐4 by baculovirus infection of Sf9 cells and describe the biochemical analysis suggesting it has a hexameric quaternary configuration. We show the detergent perfluoro‐octanoic acid is able to maintain oligomeric claudin species. Sucrose velocity centrifugation and laser light scattering are also used to investigate the oligomeric state of claudin‐4. In contrast to proteins of similar topology, such as gap junction family connexins, the oligomeric state of claudins appears more dynamic. These data suggest the structural organization of claudins in tight junction pores is unique.
Structural composition of βI‐ and βII‐proteins
Authors: Sreerama Narasimha , Woody Robert W. 
Published: January 2003

AbstractCircular dichroism spectra of proteins are sensitive to protein secondary structure. The CD spectra of α‐rich proteins are similar to those of model α‐helices, but β‐rich proteins exhibit CD spectra that are reminiscent of CD spectra of either model β‐sheets or unordered polypeptides. The existence of these two types of CD spectra for β‐rich proteins form the basis for their classification as βI‐ and βII‐proteins. Although the conformation of β‐sheets is largely responsible for the CD spectra of βI‐proteins, the source of βII‐protein CD, which resembles that of unordered polypeptides, is not completely understood. The CD spectra of unordered polypeptides are similar to that of the poly(Pro)II helix, and the poly(Pro)II‐type (P2) structure forms a significant fraction of the unordered conformation in globular proteins. We have compared the β‐sheet and P2 structure contents in β‐rich proteins to understand the origin of βII‐protein CD. We find that βII‐proteins have a ratio of P2 to β‐sheet content greater than 0.4, whereas for βI‐proteins this ratio is less than 0.4. The β‐sheet content in βI‐proteins is generally higher than that in βII‐proteins. The origin of two classes of CD spectra for β‐rich proteins appears to lie in their relative β‐sheet and P2 structure contents.
Influence of Extracellular Bactericidal Agents on Bacteria within Macrophages
Authors: Hamrick Terri S. , Diaz Adam H. , Havell Edward A. , Horton John R. , Orndorff Paul E. 
Published: January 2003

ABSTRACT

We employed gentamicin-sensitive and -resistant derivatives of
Escherichia coli
in a macrophage phagocytosis assay that compared λ bacteriophage and gentamicin as extracellular bactericidal agents. Colony counts and direct microscopic examination of phagocytized
E. coli
supported the conclusion that gentamicin entered macrophages, even at low concentrations, and contributed to their bactericidal activity. Also, two
E. coli
strains differing in the ability to express the adhesin of type 1 pili (FimH) were distinguishably different in intracellular survival when λ was used as the extracellular killing agent but were indistinguishable when gentamicin was employed.
Molecular and Phenotypic Analysis of the CS54 Island ofSalmonella entericaSerotype Typhimurium: Identification of Intestinal Colonization and Persistence Determinants
Authors: Kingsley Robert A. , Humphries Andrea D. , Weening Eric H. , de Zoete Marcel R. , Winter Sebastian , Papaconstantinopoulou Anastasia , Dougan Gordon , Bäumler Andreas J. 
Published: January 2003

ABSTRACTTheshdAgene is carried on a 25-kb genetic island at centisome 54 (CS54 island) of theSalmonella entericaserotype Typhimurium chromosome. In addition toshdA, the CS54 island ofSalmonellaserotype Typhimurium strain LT2 contains four open reading frames designatedratA,ratB,sivI, andsivH. DNA hybridization analysis revealed that the CS54 island is comprised of two regions with distinct phylogenetic distribution within the genusSalmonella. Homologues ofshdAandratBwere detected only in serotypes ofSalmonella entericasubsp. I. In contrast, sequences hybridizing withratA,sivI, andsivHwere present inS. entericasubsp. II andS. bongoriin addition toS. entericasubsp. I. Deletion of theratAandsivIgenes did not alter the ability ofSalmonellaserotype Typhimurium to colonize the organs of mice. Insertional inactivation of thesivHgene resulted in defective colonization of the Peyer's patches of the terminal ileum but normal colonization of the cecum, mesenteric lymph nodes, and spleen. Deletion of theshdAgene resulted in decreased colonization of the cecum and Peyer's patches of the terminal ileum and colonization to a lesser degree in the mesenteric lymph nodes and spleen 5 days post-oral inoculation of mice. A strain containing a deletion in theratBgene exhibited a defect for the colonization of the cecum but not of the Peyer's patches, mesenteric lymph nodes, and spleen. TheshdAandratBdeletion strains exhibited a shedding defect in mice, whereas thesivHdeletion strain was shed at numbers similar to the wild type. These data suggest that colonization of the murine cecum is required for efficient fecal shedding in mice.
Availability of Complement Bound toStaphylococcus aureusTo Interact with Membrane Complement Receptors Influences Efficiency of Phagocytosis
Authors: Cunnion K. M. , Zhang H.-M. , Frank M. M. 
Published: January 2003

ABSTRACTComplement-mediated opsonization of encapsulatedStaphylococcus aureus(CP+) of the predominant capsule types, 5 and 8, remains poorly understood. Our previous work showed that complement is important for mouse survival of CP+ type 5 bacteremia and that the type 5 capsule inhibits the binding of opsonic C3 fragments to the organism. The importance of complement-mediated opsonization of CP+ was tested by neutrophil phagocytosis assays. Complement-mediated opsonization of CP+ increased phagocytosis by 57% compared to opsonization in complement-inhibited serum. Agar-grown CP+, enhancing capsule expression, was phagocytosed only one-tenth as well as the capsule-negative organisms (CP−), supporting the belief that staphylococcal polysaccharide capsules impair phagocytosis. Despite relatively poor phagocytosis of CP+ compared to CP−, complement activation increased the phagocytosis of CP+ by 103%. Thus, complement in normal human serum may have an important role in opsonizing CP+, even when capsule expression is strong. The ability of bound C3 fragments to interact with complement receptor 1 (CD35) on the membrane of human erythrocytes was tested in an immune adherence assay.S. aureuscapsule was able to mask C3 fragments on the organism from binding to complement receptor 1. The inhibition of C3 binding to CP+ and the masking of deposited C3 fragments caused by the presence of capsule was associated with markedly decreased phagocytosis. The addition of anti-capsule antibodies to normal human serum was found to markedly improve the recognition of deposited C3 fragments by complement receptor 1 even when the absolute number of C3 molecules bound toS. aureuswas not increased.
Oral Treponemes and Their Outer Membrane Extracts Activate Human Gingival Epithelial Cells through Toll-Like Receptor 2
Authors: Asai Yasuyuki , Jinno Takayoshi , Ogawa Tomohiko 
Published: January 2003

ABSTRACTOral treponemes are considered to be important in the development and progression of periodontal diseases. We investigated the mechanisms of recognition and activation of human gingival epithelial cells (HGEC) with the oral treponemesTreponema denticola,Treponema vincentii, andTreponema mediumand their outer membrane extracts (OMEs).T. vincentiiandT. mediumbut notT. denticolaproduced interleukin 8 (IL-8) in an HGEC culture. Further, all three treponemes induced IL-8 mRNA expression and NF-κB activation in HGEC. Among them,T. denticolaespecially exhibited trypsin- and chymotrypsin-like protease activities, and the addition of chymostatin, a chymotrypsin protease inhibitor, resulted in detectable IL-8 production by HGEC cultured withT. denticola. Additionally, IL-8 mRNA expression in HGEC cultured with the three treponemes and their OMEs was definitely inhibited by the mouse anti-human Toll-like receptor 2 (TLR2) monoclonal antibody TL2.1. These findings suggest that oral treponemes and their OMEs activate HGEC through TLR2.
Heterogeneity of Mycolactones Produced by Clinical Isolates of
Mycobacterium ulcerans
: Implications for Virulence
Authors: Mve-Obiang Armand , Lee Richard E. , Portaels Françoise , Small P. L. C. 
Published: January 2003

ABSTRACT

Mycobacterium ulcerans
is the causative agent of Buruli ulcer, a severe necrotizing skin disease endemic in tropical countries. Clinical evidence suggests that
M. ulcerans
isolates from Asia, Mexico, and Australia may be less virulent than isolates from Africa. In vivo studies suggest that mycolactone, a polyketide-derived macrolide toxin, plays a major role in the tissue destruction and immune suppression which occur in cases of Buruli ulcer. Mycolactones were extracted from 34 isolates of
M. ulcerans
representing strains from Africa, Malaysia, Asia, Australia, and Mexico. Thin-layer chromatography, mass spectroscopic analysis, and cytopathic assays of partially purified mycolactones from these isolates revealed that
M. ulcerans
produces a heterogeneous mixture of mycolactone variants. Mycolactone A/B, the most biologically active mycolactone species, was identified by mass spectroscopy as [M
+
Na]
+
at
m/z
765.5 in all cytotoxic isolates except for those from Mexico. Mycolactone C [M+Na]
+
at
m/z
726.3 was the dominant mycolactone species in eight Australian isolates, and mycolactone D [M+Na]
+
m/z
781.2 was characteristic of two Asian strains. Mycolactone species are conserved within specific geographic areas, suggesting that there may be a correlation between mycolactone profile and virulence. In addition, the core lactone, [M+Na]
+
m/z
447.4, was identified as a minor species, supporting the hypothesis that mycolactones are synthesized by two polyketide synthases. A cytopathic assay of the core lactone showed that this molecule is sufficient for cytotoxicity, although it is much less potent than the complete mycolactone.
Use of a Promoter Trap System in
Bacillus anthracis
and
Bacillus subtilis
for the Development of Recombinant Protective Antigen-Based Vaccines
Authors: Gat O. , Inbar I. , Aloni-Grinstein R. , Zahavy E. , Kronman C. , Mendelson I. , Cohen S. , Velan B. , Shafferman A. 
Published: January 2003

ABSTRACT

We have recently reported
Bacillus anthracis
attenuated live vaccine strains efficiently expressing recombinant protective antigen (rPA) and have shown a direct correlation between the level of rPA secreted by these cells and efficacy (S. Cohen, I. Mendelson, Z. Altboum, D. Kobiler, E. Elhanany, T. Bino, M. Leitner, I. Inbar, H. Rosenberg, Y. Gozes, R. Barak, M. Fisher, C. Kronman, B. Velan, and A. Shafferman, Infect. Immun.
68:
4549-4558, 2000). To isolate more potent
Bacillus
promoters for a further increase in the production of rPA, we developed a promoter trap system based on various
gfp
reporter genes adapted for use in both
Bacillus subtilis
and
B. anthracis
backgrounds. Accordingly, a
B. anthracis
library of 6,000 clones harboring plasmids with chromosomal
B. anthracis
DNA fragments inserted upstream from
gfpuv
was constructed. Based on fluorescence intensity, 57 clones carrying potentially strong promoters were identified, some of which were DNA sequenced. The most potent
B. anthracis
promoter identified (P
ntr
; 271 bp) was 500 times more potent than the native
pagA
promoter and 70 times more potent than the α-amylase promoter (P
amy
). This very potent promoter was tested along with the other promoters (which are three, six, and eight times more potent than P
amy
) for the ability to drive expression of rPA in either
B. subtilis
or
B. anthracis
. The number of cell-associated pre-PA molecules in
B. anthracis
was found to correlate well with the strength of the promoter. However, there appeared to be an upper limit to the amount of mature PA secreted into the medium, which did not exceed that driven by P
amy
. Furthermore, the rPA constructs fused to the very potent promoters proved to be deleterious to the bacterial hosts and consequently led to genetic instability of the PA expression plasmid. Immunization with attenuated
B. anthracis
expressing rPA under the control of promoters more potent than P
amy
was less efficient in eliciting anti-PA antibodies than that attained with P
amy
. The results are consistent with the notion that overexpression of PA leads to severe secretion stress and have practical implications for the design of second-generation rPA-based vaccines.
Application of a
Saccharomyces cerevisiae
Model To Study Requirements for Trafficking of
Yersinia pestis
YopM in Eucaryotic Cells
Authors: Skrzypek Elżbieta , Myers-Morales Tanya , Whiteheart Sidney W. , Straley Susan C. 
Published: January 2003

ABSTRACT

YopM is a leucine-rich repeat (LRR) virulence protein that is delivered into host cells when any of the three human-pathogenic species of
Yersinia
binds to mammalian cells. It exhibits heterogeneity of size and sequence among the yersiniae, but the functional consequences of this variability are not yet known.
Yersinia pestis
YopM was previously shown to accumulate in the nuclei of infected HeLa cells by a mechanism that requires vesicular trafficking. In this study, we characterized the trafficking of
Y. pestis
YopM in a
Saccharomyces cerevisiae
model previously found to support nuclear localization of YopM from an enteropathogenic
Yersinia
strain (C. F. Lesser and S. I. Miller, EMBO J. 20:1840-1849, 2001).
Y. pestis
YopM was N-terminally fused to the yeast enhanced green fluorescent protein (yEGFP) and inducibly expressed in the cytoplasm. yEGFP-YopM localized to the yeast nucleus, showing that this property is conserved for YopMs so far tested and that infection and the presence of other Yops are not required for its trafficking. When expressed in
S. cerevisiae
that is temperature sensitive for vesicular transport, YopM failed to accumulate in the nucleus at the nonpermissive temperature but did accumulate when the permissive temperature was restored. This shows that vesicular trafficking also is required in yeast for normal localization of YopM. YopM consists of a 71-residue leader sequence, 15 LRRs, and a 32-residue tail. Deletion analysis revealed that the leader sequence or tail is alone insufficient to direct YopM to the nucleus, showing that the LRR structure is required. Both the N-terminal and C-terminal halves of YopM localized to the nucleus, indicating the possible presence of two nuclear localization signals (NLSs) in YopM or domains in YopM where an NLS-containing protein might bind; this fits with the presence of two highly conserved regions among
Yersinia
YopMs. yEGFP-YopM lacking LRRs 4 to 7 or 7 to 10 accumulated in the nucleus in yeast, and YopM lacking these LRRs concentrated normally in the HeLa cell nucleus after delivery by
Yersinia
infection, showing that these LRRs are not essential for YopM trafficking in eucaryotic cells. However, because
Y. pestis
carrying either of these YopMs is strongly compromised in virulence in mice, these findings revealed that LRRs 4 to 10 map a region of YopM or support a conformation of YopM that is necessary for a pathogenic effect.
DNA Alkylation Damage as a Sensor of Nitrosative Stress in
Mycobacterium tuberculosis
Authors: Durbach Steven I. , Springer Burkhard , Machowski Edith E. , North Robert J. , Papavinasasundaram K. G. , Colston M. Jo , Böttger Erik C. , Mizrahi Valerie 
Published: January 2003

ABSTRACT

One of the cellular consequences of nitrosative stress is alkylation damage to DNA. To assess whether nitrosative stress is registered on the genome of
Mycobacterium tuberculosis
, mutants lacking an alkylation damage repair and reversal operon were constructed. Although hypersensitive to the genotoxic effects of
N
-methyl-
N
′-nitro-
N
-nitrosoguanidine in vitro, the mutants displayed no phenotype in vivo, suggesting that permeation of nitrosative stress to the level of cytotoxic DNA damage is restricted.