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PubMed2005

Transcriptional profiles and structural models of the Synechocystis sp. PCC 6803 Deg proteases.

The Synechocystis sp. PCC 6803 genome harbours a deg gene family consisting of three members, degP (htrA, slr1204), degQ (hhoA, sll1679) and degS (hhoB, sll1427). We studied the environmental regulation of the Synechocystis sp. PCC 6803 deg genes at the level of transcription and protein structures of the gene products to evaluate their hypothetical role in D1 protein turnover. Northern blotting showed that transcription of the deg genes is differentially regulated, supporting a view of distinct roles of Degs in cellular processes. The oligomerization state as well as the three dimensional structures of the Synechocystis sp. PCC 6803 Deg proteases were predicted based on an amino acid sequence alignment and comparison of the Deg crystal structures from human, Escherichia coli and Thermotoga maritima. The structures of the Synechocystis sp. PCC 6803 Degs resemble more the Thermotoga maritima Deg enzyme structure than the Escherichia coli one. Moreover, the structures of the LA-loops hint towards a homotrimeric form of the Synechocystis sp. PCC 6803 Deg proteases.

Amino Acid Sequence↗
PubMed1987

Lecithin:cholesterol acyltransferase. Functional regions and a structural model of the enzyme.

The amino acid sequence of human lecithin:cholesterol acyltransferase has been determined by degradation and alignment of peptides obtained from tryptic and staphylococcal digestions and the cleavage with cyanogen bromide and consisted of 416 amino acid residues. All of the tryptic peptides of lecithin:cholesterol acyltransferase were isolated and sequenced. Peptides resulting from digestion by staphylococcal protease, cyanogen bromide cleavage, or the combination of the two methods were employed to find overlapping segments. The N terminus of human lecithin:cholesterol acyltransferase was determined to be phenylalanine by sequencing the whole protein up to 40 residues while the C terminus was identified as glutamic acid through carboxypeptidase Y cleavage. Cys50 and Cys74 and Cys313 and Cys356 were identified as the two disulfide bridges while the free sulfhydryl groups were located at positions 31 and 184. The N-glycosylated sites of the protein were assigned to asparagines at positions 20, 84, 272, and 384. The active site of lecithin:cholesterol acyltransferase was identified as serine on position 181 according to its homology with other serine-type esterases which have a common structure of glycine-variable amino acid-active serine-variable amino acid-glycine (Gly-X-Ser-X-Gly) with the variable amino acids disrupting the homology. No long internal repeats or homologies with apolipoproteins were found. The secondary structure is consistent with the results of predictive algorithms. A simple model of the enzyme is proposed on the basis of available chemical data and predictive methods.

Amino Acid Sequence↗
PubMed2005

A structural model for maturation of the hepatitis B virus core.

Hepatitis B virus, a widespread and serious human pathogen, replicates by reverse transcription of an RNA intermediate. The virus consists of an inner nucleocapsid or core, surrounded by a lipid envelope containing virally encoded surface proteins. Using electron cryomicroscopy, we compare the structures of the bacterially expressed RNA-containing core particle and the mature DNA-containing core particle extracted from virions. We show that the mature core contains 240 subunits in a T = 4 arrangement similar to that in expressed core (T is the triangulation number and the icosahedral shell contains 60 T subunits). During the infective cycle, the core assembles in an immature state around a complex of viral pregenomic RNA and polymerase. After reverse transcription with concomitant degradation of the RNA, the now mature core buds through a cellular membrane containing the surface proteins to become enveloped. Envelopment must not happen before reverse transcription is completed, so it has been hypothesized that a change in capsid structure may signal maturation. Our results show significant differences in structure between the RNA- and DNA-containing cores. One such difference is in a hydrophobic pocket, formed largely from residues that, on mutation, lead to abnormal secretion. We suggest that the changes we see are related to maturation and control of envelopment, and we propose a mechanism based on DNA synthesis for their triggering.

Cryoelectron Microscopy↗
PubMed1983

The modeled structure of the IgG Gar VL region and its implications for anti-flavin and anti-DNP fine specificities.

The variable domain (V) sequence of the lambda-light chain of the Gar IgG2 human myeloma protein was determined from enzymatically generated peptides that were isolated, characterized, and ordered by overlap and/or by subgroup homology. The sequence consisted of the amino terminal 107 residues of the light chain, and shared subgroup V lambda III-specific residues as well as a pattern of CDR deletions unique to most V lambda III sequences. The structural role of the light V region in the high affinity for flavins and the low affinity for 2,4-dinitrophenyl haptens characteristic of the intact IgG molecule was coarsely assessed by way of model building and by comparison with the refined combining site model of MOPC 315, an Ig with relative affinity values for DNP and riboflavin the converse of those observed in Gar.

Amino Acid Sequence↗
PubMed2003

A "dock, lock, and latch" structural model for a staphylococcal adhesin binding to fibrinogen.

Gram-positive pathogens such as staphylococci contain multiple cell wall-anchored proteins that serve as an interface between the microbe and its environment. Some of these proteins act as adhesins and mediate bacterial attachment to host tissues. SdrG is a cell wall-anchored adhesin from Staphylococcus epidermidis that binds to the Bbeta chain of human fibrinogen (Fg) and is necessary and sufficient for bacterial attachment to Fg-coated biomaterials. Here, we present the crystal structures of the ligand binding region of SdrG as an apoprotein and in complex with a synthetic peptide analogous to its binding site in Fg. Analysis of the crystal structures, along with mutational studies of both the protein and of the peptide, reveals that SdrG binds to its ligand with a dynamic "dock, lock, and latch" mechanism. We propose that this mechanism represents a general mode of ligand binding for structurally related cell wall-anchored proteins of gram-positive bacteria.

Adhesins, Bacterial↗
PubMed1985

The usefulness, in pharmacological classification, of complementary pattern-recognition techniques and structure modelling as afforded by the iterative collation of multiple-trial data in data banks.

In this emerging information age no significant limits can be envisaged to the immense resources of knowledge that pharmacological sciences can draw upon by systematically applying multivariate pattern-recognition techniques to those data banks which can be organized internationally with better standardization of descriptors, i.e. by parametrizing observations and evaluating monitoring in experimental, biological assays, clinical trials and postmarketing surveillance. Even conventionally or habitually adopted references and communalities such as traditional drug profiles and receptor models may be iteratively re-checked and suitably adapted so as to take account of more adequate, up-to-date analytical techniques, fresh biological ideas and new advances in terms of physiological refinements. An attempt may also be made to modify the chemicophysical relationships and patterns currently traced on the basis of what are held to be quantitative structure-activity oversimplifications. The present paper focuses upon specifying a number of standardization criteria in conventional assays and upon submitting multiple biological features to monitoring; in addition, it gives some picture of the new trends the approach can offer and draws attention to the more relevant, innovative literature references.

Animals↗
PubMed2004

Structural model for an AxxxG-mediated dimer of surfactant-associated protein C.

The pulmonary surfactant prevents alveolar collapse and is required for normal pulmonary function. One of the important components of the surfactant besides phospholipids is surfactant-associated protein C (SP-C). SP-C shows complex oligomerization behavior and a transition to beta-amyloid-like fibril structures, which are not yet fully understood. Besides this nonspecific oligomerization, MS and chemical cross-linking data combined with CD spectra provide evidence of a specific, mainly alpha-helical, dimer at low to neutral pH. Furthermore, resistance to CNBr cleavage and dual NMR resonances of porcine and human recombinant SP-C with Met32 replaced by isoleucine point to a dimerization site located at the C-terminus of the hydrophobic alpha-helix of SP-C, where a strictly conserved heptapeptide sequence is found. Computational docking of two SP-C helices, described here, reveals a dimer with a helix-helix interface that strikingly resembles that of glycophorin A and is mediated by an AxxxG motif similar to the experimentally determined GxxxG pattern of glycophorin A. It is highly likely that mature SP-C adopts such a dimeric structure in the lamellar bilayer systems found in the surfactant. Dimerization has been shown in previous studies to have a role in sorting and trafficking of SP-C and may also be important to the surfactant function of this protein.

Amino Acid Motifs↗
PubMed2006

Structural model of full-length human Ku70-Ku80 heterodimer and its recognition of DNA and DNA-PKcs.

Recognition of DNA double-strand breaks during non-homologous end joining is carried out by the Ku70-Ku80 protein, a 150 kDa heterodimer that recruits the DNA repair kinase DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to the lesion. The atomic structure of a truncated Ku70-Ku80 was determined; however, the subunit-specific carboxy-terminal domain of Ku80--essential for binding to DNA-PKcs--was determined only in isolation, and the C-terminal domain of Ku70 was not resolved in its DNA-bound conformation. Both regions are conserved and mediate protein-protein interactions specific to mammals. Here, we reconstruct the three-dimensional structure of the human full-length Ku70-Ku80 dimer at 25 A resolution, alone and in complex with DNA, by using single-particle electron microscopy. We map the C-terminal regions of both subunits, and their conformational changes after DNA and DNA-PKcs binding to define a molecular model of the functions of these domains during DNA repair in the context of full-length Ku70-Ku80 protein.

Antigens, Nuclear↗
PubMed2006

Molecular genetic and structural modeling studies of Staphylococcus aureus RNA polymerase and the fitness of rifampin resistance genotypes in relation to clinical prevalence.

The adaptive and further evolutionary responses of Staphylococcus aureus to selection pressure with the antibiotic rifampin have not been explored in detail. We now present a detailed analysis of these systems. The use of rifampin for the chemotherapy of infections caused by S. aureus has resulted in the selection of mutants with alterations within the beta subunit of the target enzyme, RNA polymerase. Using a new collection of strains, we have identified numerous novel mutations in the beta subunits of both clinical and in vitro-derived resistant strains and established that additional, undefined mechanisms contribute to expression of rifampin resistance in clinical isolates of S. aureus. The fitness costs associated with rifampin resistance genotypes were found to have a significant influence on their clinical prevalence, with the most common clinical genotype (H481N, S529L) exhibiting no fitness cost in vitro. Intragenic mutations which compensate for the fitness costs associated with rifampin resistance in clinical strains of S. aureus were identified for the first time. Structural explanations for rifampin resistance and the loss of fitness were obtained by molecular modeling of mutated RNA polymerase enzymes.

DNA, Bacterial↗
PubMed2004

Analysis of mupirocin resistance and fitness in Staphylococcus aureus by molecular genetic and structural modeling techniques.

Chromosomal resistance to mupirocin in clinical isolates of Staphylococcus aureus arises from V(588)F or V(631)F mutations in isoleucyl-tRNA synthetase (IRS). Whether these are the only IRS mutations that confer mupirocin resistance or simply those that survive in the clinic is unknown. Mupirocin-resistant mutants of S. aureus 8325-4 were therefore generated to examine their ileS genotypes and the in vitro and in vivo fitness costs associated with them before and after compensatory evolution. Most spontaneous first-step mupirocin-resistant mutants carried V(588)F or V(631)F mutations in IRS, but a new mutation (G(593)V) was also identified. Second-step mutants carried combinations of previously identified IRS mutations (e.g., V(588)F/V(631)F and G(593)V/V(631)F), but additional combinations also occurred involving novel mutations (R(816)C, H(67)Q, and F(563)L). First-step mupirocin-resistant mutants were not associated with substantial fitness costs, a finding that is consistent with the occurrence of V(588)F or V(631)F mutations in the IRS of clinical strains. Second-step mutants were unfit, but fitness could be restored by subculture in the absence of mupirocin. In most cases, this was the result of compensatory mutations that also suppressed mupirocin resistance (e.g., A(196)V, E(190)K, and E(195)K), despite retention of the original mutations conferring resistance. Structural explanations for mupirocin resistance and loss of fitness were obtained by molecular modeling of mutated IRS enzymes, which provided data on mupirocin binding and interaction with the isoleucyl-AMP reactive intermediate.

Adenosine Monophosphate↗
PubMed1981

A general secondary-structure model for procaryotic and eucaryotic RNAs from the small ribosomal subunits.

A consensus on the folding of the Escherichia coli 16-S ribosomal RNA is emerging and several complete nucleotide sequences of small ribosomal subunit RNAs, covering diverse types of organisms and organelles, are now available. We therefore investigated the extent of both nucleotide sequence and secondary structure conservation that may exist between the E. coli 16-S RNA and other ribosomal RNAs. All the RNA molecules examined could be folded into secondary structure schemes that illustrated remarkable preservation of many structural motifs as well as striking nucleotide sequence conservation compared with the E. coli molecule. This study presents a unitary scheme for the structural organization of the small ribosomal subunit RNAs. The evolutionary constraints on both primary and secondary structures most likely reveal the basic role of some restricted RNA regions in the function of the ribosome.

Animals↗
PubMed2003

A structural model for the assembly of the 30S subunit of the ribosome.

The order in which proteins bind to 16S rRNA, the assembly map, was determined by Nomura and co-workers in the early 1970s. The assembly map shows the dependencies of binding of successive proteins but fails to address the relationship of these dependencies to the three-dimensional folding of the ribosome. Here, using molecular mechanics techniques, we rationalize the order of protein binding in terms of ribosomal folding. We determined the specific contacts between the ribosomal proteins and 16S rRNA from a crystal structure of the 30S subunit (1FJG). We then used these contacts as restraints in a rigid body Monte-Carlo simulation with reduced-representation models of the RNA and proteins. Proteins were added sequentially to the RNA in the order that they appear in the assembly map. Our results show that proteins nucleate the folding of the head, platform, and body domains, but they do not strongly restrict the orientations of the domains relative to one another. We also examined the contributions of individual proteins to the formation of binding sites for sequential proteins in the assembly process. Binding sites for the primary binding proteins are generally more ordered in the naked RNA than those for other proteins. Furthermore, we examined one pathway in the assembly map and found that the addition of early binding proteins helps to organize the RNA around the binding sites of proteins that bind later. It appears that the order of assembly depends on the degree of pre-organization of each protein's binding site at a given stage of assembly, and the impact that the binding of each protein has on the organization of the remaining unoccupied binding sites.

Binding Sites↗
PubMed1993

Peptide nucleic acid (PNA): a model structure for the primordial genetic material?

It is proposed that the primordial genetic material could have been peptide nucleic acids, i.e., DNA analogues having a peptide backbone. PNA monomers based on the amino acid, alpha, gamma-diaminobutyric acid or ornithine are suggested as compounds that could have been formed in the prebiotic soup. Finally, the possibility of a PNA/RNA world is presented, in which PNA constitutes the stable genetic material, while RNA which may be polymerized using the PNA as template accounts for enzymatic activities including PNA replication.

Base Sequence↗
PubMed2024

Structural modelling and preventive strategy targeting of WSSV hub proteins to combat viral infection in shrimp Penaeus monodon.

White spot syndrome virus (WSSV) presents a considerable peril to the aquaculture sector, leading to notable financial consequences on a global scale. Previous studies have identified hub proteins, including WSSV051 and WSSV517, as essential binding elements in the protein interaction network of WSSV. This work further investigates the functional structures and potential applications of WSSV hub complexes in managing WSSV infection. Using computational methodologies, we have successfully generated comprehensive three-dimensional (3D) representations of hub proteins along with their three mutual binding counterparts, elucidating crucial interaction locations. The results of our study indicate that the WSSV051 hub protein demonstrates higher binding energy than WSSV517. Moreover, a unique motif, denoted as "S-S-x(5)-S-x(2)-P," was discovered among the binding proteins. This pattern perhaps contributes to the detection of partners by the hub proteins of WSSV. An antiviral strategy targeting WSSV hub proteins was demonstrated through the oral administration of dual hub double-stranded RNAs to the black tiger shrimp, Penaeus monodon, followed by a challenge assay. The findings demonstrate a decrease in shrimp mortality and a cessation of WSSV multiplication. In conclusion, our research unveils the structural features and dynamic interactions of hub complexes, shedding light on their significance in the WSSV protein network. This highlights the potential of hub protein-based interventions to mitigate the impact of WSSV infection in aquaculture.

Animals↗
PubMed2002

Vanadate inhibits the ATPase activity and DNA binding capability of bacterial MutS. A structural model for the vanadate-MutS interaction at the Walker A motif.

MutS, a member of the ABC ATPases superfamily, is a mismatch DNA-binding protein constituent of the DNA post-replicative mismatch repair system (MMRS). In this work, it is shown that the ATPase activity of Pseudomonas aeruginosa and Escherichia coli MutS is inhibited by ortho- and decavanadate. Structural comparison of the region involved in the ATP binding of E.coli MutS with the corresponding region of other ABC ATPases inhibited by vanadate, including the myosin- orthovanadate-Mg complex, showed that they are highly similar. From these results it is proposed that the orthovanadate inhibition of MutS ATPase can take place by a similar mechanism to that described for other ATPases. Docking of decavanadate on the ATP-binding region of MutS showed that the energetically more favorable interaction of this compound would take place with the complex MutS- ADP-Mg, suggesting that the inhibitory effect could be produced by a steric impediment of the protein ATP/ADP exchange. Besides the effect observed on the ATPase activity, vanadate also affects the DNA-binding capability of the protein, and partially inhibits the oligomerization of MutS and the temperature-induced inactivation of the protein. From the results obtained, and considering that vanadate is an intracellular trace component, this compound could be considered as a new modulator of the MMRS.

Adenosine Diphosphate↗
PubMed1987

Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites.

Virusoids are circular single-stranded RNAs dependent on plant viruses for replication and encapsidation. Virusoid replication appears to involve longer-than-unit-length plus and minus RNAs, indicating that unit-length plus RNA is generated by specific cleavage reactions. Here, we synthesize plus and minus partial-length RNAs of the 324-nucleotide virusoid from lucerne transient streak virus in vitro. Both RNAs self-cleave at a unique site in the presence of magnesium ions to give 5' hydroxyl and 2',3' cyclic phosphodiester termini. Conformations other than the native structures are necessary for cleavage. Similar secondary structures with considerable sequence homology are proposed for the active sites of these and other plant pathogenic RNAs. Our results are consistent with certain rolling-circle replication models.

Binding Sites↗