The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.
Rck is encoded on the Salmonella typhimurium virulence plasmid and is a member of a family of related 17to 19-kDa outer membrane proteins of Enterobacteriaceae, including Ail (Yersinia enterocolitica) and PagC (S. typhimurium). Structural models for these proteins predict eight membrane-spanning domains alternating with hydrophilic inner and outer loops. When expressed in Escherichia coli, Rck and Ail, but not PagC, confer highlevel resistance to the bactericidal activity of complement as well as the ability to adhere to and invade mammalian cell lines. To identify functional domains of Rck, we made and screened random mutations in Rck for decreased bioactivity. We found that a single amino acid substitution (glycine to aspartic acid) in the putative third outer loop greatly reduced Rck-mediated serum resistance and eukaryotic cell invasion. We then constructed two chimeric proteins between Rck and PagC. Substitution of the C-terminal half of Rck with the corresponding PagC fragment containing both the third and the fourth outer loops abolishes the Rck-mediated serum resistance and invasion phenotypes. Substitution of Rck with a smaller C-terminal portion of PagC containing the fourth outer loop did not affect the invasive phenotype or serum resistance. These data reveal that the third putative outer membrane loop region is important for the virulence-associated properties of the Rck protein and suggest a similarity between the mechanism of serum resistance and epithelial cell invasion involving the same domain of Rck.
Introduction. The TM0096 gene from Thermotoga maritima is distributed widely among microorganisms, suggesting that it performs a function indispensable for life and/or virulence. Homologs of the TM0096 protein are found in prokaryotes as well as eukaryotes, including the pathogens Yersinia pestis, Listeria, Clostridium, and Bacillus anthracis.TM0096 belongs to the NIFR3 family of proteins, named after the nifR3 gene from Rhodobacter. The biochemical functions of NIFR3 family proteins are unclear, but limited research points to a role in nitrogen metabolism. For example, the photosynthetic purple bacteria Rhodobacter capsulatus experiences an order of magnitude increase in NIFR3 expression under limiting nitrogen conditions.
Introduction S. aureus is a major cause of morbidity globally, and in the United States it contributes significantly to both hospital admissions and in-hospital morbidity [1, 2]. The increasing incidence of antibiotic-resistant strains increases the urgency of understanding the mechanisms by which this infection exerts its toxic acute effects, as well as potential longterm impact on infected patients, especially those with comorbid conditions. The major virulent toxin secreted by S. aureus is α-hemolysin (Hla). A Disintegrin And Metalloproteinase domain-containing protein-10 (ADAM10), which is involved in ectodomain shedding, is the eukaryotic receptor for Hla [3-5], and mediates vascular injury caused by Hla [6]. Almost all isolates of S. aureus express Hla, including methicillinresistant strains [7]. Recently, Hla has been shown to mediate VE-cadherin degradation in endothelial cells (EC) via ADAM10, affecting permeability [6]. Importantly, the Notch1 and 2 receptors are known ADAM10 targets [8]. Notch proteins are highly evolutionarily conserved. In mammals, the Notch pathway is comprised of the Jagged and Delta-like ligands, and the receptors Notch1 through Notch4. Both ligands and receptors are membrane-bound: in order for activation to take place, the ligand and receptor must be expressed in adjacent cells. Notch ligands are cleaved at Site 1 (S1) and can be post-translationally modified by glycosyltransferases, such as Fringe. Upon
Using compartmentalized self-replication (CSR), we evolved a version of Pyrococcus furiosus (Pfu) DNA polymerase that tolerates modification of the γ-phosphate of an incoming nucleotide. A Q484R mutation in α-helix P of the fingers domain, coupled with an unintended translational termination-reinitiation (split) near the finger tip, dramatically improve incorporation of a bulky γ-phosphate-O-linker-dabcyl substituent. Whether synthesized by coupled translation from a bicistronic (−1 frameshift) clone, or reconstituted from separately expressed and purified fragments, split Pfu mutant behaves identically to wild-type DNA polymerase with respect to chromatographic behavior, steady-state kinetic parameters (for dCTP), and PCR performance. Although naturally-occurring splits have been identified previously in the finger tip region of T4 gp43 variants, this is the first time a split (in combination with a point mutation) has been shown to broaden substrate utilization. Moreover, this latest example of a split hyperthermophilic archaeal DNA polymerase further illustrates the modular nature of the Family B DNA polymerase structure.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.