Correction to ‘The basic keratin 10-binding domain of the virulence-associated pneumococcal serine-rich protein PsrP adopts a novel MSCRAMM fold’
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PROVIDER: S-EPMC4221896 | biostudies-literature | 2014 Oct
REPOSITORIES: biostudies-literature
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Project description:Streptococcus pneumoniae is a major human pathogen, and a leading cause of disease and death worldwide. Pneumococcal invasive disease is triggered by initial asymptomatic colonization of the human upper respiratory tract. The pneumococcal serine-rich repeat protein (PsrP) is a lung-specific virulence factor whose functional binding region (BR) binds to keratin-10 (KRT10) and promotes pneumococcal biofilm formation through self-oligomerization. We present the crystal structure of the KRT10-binding domain of PsrP (BR187-385) determined to 2.0 Å resolution. BR187-385 adopts a novel variant of the DEv-IgG fold, typical for microbial surface components recognizing adhesive matrix molecules adhesins, despite very low sequence identity. An extended ?-sheet on one side of the compressed, two-sided barrel presents a basic groove that possibly binds to the acidic helical rod domain of KRT10. Our study also demonstrates the importance of the other side of the barrel, formed by extensive well-ordered loops and stabilized by short ?-strands, for interaction with KRT10.
| S-EPMC3909270 | biostudies-literature
Project description:Protein O-glycosylation is an important post-translational modification in all organisms, but deciphering the specific functions of these glycans is difficult due to their structural complexity. Understanding the glycosylation of mucin-like proteins presents a particular challenge as they are modified numerous times with both the enzymes involved and the glycosylation patterns being poorly understood. Here we systematically explored the O-glycosylation pathway of a mucin-like serine-rich repeat protein PsrP from the human pathogen Streptococcus pneumoniae TIGR4. Previous works have assigned the function of 3 of the 10 glycosyltransferases thought to modify PsrP, GtfA/B, and Gtf3 as catalyzing the first two reactions to form a unified disaccharide core structure. We now use in vivo and in vitro glycosylation assays combined with hydrolytic activity assays to identify the glycosyltransferases capable of decorating this core structure in the third and fourth steps of glycosylation. Specifically, the full-length GlyE and GlyG proteins and the GlyD DUF1792 domain participate in both steps, whereas full-length GlyA and the GlyD GT8 domain catalyze only the fourth step. Incorporation of different sugars to the disaccharide core structure at multiple sites along the serine-rich repeats results in a highly polymorphic product. Furthermore, crystal structures of apo- and UDP-complexed GlyE combined with structural analyses reveal a novel Rossmann-fold "add-on" domain that we speculate to function as a universal module shared by GlyD, GlyE, and GlyA to forward the peptide acceptor from one enzyme to another. These findings define the complete glycosylation pathway of a bacterial glycoprotein and offer a testable hypothesis of how glycosyltransferase coordination facilitates glycan assembly.
| S-EPMC5391752 | biostudies-literature
Project description:The pneumococcal serine rich repeat protein (PsrP) is displayed on the surface of Streptococcus pneumoniae with a suggested role in colonization in the human upper respiratory tract. Full-length PsrP is a 4000 residue-long multi-domain protein comprising a positively charged functional binding region (BR) domain for interaction with keratin and extracellular DNA during pneumococcal adhesion and biofilm formation, respectively. The previously determined crystal structure of the BR domain revealed a flat compressed barrel comprising two sides with an extended β-sheet on one side, and another β-sheet that is distorted by loops and β-turns on the other side. Crystallographic B-factors indicated a relatively high mobility of loop regions that were hypothesized to be important for binding. Furthermore, the crystal structure revealed an inter-molecular β-sheet formed between edge strands of two symmetry-related molecules, which could promote bacterial aggregation during biofilm formation. Here we report the near complete 15N/13C/1H backbone resonance assignment of the BR domain of PsrP, revealing a secondary structure profile that is almost identical to the X-ray structure. Dynamic 15N-T1, T2 and NOE data suggest a monomeric and rigid structure of BR with disordered residues only at the N- and C-termini. The presented peak assignment will allow us to identify BR residues that are crucial for ligand binding.
| S-EPMC7462905 | biostudies-literature
Project description:The pneumococcal serine-rich repeat protein (PsrP) is a high-molecular-weight, glycosylated adhesin that promotes the attachment of Streptococcus pneumoniae to host cells. PsrP, its associated glycosyltransferases (GTs), and dedicated secretion machinery are encoded in a 37-kb genomic island that is present in many invasive clinical isolates of S. pneumoniae PsrP has been implicated in establishment of lung infection in murine models, although specific roles of the PsrP glycans in disease progression or bacterial physiology have not been elucidated. Moreover, enzymatic specificities of associated glycosyltransferases are yet to be fully characterized. We hypothesized that the glycosyltransferases that modify PsrP are critical for the adhesion properties and infectivity of S. pneumoniae Here, we characterize the putative S. pneumoniaepsrP locus glycosyltransferases responsible for PsrP glycosylation. We also begin to elucidate their roles in S. pneumoniae virulence. We show that four glycosyltransferases within the psrP locus are indispensable for S. pneumoniae biofilm formation, lung epithelial cell adherence, and establishment of lung infection in a mouse model of pneumococcal pneumonia.IMPORTANCE PsrP has previously been identified as a necessary virulence factor for many serotypes of S. pneumoniae and studied as a surface glycoprotein. Thus, studying the effects on virulence of each glycosyltransferase (GT) that builds the PsrP glycan is of high importance. Our work elucidates the influence of GTs in vivo We have identified at least four GTs that are required for lung infection, an indication that it is worthwhile to consider glycosylated PsrP as a candidate for serotype-independent pneumococcal vaccine design.
| S-EPMC8088515 | biostudies-literature
Project description:Pneumococcal serine-rich repeat protein (PsrP) is a glycoprotein that mediates Streptococcus pneumoniae attachment to lung cells and promotes biofilm formation. Herein, we investigated the transcriptional organization of psrP-secY2A2, the 37-kbp pathogenicity island encoding PsrP and its accessory genes. PCR amplification of cDNA and RNA-seq analysis found psrP-secY2A2 to be minimally composed of three operons: psrP-glyA, glyB, and glyC-asp5. Transcription of all three operons was greatest during biofilm growth and immunoblot analyses confirmed increased PsrP production by biofilm pneumococci. Using gas chromatography-mass spectrometry we identified monomeric N-acetylglucosamine as the primary glycoconjugate present on a recombinant intracellular version of PsrP, i.e. PsrP1-734. This finding was validated by immunoblot using lectins with known carbohydrate specificities. We subsequently deleted gtfA and gtfB, the GTFs thought to be responsible for addition of O-linked N-acetylglucosamine, and tested for PsrP and its associated virulence properties. These deletions negatively affected our ability to detect PsrP1-734 in bacterial whole cell lysates. Moreover, S. pneumoniae mutants lacking these genes pheno-copied the psrP mutant and were attenuated for: biofilm formation, adhesion to lung epithelial cells, and pneumonia in mice. Our studies identify the transcriptional organization of psrP-secY2A2 and show the indispensable role of GtfA and GtfB on PsrP-mediated pneumococcal virulence.
| S-EPMC5581956 | biostudies-literature
Project description:Presenilin is an integral membrane aspartate protease that regulates cellular processes by cleaving proteins within the cell membrane. The recent crystal structure of presenilin reveals a conspicuous pore in a bundle of nine α-helices, which was originally thought to adopt a novel protein fold. However, here I show that the presenilin fold is a variant of the ClC chloride channel/transporter fold. This observation may have important implications for presenilin's postulated biological role as a calcium leak channel.
| S-EPMC4918416 | biostudies-literature
Project description:Pneumococcal serine-rich repeat protein (PsrP) is a pathogenicity island-encoded adhesin that mediates attachment to lung cells. It is a member of the serine-rich repeat protein family and the largest bacterial protein known. PsrP production by S. pneumoniae was confirmed by immunoblotting and a truncated version of the protein was determined to be glycosylated. Using isogenic psrP mutants complemented with various PsrP constructs and competitive inhibition assays with recombinant proteins, we determined that PsrP requires an extended SRR2 domain for function and that adhesion is mediated through amino acids 273-341 of its basic region (BR) domain. Affinity chromatography, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), fluorescent-activated cell sorting (FACS) and immunofluorescent colocalization studies determined that PsrP binds to Keratin 10 (K10) on the surface of lung but not nasopharyngeal epithelial cells. Unglycosylated K10 bound to wild type but not psrP deficient pneumococci; suggesting that unlike other serine-rich repeat proteins, PsrP-mediated adhesion is independent of lectin activity. Finally, mice immunized with recombinant (r)PsrP(BR) had significantly less bacteria in their blood and improved survival versus controls following intranasal challenge. We conclude that the BR domain of PsrP binds to K10 in a lectin-independent manner, that K10 is expressed on lung cells and that vaccination with rPsrP(BR) is protective against pneumococcal disease.
| S-EPMC2753542 | biostudies-literature
Project description:Pneumococcal pneumonia remains a WHO high-priority disease despite multivalent conjugate vaccines administered in clinical practice worldwide. A protein-based, serotype-independent vaccine has long-promised comprehensive coverage of most clinical isolates of the pneumococcus. Along with numerous pneumococcal surface protein immunogens, the pneumococcal serine-rich repeat protein (PsrP) has been investigated as a potential vaccine target due to its surface exposure and functions toward bacterial virulence and lung infection. Three critical criteria for its vaccine potential - the clinical prevalence, serotype distribution, and sequence homology of PsrP - have yet to be well characterized. Here, we used genomes of 13,454 clinically isolated pneumococci from the Global Pneumococcal Sequencing project to investigate PsrP presence among isolates, distribution among serotypes, and interrogate its homology as a protein across species. These isolates represent all age groups, countries worldwide, and types of pneumococcal infection. We found PsrP present in at least 50% of all isolates across all determined serotypes and nontypeable (NT) clinical isolates. Using a combination of peptide matching and HMM profiles built on full-length and individual PsrP domains, we identified novel variants that expand PsrP diversity and prevalence. We also observed sequence variability in its basic region (BR) between isolates and serotypes. PsrP has a strong vaccine potential due to its breadth of coverage, especially in nonvaccine serotypes (NVTs) when exploiting its regions of conservation in vaccine design. IMPORTANCE An updated outlook on PsrP prevalence and serotype distribution sheds new light on the comprehensiveness of a PsrP-based protein vaccine. The protein is present in all vaccine serotypes and highly present in the next wave of potentially disease-causing serotypes not included in the current multivalent conjugate vaccines. Furthermore, PsrP is strongly correlated with clinical isolates harboring pneumococcal disease as opposed to pneumococcal carriage. PsrP is also highly present in strains and serotypes from Africa, where the need for a protein-based vaccine is the greatest, giving new reasoning to pursue PsrP as a protein vaccine.
| S-EPMC10269691 | biostudies-literature
Project description:Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.
| S-EPMC8342540 | biostudies-literature
Project description:To fight for essential metal ions, human pathogens secrete virulence-associated siderophores and retake the metal-chelated siderophores through a subfamily of adenosine triphosphate (ATP)-binding cassette (ABC) importer, whose molecular mechanisms are completely unknown. We have determined multiple structures of the yersiniabactin importer YbtPQ from uropathogenic Escherichia coli (UPEC) at inward-open conformation in both apo and substrate-bound states by cryo-electron microscopy. YbtPQ does not adopt any known fold of ABC importers but surprisingly adopts the fold of type IV ABC exporters. To our knowledge, it is the first time an exporter fold of ABC importer has been reported. We have also observed two unique features in YbtPQ: unwinding of a transmembrane helix in YbtP upon substrate release and tightly associated nucleotide-binding domains without bound nucleotides. Together, our study suggests that siderophore ABC importers have a distinct transport mechanism and should be classified as a separate subfamily of ABC importers.
| S-EPMC7002159 | biostudies-literature