Structural and Functional Variation within the Alanine-Rich Repetitive Domain of Streptococcal Antigen I/II

ABSTRACT Members of the antigen I/II family of cell surface proteins are highly conserved, multifunctional adhesins that mediate interactions of oral streptococci with other oral bacteria, with cell matrix proteins (e.g., type I collagen), and with salivary glycoproteins, e.g., gp340. The interaction of gp340 (formerly designated salivary agglutinin) with Streptococcus mutans requires an alanine-rich repetitive domain (A region) of antigen I/II that is highly conserved in all members of this family of proteins. In this report, we show that the A regions from the two Streptococcus gordonii M5 antigen I/II proteins (SspA and SspB) interact differently with the salivary gp340 glycoprotein and appear to be structurally distinct. Recombinant polypeptides encompassing the A region of SspA or from a highly related S. mutans antigen I/II protein (SpaP) competitively inhibited the interaction of gp340 with intact S. gordonii and S. mutans cells, respectively. In contrast, an A region polypeptide from SspB was inactive, and furthermore, it did not bind to purified gp340 in vitro. Circular dichroism spectra suggested that all three polypeptides were highly α-helical and may form coiled-coil structures. However, the A region of SspB underwent a conformational change and exhibited reduced α-helical structure at pH 8.5, whereas the A region polypeptides from SspA and SpaP were relatively stable under these conditions. Melt curves also indicated that at physiological pH, the A region of SspB lost α-helical structure more rapidly than that of SspA or SpaP when the temperature was increased from 10 to 40°C. Furthermore, the SspB A region polypeptide denatured completely at a temperature that was 7 to 9°C lower than that required for the A region polypeptide of SspA or SpaP. The full-length SspB protein and the three A region peptides migrated in native gel electrophoresis and column chromatography with apparent molecular masses that were approximately 2- to 2.5-fold greater than their predicted molecular masses. However, sedimentation equilibrium ultracentrifugation data showed that the A region peptides sedimented as monomers, suggesting that the peptides may form nonglobular intramolecular coiled-coil structures under the experimental conditions used. Taken together, our results suggest that the A region of SspB is less stable than the corresponding A regions of SspA and SpaP and that this structural difference may explain, at least in part, the functional variation observed in their interactions with salivary gp340.

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Examination of a Fusogenic Hexameric Core from Human Metapneumovirus and Identification of a Potent Synthetic Peptide Inhibitor from the Heptad Repeat 1 Region

Journal of Virology ◽

10.1128/jvi.01243-06 ◽

2006 ◽

Vol 81 (1) ◽

pp. 141-149 ◽

Cited By ~ 22

Author(s):

Scott A. Miller ◽

Sharon Tollefson ◽

James E. Crowe ◽

John V. Williams ◽

David W. Wright

Keyword(s):

Membrane Proteins ◽

Coiled Coil ◽

Helical Structure ◽

Human Metapneumovirus ◽

Integral Membrane Proteins ◽

Heptad Repeat ◽

Type I ◽

Viral Fusion ◽

Fusion Inhibitors ◽

Major Player

ABSTRACT Paramyxoviruses are a leading cause of childhood illness worldwide. A recently discovered paramyxovirus, human metapneumovirus (hMPV), has been studied by our group in order to determine the structural relevance of its fusion (F) protein to other well-characterized viruses utilizing type I integral membrane proteins as fusion aids. Sequence analysis and homology models suggested the presence of requisite heptad repeat (HR) regions. Synthetic peptides from HR regions 1 and 2 (HR-1 and -2, respectively) were induced to form a thermostable (melting temperature, ∼90°C) helical structure consistent in mass with a hexameric coiled coil. Inhibitory studies of hMPV HR-1 and -2 indicated that the synthetic HR-1 peptide was a significant fusion inhibitor with a 50% inhibitory concentration and a 50% effective concentration of ∼50 nM. Many viral fusion proteins are type I integral membrane proteins utilizing the formation of a hexameric coiled coil of HR peptides as a major driving force for fusion. Our studies provide evidence that hMPV also uses a coiled-coil structure as a major player in the fusion process. Additionally, viral HR-1 peptide sequences may need further investigation as potent fusion inhibitors.

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Modulatory Effect of Glycated Collagen on Oral Streptococcal Nanoadhesion

Journal of Dental Research ◽

10.1177/0022034520946320 ◽

2020 ◽

Vol 100 (1) ◽

pp. 82-89

Author(s):

C.M.A.P. Schuh ◽

B. Benso ◽

P.A. Naulin ◽

N.P. Barrera ◽

L. Bozec ◽

...

Keyword(s):

Dental Caries ◽

Biofilm Formation ◽

Type I Collagen ◽

Type I ◽

Oral Streptococci ◽

Oral Diseases ◽

Collagen Crosslinking ◽

Initial Adhesion ◽

Collagen Glycation ◽

The Impact

Biofilm-mediated oral diseases such as dental caries and periodontal disease remain highly prevalent in populations worldwide. Biofilm formation initiates with the attachment of primary colonizers onto surfaces, and in the context of caries, the adhesion of oral streptococci to dentinal collagen is crucial for biofilm progression. It is known that dentinal collagen suffers from glucose-associated crosslinking as a function of aging or disease; however, the effect of collagen crosslinking on the early adhesion and subsequent biofilm formation of relevant oral streptococci remains unknown. Therefore, the aim of this work was to determine the impact of collagen glycation on the initial adhesion of primary colonizers such as Streptococcus mutans UA159 and Streptococcus sanguinis SK 36, as well as its effect on the early stages of streptococcal biofilm formation in vitro. Type I collagen matrices were crosslinked with either glucose or methylglyoxal. Atomic force microscopy nanocharacterization revealed morphologic and mechanical changes within the collagen matrix as a function of crosslinking, such as a significantly increased elastic modulus in crosslinked fibrils. Increased nanoadhesion forces were observed for S. mutans on crosslinked collagen surfaces as compared with the control, and retraction curves obtained for both streptococcal strains demonstrated nanoscale unbinding behavior consistent with bacterial adhesin-substrate coupling. Overall, glucose-crosslinked substrates specifically promoted the initial adhesion, biofilm formation, and insoluble extracellular polysaccharide production of S. mutans, while methylglyoxal treatment reduced biofilm formation for both strains. Changes in the adhesion behavior and biofilm formation of oral streptococci as a function of collagen glycation could help explain the biofilm dysbiosis seen in older people and patients with diabetes. Further studies are necessary to determine the influence of collagen crosslinking on the balance between acidogenic and nonacidogenic streptococci to aid in the development of novel preventive and therapeutic treatment against dental caries in these patients.

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Conserved DNA binding and self-association domains of the Drosophila zeste protein

Molecular and Cellular Biology ◽

10.1128/mcb.12.2.598-608.1992 ◽

1992 ◽

Vol 12 (2) ◽

pp. 598-608

Author(s):

J D Chen ◽

C S Chan ◽

V Pirrotta

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Amino Acid Sequence ◽

Coiled Coil ◽

Helical Structure ◽

Binding Activity ◽

Binding Domain ◽

Drosophila Virilis ◽

Predicted Amino Acid Sequence ◽

Dna Binding Domain

The zeste gene product is involved in two types of genetic effects dependent on chromosome pairing: transvection and the zeste-white interaction. Comparison of the predicted amino acid sequence with that of the Drosophila virilis gene shows that several blocks of amino acid sequence have been very highly conserved. One of these regions corresponds to the DNA binding domain. Site-directed mutations in this region indicate that a sequence resembling that of the homeodomain DNA recognition helix is essential for DNA binding activity. The integrity of an amphipathic helical region is also essential for binding activity and is likely to be responsible for dimerization of the DNA binding domain. Another very strongly conserved domain of zeste is the C-terminal region, predicted to form a long helical structure with two sets of heptad repeats that constitute two long hydrophobic ridges at opposite ends and on opposite faces of the helix. We show that this domain is responsible for the extensive aggregation properties of zeste that are required for its role in transvection phenomena. A model is proposed according to which the hydrophobic ridges induce the formation of open-ended coiled-coil structures holding together many hundreds of zeste molecules and possibly anchoring these complexes to other nuclear structures.

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The N-Terminal Region of the Human Autophagy Protein ATG16L1 Contains a Domain That Folds into a Helical Structure Consistent with Formation of a Coiled-Coil

PLoS ONE ◽

10.1371/journal.pone.0076237 ◽

2013 ◽

Vol 8 (9) ◽

pp. e76237 ◽

Cited By ~ 10

Author(s):

Rhiannon Parkhouse ◽

Ima-Obong Ebong ◽

Carol V. Robinson ◽

Tom P. Monie

Keyword(s):

Coiled Coil ◽

Helical Structure ◽

Terminal Region

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P029 TRIM 21 protects against ulcerative colitis and colitic cancer via smad7 inhibition

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjz203.158 ◽

2020 ◽

Vol 14 (Supplement_1) ◽

pp. S146-S146

Author(s):

K B Hahm

Keyword(s):

Ulcerative Colitis ◽

Coiled Coil ◽

Type I ◽

Typical Structure ◽

Th17 Cell Differentiation ◽

Bowel Diseases ◽

Colitic Cancer ◽

Β Receptor ◽

Trim Proteins

Abstract Background Proteins of the tripartite motif-containing (TRIM) superfamily are critical in a variety of biological processes in either innate immunity or eliminating invading pathogens, by which had been implicated in pathogenesis of autoimmune diseases including inflammatory bowel diseases. The typical structure of TRIM proteins contains a RING motif in the N-terminal end, followed by a B-box motif, a coiled-coil domain and a B30.2/PRYSPRY region in the C-terminal end led to the regulation of TGF-β anti-inflammatory cytokines, by which TRIM21 has been reported to regulate IBD negatively through inhibiting Th1/Th17 cell differentiation. Methods Since antisense oligonucleotide targeting smad7 was withdrawn from clinical trial due to insufficient efficacy, in this study, we generated TRIM21 overexpressed cell lines to study the binding of TRIM21 to smad7 as well as the regulation of consequent TGF-β receptor. Results TRIM21 significantly binds to smad7 as well as repressed levels of TGF-b type I/II receptor. SBE-luc and 3TP-luc assay showed significantly decreased activities under TRIM21 + TGF-β. Since TRIM21 contains ubiquitin ligase, PRYSPRY, TRIM21 with TGF-β significantly decreased TGFRII via UPL. These in vitro evidences that TRIM21 significantly repressed TGF-β after binding smad7 were validated with DSS-induced colitis and colitic cancer model. TRIM21 was significantly decreased in DSS-induced ulcerative colitis, whereas ameliorated colitis showed significant restoration of TRIM21 Conclusion Leading to conclusion that loss of TRIM21 led to significant bout of IBD.

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Extrachromosomal Histone H2B Mediates Innate Antiviral Immune Responses Induced by Intracellular Double-Stranded DNA

Journal of Virology ◽

10.1128/jvi.01339-09 ◽

2009 ◽

Vol 84 (2) ◽

pp. 822-832 ◽

Cited By ~ 30

Author(s):

Kouji Kobiyama ◽

Fumihiko Takeshita ◽

Nao Jounai ◽

Asako Sakaue-Sawano ◽

Atsushi Miyawaki ◽

...

Keyword(s):

Immune Responses ◽

Rna Virus ◽

Cellular Signaling ◽

Helical Structure ◽

Human Cells ◽

Type I Interferons ◽

Type I ◽

Histone H2b ◽

Dna Viruses ◽

Double Stranded Dna

ABSTRACT Fragments of double-stranded DNA (dsDNA) forming a right-handed helical structure (B-DNA) stimulate cells to produce type I interferons (IFNs). While an adaptor molecule, IFN-β promoter stimulator 1 (IPS-1), mediates dsDNA-induced cellular signaling in human cells, the underlying molecular mechanism is not fully understood. Here, we demonstrate that the extrachromosomal histone H2B mediates innate antiviral immune responses in human cells. H2B physically interacts with IPS-1 through the association with a newly identified adaptor, CIAO (COOH-terminal importin 9-related adaptor organizing histone H2B and IPS-1), to transmit the cellular signaling for dsDNA but not immunostimulatory RNA. Extrachromosomal histone H2B was biologically crucial for cell-autonomous responses to protect against multiplication of DNA viruses but not an RNA virus. Thus, the present findings provide evidence indicating that the extrachromosomal histone H2B is engaged in the signaling pathway initiated by dsDNA to trigger antiviral innate immune responses.

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Dislocation of a Type I Membrane Protein Requires Interactions between Membrane-spanning Segments within the Lipid Bilayer

Molecular Biology of the Cell ◽

10.1091/mbc.e03-03-0192 ◽

2003 ◽

Vol 14 (9) ◽

pp. 3690-3698 ◽

Cited By ~ 32

Author(s):

Brendan N. Lilley ◽

Domenico Tortorella ◽

Hidde L. Ploegh

Keyword(s):

Membrane Protein ◽

Heavy Chain ◽

Transmembrane Domain ◽

Helical Structure ◽

Type I ◽

Rapid Degradation ◽

Heavy Chains ◽

Histocompatibility Complex ◽

Subsequent Degradation ◽

Membrane Spanning

The human cytomegalovirus gene product US11 causes rapid degradation of class I major histocompatibility complex (MHCI) heavy chains by inducing their dislocation from the endoplasmic reticulum (ER) and subsequent degradation by the proteasome. This set of reactions resembles the endogenous cellular quality control pathway that removes misfolded or unassembled proteins from the ER. We show that the transmembrane domain (TMD) of US11 is essential for MHCI heavy chain dislocation, but dispensable for MHCI binding. A Gln residue at position 192 in the US11 TMD is crucial for the ubiquitination and degradation of MHCI heavy chains. Cells that express US11 TMD mutants allow formation of MHCI-β2m complexes, but their rate of egress from the ER is significantly impaired. Further mutagenesis data are consistent with the presence of an alpha-helical structure in the US11 TMD essential for MHCI heavy chain dislocation. The failure of US11 TMD mutants to catalyze dislocation is a unique instance in which a polar residue in the TMD of a type I membrane protein is required for that protein's function. Targeting of MHCI heavy chains for dislocation by US11 thus requires the formation of interhelical hydrogen bonds within the ER membrane.

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4138 Development of an Antibiofilm Resorbable Membrane for Treating Peri-implantitis

Journal of Clinical and Translational Science ◽

10.1017/cts.2020.364 ◽

2020 ◽

Vol 4 (s1) ◽

pp. 121-121

Author(s):

Zhou Ye ◽

Joseph R. Rahimi ◽

Nicholas G. Fischer ◽

Hooi Pin Chew ◽

Conrado Aparicio

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Peptide ◽

Statistical Significance ◽

Oral Bacteria ◽

Collagen Fibers ◽

Collagen Membrane ◽

Type I ◽

Success Rates ◽

Hydrophobic Membrane ◽

Water Contact

OBJECTIVES/GOALS: Peri-implantitis is the inflammation of peri-implant mucosa and subsequent loss of supporting bone. Its treatment is only <40% successful mainly due to persistent bacterial infection. The goal of this project is to increase success rates by developing a robust antibiofilm multi-biomolecular membrane that can be placed around implant surfaces. METHODS/STUDY POPULATION: A collagen membrane was soaked in the antimicrobial peptide GL13K solution overnight to form an interpenetrating fibrillary network. The nanostructure of the membrane was imaged with scanning electron microscope (SEM). The hydrophobicity of the membrane was analyzed by water contact angle (WCA) measurements. The biodegradability was tested in a 0.01 mg/mL Type I collagenase solution for up to 5 weeks. The antimicrobial activity of the membrane was assessed with Gram-positive oral bacteria Streptococcus gordonii. The cytotoxicity was evaluated by culturing human gingival fibroblasts (HGF), and the osteogenesis was assessed using preosteoblasts MC3T3. Pure collagen membrane was used as the control. Statistical significance (p<0.05) was determined by one-way ANOVA with Tukey’s HSD test. RESULTS/ANTICIPATED RESULTS: The antimicrobial peptide GL13K self-assembled to short fibrils (< 1 µm long), which entangled with the larger collagen fibers (around 200 nm in diameter). The collagen fibers presented characteristic periodic banding structures, which provided biomimetic cues for cell behavior as extracellular matrix. The interpenetrated GL13K fibrils turned the highly hydrophilic collagen membrane to a hydrophobic membrane (WCA = 135 °) and significantly reduced the rate of degradation by collagenases. The developed membrane was efficient in preventing the attachment of S. gordonii. A large portion of the attached bacteria was killed on the surface of the membrane. The incorporation of GL13K did not affect the cytocompatibility of the membrane for HGF. DISCUSSION/SIGNIFICANCE OF IMPACT: We developed an antibiofilm membrane with interpenetrating collagen and antimicrobial peptide fibrils. The strong antimicrobial activity and low cytotoxicity support its further translational evaluation as scaffolds for increasing success rate in treating peri-implantitis.

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Conserved DNA binding and self-association domains of the Drosophila zeste protein.

Molecular and Cellular Biology ◽

10.1128/mcb.12.2.598 ◽

1992 ◽

Vol 12 (2) ◽

pp. 598-608 ◽

Cited By ~ 15

Author(s):

J D Chen ◽

C S Chan ◽

V Pirrotta

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Amino Acid Sequence ◽

Coiled Coil ◽

Helical Structure ◽

Binding Activity ◽

Binding Domain ◽

Drosophila Virilis ◽

Predicted Amino Acid Sequence ◽

Dna Binding Domain

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Effect of hydroxylysine-O-glycosylation on the structure of type I collagen molecule: A computational study

Glycobiology ◽

10.1093/glycob/cwaa026 ◽

2020 ◽

Vol 30 (10) ◽

pp. 830-843

Author(s):

Ming Tang ◽

Xiaocong Wang ◽

Neha S Gandhi ◽

Bethany Lachele Foley ◽

Kevin Burrage ◽

...

Keyword(s):

Force Field ◽

Type I Collagen ◽

Computational Study ◽

Experimental Studies ◽

Helical Structure ◽

Atomic Level ◽

Collagen Molecule ◽

Type I ◽

Dynamic Simulations ◽

Force Field Parameters

Abstract Collagen undergoes many types of post-translational modifications (PTMs), including intracellular modifications and extracellular modifications. Among these PTMs, glycosylation of hydroxylysine (Hyl) is the most complicated. Experimental studies demonstrated that this PTM ceases once the collagen triple helix is formed and that Hyl-O-glycosylation modulates collagen fibrillogenesis. However, the underlying atomic-level mechanisms of these phenomena remain unclear. In this study, we first adapted the force field parameters for O-linkages between Hyl and carbohydrates and then investigated the influence of Hyl-O-glycosylation on the structure of type I collagen molecule, by performing comprehensive molecular dynamic simulations in explicit solvent of collagen molecule segment with and without the glycosylation of Hyl. Data analysis demonstrated that (i) collagen triple helices remain in a triple-helical structure upon glycosylation of Hyl; (ii) glycosylation of Hyl modulates the peptide backbone conformation and their solvation environment in the vicinity and (iii) the attached sugars are arranged such that their hydrophilic faces are well exposed to the solvent, while their hydrophobic faces point towards the hydrophobic portions of collagen. The adapted force field parameters for O-linkages between Hyl and carbohydrates will aid future computational studies on proteins with Hyl-O-glycosylation. In addition, this work, for the first time, presents the detailed effect of Hyl-O-glycosylation on the structure of human type I collagen at the atomic level, which may provide insights into the design and manufacture of collagenous biomaterials and the development of biomedical therapies for collagen-related diseases.

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