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Structural studies of the surface adhesin SspB from Streptococcus gordonii
Umeå University, Faculty of Medicine, Odontology, Cariology. (Karina Persson)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Surface proteins on microorganisms that build up the oral biofilm are key players in the formation of the biofilm. Antigen I/II proteins are surface adhesins found on virtually all oral streptococci and share a conserved multi-domain architecture. These adhesins bind surface components on other bacteria and on host cells. Thus, they are crucial for the development of the biofilm.    

The objective of this thesis work is the structural characterization of the large multi-domain Antigen I/II protein SspB from the primary colonizing commensal bacterium Streptococcus gordonii.

The crystal structure of the variable domain of SspB was determined to 2.3 Å resolution. The domain comprises a β-supersandwich and a putative binding cleft stabilized by a calcium ion. Despite high similarity in the overall structure, the cleft within SspB is significantly smaller than the cleft within the homologous protein from Streptococcus mutans, indicating that different substrates may bind in the clefts. A screen for carbohydrate binding resulted in no hits for interaction with the SspB variable domain suggesting that the cleft may not be suitable for binding sugars.

This thesis also presents the high resolution 1.5 Å structure of a truncated C-terminal domain of SspB, the first of an Antigen I/II C-domain. The structure contains two structurally related domains, each containing one calcium ion and one intramolecular isopeptide bond. The SspB protein shares the feature of intramoleular isopeptide bonds with other surface proteins from Gram positive bacteria, such as pili from Streptococcus pyogenes and Corynebacterium diphtheriae. Intramolecular isopeptide bonds are suggested to be a common feature for retaining stability in a harsh environment. The SspB adherence region, shown to be the recognition motif for Porphyromonas gingivalis attachment to S. gordonii, protrudes from the core protein as a handle available for recognition.

In conclusion, this thesis work has provided new knowledge about the SspB protein and increased the understanding of the common structure of AgI/II proteins.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2010. , 41 p.
Series
Umeå University odontological dissertations, ISSN 0345-7532 ; 111
Keyword [en]
Streptococcus gordonii, X-ray crystallography, surface adhesin, dental plaque, structural biology
National Category
Structural Biology Dentistry
Research subject
Odontology
Identifiers
URN: urn:nbn:se:umu:diva-32910ISBN: 978-91-7264-955-2 (print)OAI: oai:DiVA.org:umu-32910DiVA: diva2:307160
Public defence
2010-04-23, Sal D, By 1D, Tandläkarhögskolan, NUS, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2010-04-01 Created: 2010-03-30 Last updated: 2010-04-01Bibliographically approved
List of papers
1. Crystal structure of the variable domain of the Streptococcus gordonii surface protein SspB
Open this publication in new window or tab >>Crystal structure of the variable domain of the Streptococcus gordonii surface protein SspB
2009 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 18, no 9, 1896-1905 p.Article in journal (Refereed) Published
Abstract [en]

The Antigen I/II (AgI/II) family of proteins are cell wall anchored adhesins expressed on the surface of oral streptococci. The AgI/II proteins interact with molecules on other bacteria, on the surface of host cells, and with salivary proteins. Streptococcus gordonii is a commensal bacterium, and one of the primary colonizers that initiate the formation of the oral biofilm. S. gordonii expresses two AgI/II proteins, SspA and SspB that are closely related. One of the domains of SspB, called the variable (V-) domain, is significantly different from corresponding domains in SspA and all other AgI/II proteins. As a first step to elucidate the differences among these proteins, we have determined the crystal structure of the V-domain from S. gordonii SspB at 2.3 A resolution. The domain comprises a beta-supersandwich with a putative binding cleft stabilized by a metal ion. The overall structure of the SspB V-domain is similar to the previously reported V-domain of the Streptococcus mutans protein SpaP, despite their low sequence similarity. In spite of the conserved architecture of the binding cleft, the cavity is significantly smaller in SspB, which may provide clues about the difference in ligand specificity. We also verified that the metal in the binding cleft is a calcium ion, in concurrence with previous biological data. It was previously suggested that AgI/II V-domains are carbohydrate binding. However, we tested that hypothesis by screening the SspB V-domain for binding to over 400 glycoconjucates and found that the domain does not interact with any of the carbohydrates.

Keyword
crystal structure, protein structure, oral biofilm, surface adhesin
National Category
Biochemistry and Molecular Biology Structural Biology
Research subject
Biochemistry; Odontology
Identifiers
urn:nbn:se:umu:diva-25828 (URN)10.1002/pro.200 (DOI)19609934 (PubMedID)
Available from: 2009-09-04 Created: 2009-09-04 Last updated: 2017-12-13
2. A crystallizable form of the Streptococcus gordonii surface antigen SspB C-domain obtained by limited proteolysis
Open this publication in new window or tab >>A crystallizable form of the Streptococcus gordonii surface antigen SspB C-domain obtained by limited proteolysis
2009 (English)In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 65, no 7, 712-714 p.Article in journal (Refereed) Published
Abstract [en]

SspB is a 1500-residue adhesin expressed on the surface of the oral bacterium Streptococcus gordonii. Its interaction with other bacteria and host cells initiates the development of dental plaque. The full-length C-terminal domain of SspB was cloned, overexpressed in Escherichia coli and purified. However, the protein could not be crystallized. Limited proteolysis of the full-length C-domain identified a core fragment. The proteolysis product was cloned, expressed and purified. The protein was crystallized using the hanging-drop vapour-diffusion method. X-ray data were collected and processed to a maximum resolution of 2.1 A with 96.4% completeness. The crystals belonged to space group P2(1), with one molecule in the asymmetric unit, a solvent content of 33.7% and a corresponding Matthews coefficient of 1.85 A(3) Da(-1).

Keyword
protein crystal, limited proteolysis, adhesin
National Category
Dentistry Dentistry
Research subject
Odontology; Molecular Biology
Identifiers
urn:nbn:se:umu:diva-26212 (URN)10.1107/S1744309109021046 (DOI)19574647 (PubMedID)
Available from: 2009-10-13 Created: 2009-09-30 Last updated: 2017-12-13Bibliographically approved
3. Two intramolecular isopeptide bonds are identified in the crystal structure of the Streptococcus gordonii SspB c-terminal domain
Open this publication in new window or tab >>Two intramolecular isopeptide bonds are identified in the crystal structure of the Streptococcus gordonii SspB c-terminal domain
2010 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 397, no 3, 740-751 p.Article in journal (Refereed) Published
Abstract [en]

Streptococcus gordonii is a primary colonizer and is involved in the formation of dental plaque. This bacterium expresses several surface proteins. One of them is the adhesin SspB, which is a member of the Antigen I/II family of proteins. SspB is a large multi-domain protein that has interactions with surface molecules on other bacteria and on host cells, and is thus a key factor in the formation of biofilms. Here, we report the crystal structure of a truncated form of the SspB C-terminal domain, solved by single-wavelength anomalous dispersion to 1.5Å resolution. The structure represents the first of a C-terminal domain from a streptococcal Antigen I/II protein and is comprised of two structurally related β-sandwich domains, C2 and C3, both with a Ca2+ bound in equivalent positions. In each of the domains, a covalent isopeptide bond is observed between a lysine and an asparagine, a feature that is believed to be a common stabilization mechanism in Gram-positive surface proteins. S. gordonii biofilms contain attachment sites for the periodontal pathogen Porphyromonas gingivalis and the SspB C-terminal domain has been shown to have one such recognition motif, the SspB adherence region. The motif protrudes from the protein, and serves as a handle for attachment. The structure suggests several additional putative binding surfaces, and other binding clefts may be created when the fulllength protein is folded.

Place, publisher, year, edition, pages
Academic Press, 2010
Keyword
crystal structure, X-ray crystallography, protein structure, surface adhesin, isopeptide bond
National Category
Dentistry Structural Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry; Odontology
Identifiers
urn:nbn:se:umu:diva-32907 (URN)10.1016/j.jmb.2010.01.065 (DOI)000276177300010 ()20138058 (PubMedID)
Available from: 2010-03-30 Created: 2010-03-30 Last updated: 2017-12-12Bibliographically approved

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