ジャーナル: Sci Rep / 年: 2016 タイトル: The BR domain of PsrP interacts with extracellular DNA to promote bacterial aggregation; structural insights into pneumococcal biofilm formation. 著者: Tim Schulte / Cecilia Mikaelsson / Audrey Beaussart / Alexey Kikhney / Maya Deshmukh / Sebastian Wolniak / Anuj Pathak / Christine Ebel / Jonas Löfling / Federico Fogolari / Birgitta ...著者: Tim Schulte / Cecilia Mikaelsson / Audrey Beaussart / Alexey Kikhney / Maya Deshmukh / Sebastian Wolniak / Anuj Pathak / Christine Ebel / Jonas Löfling / Federico Fogolari / Birgitta Henriques-Normark / Yves F Dufrêne / Dmitri Svergun / Per-Åke Nygren / Adnane Achour / 要旨: The major human pathogen Streptococcus pneumoniae is a leading cause of disease and death worldwide. Pneumococcal biofilm formation within the nasopharynx leads to long-term colonization and ...The major human pathogen Streptococcus pneumoniae is a leading cause of disease and death worldwide. Pneumococcal biofilm formation within the nasopharynx leads to long-term colonization and persistence within the host. We have previously demonstrated that the capsular surface-associated pneumococcal serine rich repeat protein (PsrP), key factor for biofilm formation, binds to keratin-10 (KRT10) through its microbial surface component recognizing adhesive matrix molecule (MSCRAMM)-related globular binding region domain (BR187-385). Here, we show that BR187-385 also binds to DNA, as demonstrated by electrophoretic mobility shift assays and size exclusion chromatography. Further, heterologous expression of BR187-378 or the longer BR120-378 construct on the surface of a Gram-positive model host bacterium resulted in the formation of cellular aggregates that was significantly enhanced in the presence of DNA. Crystal structure analyses revealed the formation of BR187-385 homo-dimers via an intermolecular β-sheet, resulting in a positively charged concave surface, shaped to accommodate the acidic helical DNA structure. Furthermore, small angle X-ray scattering and circular dichroism studies indicate that the aggregate-enhancing N-terminal region of BR120-166 adopts an extended, non-globular structure. Altogether, our results suggest that PsrP adheres to extracellular DNA in the biofilm matrix and thus promotes pneumococcal biofilm formation.
#1: ジャーナル: Open Biol / 年: 2014 タイトル: The basic keratin 10-binding domain of the virulence-associated pneumococcal serine-rich protein PsrP adopts a novel MSCRAMM fold. 著者: Tim Schulte / Jonas Löfling / Cecilia Mikaelsson / Alexey Kikhney / Karina Hentrich / Aurora Diamante / Christine Ebel / Staffan Normark / Dmitri Svergun / Birgitta Henriques-Normark / Adnane Achour / 要旨: 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 ...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 β-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 β-strands, for interaction with KRT10.
温度: 298 K / 手法: 蒸気拡散法 詳細: The BR187-378 dimer was concentrated to 20 mg/mL in 20 mM Na-Citrate, 500 mM NaCl, 10% (V/V) glycerol, pH 5.5. Well-diffracting crystals were obtained in 100 mM Trisodiumcitrate dihydrate pH ...詳細: The BR187-378 dimer was concentrated to 20 mg/mL in 20 mM Na-Citrate, 500 mM NaCl, 10% (V/V) glycerol, pH 5.5. Well-diffracting crystals were obtained in 100 mM Trisodiumcitrate dihydrate pH 5.6; 34-44% 2-methyl-4-pentanediole using the sitting drop vapor-diffusion method.
解像度: 2.1→48.361 Å / SU ML: 0.26 / 交差検証法: FREE R-VALUE / σ(F): 1.33 / 位相誤差: 25.97 詳細: A molecular replacement search performed using Phaser revealed three molecules of the BR187-385 monomer (PDB: 3ZGH) within the asymmetric unit. Initial rigid body and restrained refinement ...詳細: A molecular replacement search performed using Phaser revealed three molecules of the BR187-385 monomer (PDB: 3ZGH) within the asymmetric unit. Initial rigid body and restrained refinement rounds were performed in CCP4 refmac, and Coot was used for manual model rebuilding. The mFo-DFc electron density difference map clearly indicated that residues Q312-G315 had to be re-built such that residues L202-G315 of chain A were linked to residues Y316-S377 of the symmetry mate [X, -Y, -Z + (1 0 0) & {-1 0 0}), symmetry operation in Coot], and vice versa. The same procedure was applied to swap the corresponding regions between chains B and C. The model was thereafter refined using Phenix with individual isotropic ADP factors, TLS refinement and NCS torsion restraints. The final model was refined to R and Rfree values of 21.1 and 24.4, respectively, and comprised residues L202-S377, residues N203-S377 and residues N203-S376 for chains A, B and C, respectively. The electron density for the structural model of chain A was of higher quality compared to the density for both chains B and C, which was corroborated by higher values for real-space correlation coefficients. This difference in the quality of the electron density map was also true for the crucial loop region around residue S314.
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Streptococcus pneumoniae serotype 4 (肺炎レンサ球菌)
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Escherichia coli BL21(DE3) (大腸菌) / 参照: UniProt: A0A0H2URK1
分子量: 22.990 Da / 分子数: 1 / 由来タイプ: 合成 / 式: Na
分子量: 92.094 Da / 分子数: 1 / 由来タイプ: 合成 / 式: C3H8O3
分子量: 18.015 Da / 分子数: 207 / 由来タイプ: 天然 / 式: H2O
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