Structural basis for the role of serine-rich repeat proteins from Lactobacillus reuteri in gut microbe–host interactions

Lactobacillus reuteri, a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates, displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP100-23) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens, but no structural information is available in commensal bacteria. Here we report the 2.00-Å and 1.92-Å crystal structures of the binding regions (BRs) of SRRP100-23 and SRRP53608 from L. reuteri ATCC 53608, revealing a unique β-solenoid fold in this important adhesin family. SRRP53608-BR bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of SRRP53608-BR with PGA. Long molecular dynamics simulations showed that SRRP53608-BR undergoes a pH-dependent conformational change. Together, these findings provide mechanistic insights into the role of SRRPs in host–microbe interactions and open avenues of research into the use of biofilm-forming probiotics against clinically important pathogens.

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Altered Local Interactions and Long-Range Communications in UK Variant (B.1.1.7) Spike Glycoprotein

International Journal of Molecular Sciences ◽

10.3390/ijms22115464 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5464

Author(s):

Stefano Borocci ◽

Carmen Cerchia ◽

Alessandro Grottesi ◽

Nico Sanna ◽

Ingrid Guarnetti Prandi ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Structural Information ◽

Conformational Transitions ◽

Structural Basis ◽

Receptor Binding Domain ◽

Wild Type ◽

Dynamics Simulations ◽

The Uk ◽

The Impact

The COVID-19 pandemic is caused by SARS-CoV-2. Currently, most of the research efforts towards the development of vaccines and antibodies against SARS-CoV-2 were mainly focused on the spike (S) protein, which mediates virus entry into the host cell by binding to ACE2. As the virus SARS-CoV-2 continues to spread globally, variants have emerged, characterized by multiple mutations of the S glycoprotein. Herein, we employed microsecond-long molecular dynamics simulations to study the impact of the mutations of the S glycoprotein in SARS-CoV-2 Variant of Concern 202012/01 (B.1.1.7), termed the “UK variant”, in comparison with the wild type, with the aim to decipher the structural basis of the reported increased infectivity and virulence. The simulations provided insights on the different dynamics of UK and wild-type S glycoprotein, regarding in particular the Receptor Binding Domain (RBD). In addition, we investigated the role of glycans in modulating the conformational transitions of the RBD. The overall results showed that the UK mutant experiences higher flexibility in the RBD with respect to wild type; this behavior might be correlated with the increased transmission reported for this variant. Our work also adds useful structural information on antigenic “hotspots” and epitopes targeted by neutralizing antibodies.

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Role of Extracellular Transaldolase from Bifidobacterium bifidum in Mucin Adhesion and Aggregation

Applied and Environmental Microbiology ◽

10.1128/aem.08024-11 ◽

2012 ◽

Vol 78 (11) ◽

pp. 3992-3998 ◽

Cited By ~ 61

Author(s):

Irene González-Rodríguez ◽

Borja Sánchez ◽

Lorena Ruiz ◽

Francesca Turroni ◽

Marco Ventura ◽

...

Keyword(s):

Bacterial Species ◽

Bifidobacterium Bifidum ◽

Surface Proteins ◽

Proteinase K ◽

Intestinal Cell ◽

Immunogold Electron Microscopy ◽

Content Type ◽

Colonization Factor ◽

Intestinal Cell Line

ABSTRACTThe ability of bifidobacteria to establish in the intestine of mammals is among the main factors considered to be important for achieving probiotic effects. The role of surface molecules fromBifidobacterium bifidumtaxon in mucin adhesion capability and the aggregation phenotype of this bacterial species was analyzed. Adhesion to the human intestinal cell line HT29 was determined for a collection of 12B. bifidumstrains. In four of them—B. bifidumLMG13195, DSM20456, DSM20239, and A8—the involvement of surface-exposed macromolecules in the aggregation phenomenon was determined. The aggregation ofB. bifidumA8 and DSM20456 was abolished after treatment with proteinase K, this effect being more pronounced for the strain A8. Furthermore, a mucin binding assay ofB. bifidumA8 surface proteins showed a high adhesive capability for its transaldolase (Tal). The localization of this enzyme on the surface ofB. bifidumA8 was unequivocally demonstrated by immunogold electron microscopy experiments. The gene encoding Tal fromB. bifidumA8 was expressed inLactococcus lactis, and the protein was purified to homogeneity. The pure protein was able to restore the autoaggregation phenotype of proteinase K-treatedB. bifidumA8 cells. A recombinantL. lactisstrain, engineered to secrete Tal, displayed a mucin- binding level more than three times higher than the strain not producing the transaldolase. These findings suggest that Tal, when exposed on the cell surface ofB. bifidum, could act as an important colonization factor favoring its establishment in the gut.

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Molecular determinant of substrate binding and specificity of cytochrome P450 2J2

Scientific Reports ◽

10.1038/s41598-020-79284-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Liang Xu ◽

Liao Y. Chen

Keyword(s):

Arachidonic Acid ◽

Cytochrome P450 ◽

Active Site ◽

Structural Model ◽

Substrate Binding ◽

Conformational Dynamics ◽

Structural Basis ◽

Molecular Determinant ◽

Dynamics Simulations

AbstractCytochrome P450 2J2 (CYP2J2) is responsible for the epoxidation of endogenous arachidonic acid, and is involved in the metabolism of exogenous drugs. To date, no crystal structure of CYP2J2 is available, and the proposed structural basis for the substrate recognition and specificity in CYP2J2 varies with the structural models developed using different computational protocols. In this study, we developed a new structural model of CYP2J2, and explored its sensitivity to substrate binding by molecular dynamics simulations of the interactions with chemically similar fluorescent probes. Our results showed that the induced-fit binding of these probes led to the preferred active poses ready for the catalysis by CYP2J2. Divergent conformational dynamics of CYP2J2 due to the binding of each probe were observed. However, a stable hydrophobic clamp composed of residues I127, F310, A311, V380, and I487 was identified to restrict any substrate access to the active site of CYP2J2. Molecular docking of a series of compounds including amiodarone, astemizole, danazol, ebastine, ketoconazole, terfenadine, terfenadone, and arachidonic acid to CYP2J2 confirmed the role of those residues in determining substrate binding and specificity of CYP2J2. In addition to the flexibility of CYP2J2, the present work also identified other factors such as electrostatic potential in the vicinity of the active site, and substrate strain energy and property that have implications for the interpretation of CYP2J2 metabolism.

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Structural Flexibility of the Macrophage Dengue Virus Receptor CLEC5A

Journal of Biological Chemistry ◽

10.1074/jbc.m111.226142 ◽

2011 ◽

Vol 286 (27) ◽

pp. 24208-24218 ◽

Cited By ~ 34

Author(s):

Aleksandra A. Watson ◽

Andrey A. Lebedev ◽

Benjamin A. Hall ◽

Angharad E. Fenton-May ◽

Alexei A. Vagin ◽

...

Keyword(s):

Dengue Virus ◽

Structural Information ◽

Docking Studies ◽

Binding Potential ◽

Structural Flexibility ◽

Dengue Virus Serotypes ◽

Adapter Molecule ◽

Dynamics Simulations ◽

Β Sheet

The human C-type lectin-like molecule CLEC5A is a critical macrophage receptor for dengue virus. The binding of dengue virus to CLEC5A triggers signaling through the associated adapter molecule DAP12, stimulating proinflammatory cytokine release. We have crystallized an informative ensemble of CLEC5A structural conformers at 1.9-Å resolution and demonstrate how an on-off extension to a β-sheet acts as a binary switch regulating the flexibility of the molecule. This structural information together with molecular dynamics simulations suggests a mechanism whereby extracellular events may be transmitted through the membrane and influence DAP12 signaling. We demonstrate that CLEC5A is homodimeric at the cell surface and binds to dengue virus serotypes 1–4. We used blotting experiments, surface analyses, glycan microarray, and docking studies to investigate the ligand binding potential of CLEC5A with particular respect to dengue virus. This study provides a rational foundation for understanding the dengue virus-macrophage interaction and the role of CLEC5A in dengue virus-induced lethal disease.

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Role of Phosphoglucomutase of Bordetella bronchiseptica in Lipopolysaccharide Biosynthesis and Virulence

Infection and Immunity ◽

10.1128/iai.68.8.4673-4680.2000 ◽

2000 ◽

Vol 68 (8) ◽

pp. 4673-4680 ◽

Cited By ~ 35

Author(s):

Nicholas P. West ◽

Heidrun Jungnitz ◽

John T. Fitter ◽

Jason D. McArthur ◽

Carlos A. Guzmán ◽

...

Keyword(s):

Bacterial Resistance ◽

Bacterial Species ◽

Bordetella Bronchiseptica ◽

Insertion Mutant ◽

Wild Type ◽

Gene Encoding ◽

High Identity ◽

Lipopolysaccharide Biosynthesis

ABSTRACT The phosphoglucomutase (PGM)-encoding gene of Bordetella bronchiseptica is required for lipopolysaccharide (LPS) biosynthesis. An insertion mutant of the wild-type B. bronchiseptica strain BB7865 which disrupted LPS biosynthesis was created and characterized (BB7865pgm). Genetic analysis of the mutated gene showed it shares high identity with PGM genes of various bacterial species and forms part of an operon which also encompasses the gene encoding phosphoglucose isomerase. Functional assays for PGM revealed that enzyme activity is expressed in bothbvg-positive and bvg-negative strains ofB. bronchiseptica and is substantially reduced in BB7865pgm. Complementation of the mutated PGM gene with that from BB7865 restored the wild-type condition for all phenotypes tested. The ability of the mutant BB7865pgm to survive within J774.A1 cells was significantly reduced at 2 h (40% reduction) and 24 h (56% reduction) postinfection. BB7865pgm was also significantly attenuated in its ability to survive in vivo following intranasal infection of mice, being effectively cleared from the lungs within 4 days, whereas the wild-type strain persisted at least 35 days. The activities of superoxide dismutase, urease, and acid phosphatase were unaffected in the PGM-deficient strain. In contrast, the inability to produce wild-type LPS resulted in a reduced bacterial resistance to oxidative stress and a higher susceptibility to the antimicrobial peptide cecropin P.

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Horizontal Transfer of the Salmonella enterica Serovar Infantis Resistance and Virulence Plasmid pESI to the Gut Microbiota of Warm-Blooded Hosts

mBio ◽

10.1128/mbio.01395-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 35

Author(s):

Gili Aviv ◽

Galia Rahav ◽

Ohad Gal-Mor

Keyword(s):

Multidrug Resistance ◽

Gut Microbiota ◽

Salmonella Enterica ◽

Pathogenic Bacteria ◽

Lactobacillus Reuteri ◽

Bacterial Species ◽

Microbial Evolution ◽

Virulence Plasmid ◽

Transmission Properties

ABSTRACT Salmonella enterica serovar Infantis is one of the prevalent salmonellae worldwide. Recently, we showed that the emergence of S . Infantis in Israel was facilitated by the acquisition of a unique megaplasmid (pESI) conferring multidrug resistance and increased virulence phenotypes. Here we elucidate the ecology, transmission properties, and regulation of pESI. We show that despite its large size (~280 kb), pESI does not impose a significant metabolic burden in vitro and that it has been recently fixed in the domestic S . Infantis population. pESI conjugation and the transcription of its pilus ( pil ) genes are inhibited at the ambient temperature (27°C) and by ≥1% bile but increased under temperatures of 37 to 41°C, oxidative stress, moderate osmolarity, and the microaerobic conditions characterizing the intestinal environment of warm-blooded animals. The pESI-encoded protein TraB and the oxygen homeostasis regulator Fnr were identified as transcriptional regulators of pESI conjugation. Using the mouse model, we show that following S . Infantis infection, pESI can be horizontally transferred to the gut microbiota, including to commensal Escherichia coli strains. Possible transfer, but not persistence, of pESI was also observed into Gram-positive mouse microbiota species, especially Lactobacillus reuteri . Moreover, pESI was demonstrated to further disseminate from gut microbiota to S. enterica serovar Typhimurium, in the context of gastrointestinal infection. These findings exhibit the ability of a selfish clinically relevant megaplasmid to distribute to and from the microbiota and suggest an overlooked role of the microbiota as a reservoir of mobile genetic elements and intermediator in the spread of resistance and virulence genes between commensals and pathogenic bacteria. IMPORTANCE Plasmid conjugation plays a key role in microbial evolution, enabling the acquisition of new phenotypes, including resistance and virulence. Salmonella enterica serovar Infantis is one of the ubiquitous salmonellae worldwide and a major cause of foodborne infections. Previously, we showed that the emergence of S . Infantis in Israel has involved the acquisition of a unique megaplasmid (pESI) conferring multidrug resistance and increased virulence phenotypes. Recently, the emergence of another S . Infantis strain carrying a pESI-like plasmid was identified in Italy, suggesting that the acquisition of pESI may be common to different emergent S . Infantis populations globally. Transmission of this plasmid to other strains or bacterial species is an alarming scenario. Understanding the ecology, regulation, and transmission properties of clinically relevant plasmids and the role of the microbiota in their spreading offers a new mechanism explaining the emergence of new pathogenic and resistant biotypes and may assist in the development of appropriate surveillance and prevention measures.

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Structural basis of the heterodimerization of the MST and RASSF SARAH domains in the Hippo signalling pathway

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s139900471400947x ◽

2014 ◽

Vol 70 (7) ◽

pp. 1944-1953 ◽

Cited By ~ 35

Author(s):

Eunha Hwang ◽

Hae-Kap Cheong ◽

Ameeq Ul Mushtaq ◽

Hye-Yeon Kim ◽

Kwon Joo Yeo ◽

...

Keyword(s):

Structural Information ◽

Critical Role ◽

Three Dimensional ◽

Helical Structure ◽

Structural Features ◽

Signalling Pathway ◽

Structural Basis ◽

X Ray Crystallography ◽

Hippo Signalling

Despite recent progress in research on the Hippo signalling pathway, the structural information available in this area is extremely limited. Intriguingly, the homodimeric and heterodimeric interactions of mammalian sterile 20-like (MST) kinases through the so-called `SARAH' (SAV/RASSF/HPO) domains play a critical role in cellular homeostasis, dictating the fate of the cell regarding cell proliferation or apoptosis. To understand the mechanism of the heterodimerization of SARAH domains, the three-dimensional structures of an MST1–RASSF5 SARAH heterodimer and an MST2 SARAH homodimer were determined by X-ray crystallography and were analysed together with that previously determined for the MST1 SARAH homodimer. While the structure of the MST2 homodimer resembled that of the MST1 homodimer, the MST1–RASSF5 heterodimer showed distinct structural features. Firstly, the six N-terminal residues (Asp432–Lys437), which correspond to the short N-terminal 310-helix h1 kinked from the h2 helix in the MST1 homodimer, were disordered. Furthermore, the MST1 SARAH domain in the MST1–RASSF5 complex showed a longer helical structure (Ser438–Lys480) than that in the MST1 homodimer (Val441–Lys480). Moreover, extensive polar and nonpolar contacts in the MST1–RASSF5 SARAH domain were identified which strengthen the interactions in the heterodimer in comparison to the interactions in the homodimer. Denaturation experiments performed using urea also indicated that the MST–RASSF heterodimers are substantially more stable than the MST homodimers. These findings provide structural insights into the role of the MST1–RASSF5 SARAH domain in apoptosis signalling.

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Bcl-xL dynamics and cancer-associated mutations under the lens of protein structure network and biomolecular simulations

10.1101/574699 ◽

2019 ◽

Cited By ~ 2

Author(s):

Valentina Sora ◽

Elena Papaleo

Keyword(s):

Protein Structure ◽

Structural Information ◽

Binding Pocket ◽

Missense Mutations ◽

Free Energies ◽

Allosteric Communication ◽

Explicit Solvent ◽

Biomolecular Simulations ◽

Dynamics Simulations

AbstractUnderstanding the finely orchestrated interactions leading to or preventing programmed cell death (apoptosis) is of utmost importance in cancer research since the failure of these systems could eventually lead to the onset of the disease. In this regard, the maintenance of a delicate balance between promoters and inhibitors of mitochondrial apoptosis is crucial, as demonstrated by the interplay among the Bcl-2 family members. Particularly, Bcl-xL is a target of interest due to its forefront role of its dysfunctions in cancer development. Bcl-xL prevents apoptosis by binding both the pro-apoptotic BH3-only proteins, as PUMA, and noncanonical partners such as p53 at different sites. An allosteric communication between the BH3-only proteins binding pocket and the p53 binding site has been postulated and supported by NMR and other biophysical data, mediating the release of p53 from Bcl-xL upon PUMA binding. The molecular details, especially at the residue level, of this mechanism remain unclear. In this work, we investigated the distal communication between these two sites in both Bcl-xL in its free state and bound to PUMA, and we evaluated how missense mutations of Bcl-xL found in cancer samples might impair the communication and thus the allosteric mechanism. We employed all-atom explicit solvent microsecond molecular dynamics simulations analyzed through a Protein Structure Network approach and integrated with calculations of changes in free energies upon cancer-related mutations identified by genomics studies. We found a subset of candidate residues responsible for both maintaining protein stability and for conveying structural information between the two binding sites and hypothesized possible communication routes between specific residues at both sites.

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Broadly conserved Na+-binding site in the N-lobe of prokaryotic multidrug MATE transporters

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1802080115 ◽

2018 ◽

Vol 115 (27) ◽

pp. E6172-E6181 ◽

Cited By ~ 12

Author(s):

Emel Ficici ◽

Wenchang Zhou ◽

Steven Castellano ◽

José D. Faraldo-Gómez

Keyword(s):

Binding Site ◽

Pathogenic Bacteria ◽

Structural Information ◽

Pyrococcus Furiosus ◽

Structural Basis ◽

Binding Motif ◽

Pharmacological Targets ◽

Cytotoxic Compounds ◽

Dynamics Simulations ◽

Complex Ion

Multidrug and toxic-compound extrusion (MATE) proteins comprise an important but largely uncharacterized family of secondary-active transporters. In both eukaryotes and prokaryotes, these transporters protect the cell by catalyzing the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are thus potential pharmacological targets against drug-resistant pathogenic bacteria and tumor cells. The activity of MATEs is powered by transmembrane electrochemical ion gradients, but their molecular mechanism and ion specificity are not understood, in part because high-quality structural information is limited. Here, we use computational methods to study PfMATE, from Pyrococcus furiosus, whose structure is the best resolved to date. Analysis of available crystallographic data and additional molecular dynamics simulations unequivocally reveal an occupied Na+-binding site in the N-lobe of this transporter, which had not been previously recognized. We find this site to be selective against K+ and broadly conserved among prokaryotic MATEs, including homologs known to be Na+-dependent such as NorM-VC, VmrA, and ClbM, for which the location of the Na+ site had been debated. We note, however, that the chemical makeup of the proposed Na+ site indicates it is weakly specific against H+, explaining why MATEs featuring this Na+-binding motif may be solely driven by H+ in laboratory conditions. We further posit that the concurrent coupling to H+ and Na+ gradients observed for some Na+-driven MATEs owes to a second H+-binding site, within the C-lobe. In summary, our study provides insights into the structural basis for the complex ion dependency of MATE transporters.

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Cyclin-Dependent Kinase 2 in Cellular Senescence and Cancer. A Structural and Functional Review

Current Drug Targets ◽

10.2174/1389450120666181204165344 ◽

2019 ◽

Vol 20 (7) ◽

pp. 716-726 ◽

Cited By ~ 23

Author(s):

Priscylla Andrade Volkart ◽

Gabriela Bitencourt-Ferreira ◽

André Arigony Souto ◽

Walter Filgueira de Azevedo

Keyword(s):

Binding Affinity ◽

Cellular Senescence ◽

Cell Cycle Progression ◽

Structural Information ◽

Structural Basis ◽

Cyclin Dependent Kinase ◽

New Era ◽

Functional Studies ◽

Molecular Aspects

Background: Cyclin-dependent kinase 2 (CDK2) has been studied due to its role in the cell-cycle progression. The elucidation of the CDK2 structure paved the way to investigate the molecular basis for inhibition of this enzyme, with the coordinated efforts combining crystallography with functional studies. Objective: Our goal here is to review recent functional and structural studies directed to understanding the role of CDK2 in cancer and senescence. Methods: There are over four hundreds of crystallographic structures available for CDK2, many of them with binding affinity information. We use this abundance of data to analyze the essential features responsible for the inhibition of CDK2 and its function in cancer and senescence. Results: The structural and affinity data available CDK2 makes it possible to have a clear view of the vital CDK2 residues involved in molecular recognition. A detailed description of the structural basis for ligand binding is of pivotal importance in the design of CDK2 inhibitors. Our analysis shows the relevance of the residues Leu 83 and Asp 86 for binding affinity. The recent findings revealing the participation of CDK2 inhibition in senescence open the possibility to explore the richness of structural and affinity data for a new era in the development of CDK2 inhibitors, targeting cellular senescence. Conclusion: Here, we analyzed structural information for CDK2 in combination with inhibitors and mapped the molecular aspects behind the strongest CDK2 inhibitors for which structures and ligandbinding affinity data were available. From this analysis, we identified the significant intermolecular interactions responsible for binding affinity. This knowledge may guide the future development of CDK2 inhibitors targeting cancer and cellular senescence.

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