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1.
bioRxiv ; 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38746434

RESUMO

Proteins harboring intrinsically disordered regions (IDRs) lacking stable secondary or tertiary structures are abundant across the three domains of life. These regions have not been systematically studied in prokaryotes. Our genome-wide analysis identifies extracytoplasmic serine/threonine-rich IDRs in several biologically important membrane proteins in streptococci. We demonstrate that these IDRs are O-glycosylated with glucose by glycosyltransferases GtrB and PgtC2 in Streptococcus pyogenes and Streptococcus pneumoniae, and with N-acetylgalactosamine by a Pgf-dependent mechanism in Streptococcus mutans. Absence of glycosylation leads to a defect in biofilm formation under ethanol-stressed conditions in S. mutans. We link this phenotype to the C-terminal IDR of a post-translocation secretion chaperone PrsA. O-glycosylation of the IDR protects this region from proteolytic degradation. The IDR length attenuates the efficiency of glycosylation and, consequently, the expression level of PrsA. Taken together, our data reveal that O-glycosylation of IDRs functions as a dynamic switch of protein homeostasis in streptococci.

2.
bioRxiv ; 2023 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-36747832

RESUMO

Staphylococci, whether beneficial commensals or pathogens, often colonize human skin, potentially leading to competition for the same niche. In this multidisciplinary study we investigate the structure, binding specificity, and mechanism of adhesion of the Aap lectin domain required for Staphylococcus epidermidis skin colonization and compare its characteristics to the lectin domain from the orthologous Staphylococcus aureus adhesin SasG. The Aap structure reveals a legume lectin-like fold with atypical architecture, showing specificity for N-acetyllactosamine and sialyllactosamine. Bacterial adhesion assays using human corneocytes confirmed the biological relevance of these Aap-glycan interactions. Single-cell force spectroscopy experiments measured individual binding events between Aap and corneocytes, revealing an extraordinarily tight adhesion force of nearly 900 nN and a high density of receptors at the corneocyte surface. The SasG lectin domain shares similar structural features, glycan specificity, and corneocyte adhesion behavior. We observe cross-inhibition of Aap-and SasG-mediated staphylococcal adhesion to corneocytes. Together, these data provide insights into staphylococcal interspecies competition for skin colonization and suggest potential avenues for inhibition of S. aureus colonization.

3.
Nat Chem Biol ; 17(8): 878-887, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34045745

RESUMO

In ovoid-shaped, Gram-positive bacteria, MapZ guides FtsZ-ring positioning at cell equators. The cell wall of the ovococcus Streptococcus mutans contains peptidoglycan decorated with serotype c carbohydrates (SCCs). In the present study, we identify the major cell separation autolysin AtlA as an SCC-binding protein. AtlA binding to SCC is attenuated by the glycerol phosphate (GroP) modification. Using fluorescently labeled AtlA constructs, we mapped SCC distribution on the streptococcal surface, revealing enrichment of GroP-deficient immature SCCs at the cell poles and equators. The immature SCCs co-localize with MapZ at the equatorial rings throughout the cell cycle. In GroP-deficient mutants, AtlA is mislocalized, resulting in dysregulated cellular autolysis. These mutants display morphological abnormalities associated with MapZ mislocalization, leading to FtsZ-ring misplacement. Altogether, our data support a model in which maturation of a cell wall polysaccharide provides the molecular cues for the recruitment of cell division machinery, ensuring proper daughter cell separation and FtsZ-ring positioning.


Assuntos
Parede Celular/metabolismo , Polissacarídeos/metabolismo , Streptococcus mutans/metabolismo , Divisão Celular , Parede Celular/química , Polissacarídeos/química , Streptococcus mutans/citologia
4.
J Biol Chem ; 295(36): 12706-12715, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675282

RESUMO

Mycobacterium tuberculosis has evolved numerous type VII secretion (ESX) systems to secrete multiple factors important for both growth and virulence across their cell envelope. ESX-1, ESX-3, and ESX-5 systems have been shown to each secrete a distinct set of substrates, including PE and PPE families of proteins, named for conserved Pro-Glu and Pro-Pro-Glu motifs in their N termini. Proper secretion of the PE-PPE proteins requires the presence of EspG, with each system encoding its own unique copy. There is no cross-talk between any of the ESX systems, and how each EspG recognizes its subset of PE-PPE proteins is currently unknown. The only current structural characterization of PE-PPE-EspG heterotrimers is from the ESX-5 system. Here we present the crystal structure of the PE5mt-PPE4mt-EspG3mm heterotrimer from the ESX-3 system. Our heterotrimer reveals that EspG3mm interacts exclusively with PPE4mt in a similar manner to EspG5, shielding the hydrophobic tip of PPE4mt from solvent. The C-terminal helical domain of EspG3mm is dynamic, alternating between "open" and "closed" forms, and this movement is likely functionally relevant in the unloading of PE-PPE heterodimers at the secretion machinery. In contrast to the previously solved ESX-5 heterotrimers, the PE-PPE heterodimer of our ESX-3 heterotrimer is interacting with its chaperone at a drastically different angle and presents different faces of the PPE protein to the chaperone. We conclude that the PPE-EspG interface from each ESX system has a unique shape complementarity that allows each EspG to discriminate among noncognate PE-PPE pairs.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Mycobacterium tuberculosis/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Mycobacterium tuberculosis/genética , Domínios Proteicos
5.
Proc Natl Acad Sci U S A ; 116(8): 3202-3210, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30723150

RESUMO

The human pathogen Mycobacterium tuberculosis encodes a proteasome that carries out regulated degradation of bacterial proteins. It has been proposed that the proteasome contributes to nitrogen metabolism in M. tuberculosis, although this hypothesis had not been tested. Upon assessing M. tuberculosis growth in several nitrogen sources, we found that a mutant strain lacking the Mycobacterium proteasomal activator Mpa was unable to use nitrate as a sole nitrogen source due to a specific failure in the pathway of nitrate reduction to ammonium. We found that the robust activity of the nitrite reductase complex NirBD depended on expression of the groEL/groES chaperonin genes, which are regulated by the repressor HrcA. We identified HrcA as a likely proteasome substrate, and propose that the degradation of HrcA is required for the full expression of chaperonin genes. Furthermore, our data suggest that degradation of HrcA, along with numerous other proteasome substrates, is enhanced during growth in nitrate to facilitate the derepression of the chaperonin genes. Importantly, growth in nitrate is an example of a specific condition that reduces the steady-state levels of numerous proteasome substrates in M. tuberculosis.


Assuntos
Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Chaperonina 60/genética , Proteínas de Choque Térmico/genética , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Compostos de Amônio/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Humanos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Nitrogênio/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Tuberculose/genética , Tuberculose/metabolismo , Tuberculose/patologia
6.
Sci Rep ; 9(1): 20267, 2019 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-31889085

RESUMO

Mycobacterium tuberculosis is the cause of the world's most deadly infectious disease. Efforts are underway to target the methionine biosynthesis pathway, as it is not part of the host metabolism. The homoserine transacetylase MetX converts L-homoserine to O-acetyl-L-homoserine at the committed step of this pathway. In order to facilitate structure-based drug design, we determined the high-resolution crystal structures of three MetX proteins, including M. tuberculosis (MtMetX), Mycolicibacterium abscessus (MaMetX), and Mycolicibacterium hassiacum (MhMetX). A comparison of homoserine transacetylases from other bacterial and fungal species reveals a high degree of structural conservation amongst the enzymes. Utilizing homologous structures with bound cofactors, we analyzed the potential ligandability of MetX. The deep active-site tunnel surrounding the catalytic serine yielded many consensus clusters during mapping, suggesting that MtMetX is highly druggable.


Assuntos
Acetiltransferases/química , Acetiltransferases/metabolismo , Domínio Catalítico , Metionina/biossíntese , Modelos Moleculares , Mycobacterium/enzimologia , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Descoberta de Drogas , Redes e Vias Metabólicas/efeitos dos fármacos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Relação Estrutura-Atividade
7.
J Mol Biol ; 431(2): 289-307, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30419243

RESUMO

Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG1 adopts a quasi 2-fold symmetric structure that consists of a central ß-sheet and two α-helical bundles. In addition, we describe the structures of EspG3 chaperones from four different crystal forms. Alternate conformations of the putative PE-PPE binding site are revealed by comparison of the available EspG3 structures. Analysis of EspG1, EspG3, and EspG5 chaperones using small-angle X-ray scattering reveals that EspG1 and EspG3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG3-PE5-PPE4 complex based on the small-angle X-ray scattering analysis.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Chaperonas Moleculares/metabolismo , Mycobacterium tuberculosis/metabolismo , Sistemas de Secreção Tipo VII/metabolismo , Fatores de Virulência/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/fisiologia , Conformação Proteica , Conformação Proteica em alfa-Hélice/fisiologia , Conformação Proteica em Folha beta/fisiologia
8.
Protein Sci ; 26(10): 1964-1973, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28707417

RESUMO

The accumulation associated protein (Aap) of Staphylococcus epidermidis mediates intercellular adhesion events necessary for biofilm growth. This process depends upon Zn2+ -induced self-assembly of G5 domains within the B-repeat region of the protein, forming anti-parallel, intertwined protein "ropes" between cells. Pleomorphism in the Zn2+ -coordinating residues was observed in previously solved crystal structures, suggesting that the metal binding site might accommodate other transition metals and thereby support dimerization. By use of carefully selected buffer systems and a specialized approach to analyze sedimentation velocity analytical ultracentrifugation data, we were able to analyze low-affinity metal binding events in solution. Our data show that both Zn2+ and Cu2+ support B-repeat assembly, whereas Mn2+ , Co2+ , and Ni2+ bind to Aap but do not support self-association. As the number of G5 domains are increased in longer B-repeat constructs, the total concentration of metal required for dimerization decreases and the transition between monomer and dimer becomes more abrupt. These characteristics allow Aap to function as an environmental sensor that regulates biofilm formation in response to local concentrations of Zn2+ and Cu2+ , both of which are implicated in immune cell activity.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Metais Pesados/química , Metais Pesados/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/genética , Biofilmes , Clonagem Molecular , Escherichia coli/genética , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Staphylococcus epidermidis/química , Staphylococcus epidermidis/genética , Ultracentrifugação
9.
Methods Enzymol ; 562: 187-204, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26412652

RESUMO

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has seen a resurgence in popularity as a technique for characterizing macromolecules and complexes in solution. SV-AUC is a particularly powerful tool for studying protein conformation, complex stoichiometry, and interacting systems in general. Deconvoluting velocity data to determine a sedimentation coefficient distribution c(s) allows for the study of either individual proteins or multicomponent mixtures. The standard c(s) approach estimates molar masses of the sedimenting species based on determination of the frictional ratio (f/f0) from boundary shapes. The frictional ratio in this case is a weight-averaged parameter, which can lead to distortion of mass estimates and loss of information when attempting to analyze mixtures of macromolecules with different shapes. A two-dimensional extension of the c(s) analysis approach provides size-and-shape distributions that describe the data in terms of a sedimentation coefficient and frictional ratio grid. This allows for better resolution of species with very distinct shapes that may co-sediment and provides better molar mass determinations for multicomponent mixtures. An example case is illustrated using globular and nonglobular proteins of different masses with nearly identical sedimentation coefficients that could only be resolved using the size-and-shape distribution. Other applications of this analytical approach to complex biological systems are presented, focusing on proteins involved in the innate immune response to cytosolic microbial DNA.


Assuntos
Proteínas/isolamento & purificação , Animais , Interpretação Estatística de Dados , Ensaios Enzimáticos , Humanos , Peso Molecular , Fosforilação , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Proteínas/química , Ultracentrifugação/métodos
10.
PLoS One ; 10(5): e0126420, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25997164

RESUMO

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.


Assuntos
Ultracentrifugação/métodos , Ultracentrifugação/normas , Calibragem , Reprodutibilidade dos Testes
11.
Immunity ; 39(6): 1019-31, 2013 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-24332030

RESUMO

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor mediating innate antimicrobial immunity. It catalyzes the synthesis of a noncanonical cyclic dinucleotide, 2',5' cGAMP, that binds to STING and mediates the activation of TBK1 and IRF-3. Activated IRF-3 translocates to the nucleus and initiates the transcription of the IFN-ß gene. The structure of mouse cGAS bound to an 18 bp dsDNA revealed that cGAS interacts with dsDNA through two binding sites, forming a 2:2 complex. Enzyme assays and IFN-ß reporter assays of cGAS mutants demonstrated that interactions at both DNA binding sites are essential for cGAS activation. Mutagenesis and DNA binding studies showed that the two sites bind dsDNA cooperatively and that site B plays a critical role in DNA binding. The structure of mouse cGAS bound to dsDNA and 2',5' cGAMP provided insight into the catalytic mechanism of cGAS. These results demonstrated that cGAS is activated by dsDNA-induced oligomerization.


Assuntos
DNA/metabolismo , Modelos Moleculares , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Animais , Sítios de Ligação/genética , Domínio Catalítico , Humanos , Camundongos , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/genética , Ligação Proteica , Estrutura Quaternária de Proteína
12.
Structure ; 21(7): 1137-48, 2013 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-23746807

RESUMO

Tank-binding kinase 1 (TBK1) is a serine/threonine protein-kinase mediating innate antimicrobial immunity. TBK1 is involved in the signaling of TLRs, RLRs, and STING-mediated sensing of cytosolic DNA. Stimulation of these receptors results in the activation of TBK1, which phosphorylates interferon regulatory factor (IRF)-3. Phosphorylated IRF-3 translocates into the nucleus to initiate the transcription of the interferon (IFN)-ß gene. Here, we show that TBK1 is activated by autophosphorylation at residue Ser172. Structures of TBK1 bound to two inhibitors showed that TBK1 has the IκB kinase fold with three distinct domains: the kinase domain, the ubiquitin-like domain, and the scaffold and dimerization domain. However, the overall structures of the TBK1 monomer and its dimer are different from IKKß in the arrangements of the three domains and in dimer formation. Phosphorylation of IRF-3 by TBK1 in vitro results in its oligomerization, and phosphorylation of residue Ser386 plays a key role in IRF-3 activation.


Assuntos
Proteínas Serina-Treonina Quinases/química , Sequência de Aminoácidos , Animais , Infecções Bacterianas/enzimologia , Infecções Bacterianas/imunologia , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Imunidade Inata , Indóis/química , Fator Regulador 3 de Interferon/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Oxindóis , Fosforilação , Propionatos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/química , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Pirróis/química , Homologia Estrutural de Proteína
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