RESUMO
The human pathogen Streptococcus pyogenes produces pili that are essential for adhesion to host surface receptors. Cpa, the adhesin at the pilus tip, was recently shown to have a thioester-containing domain. The thioester bond is believed to be important in adhesion, implying a mechanism of covalent attachment analogous to that used by human complement factors. Here, we have characterized a second active thioester-containing domain on Cpa, the N-terminal domain of Cpa (CpaN). Expression of CpaN in Escherichia coli gave covalently linked dimers. These were shown by x-ray crystallography and mass spectrometry to comprise two CpaN molecules cross-linked by the polyamine spermidine following reaction with the thioester bonds. This cross-linked CpaN dimer provides a model for the covalent attachment of Cpa to target receptors and thus the streptococcal pilus to host cells. Similar thioester domains were identified in cell wall proteins of other Gram-positive pathogens, suggesting that thioester domains are more widely used and provide a mechanism of adhesion by covalent bonding to target molecules on host cells that mimics that used by the human complement system to eliminate pathogens.
Assuntos
Adesinas Bacterianas/química , Fímbrias Bacterianas/química , Modelos Moleculares , Multimerização Proteica , Streptococcus pyogenes/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Sequência de Bases , Proteínas do Sistema Complemento/química , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/metabolismo , Cristalografia por Raios X , Escherichia coli , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Humanos , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidadeRESUMO
Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in llamas is cumbersome and costly. Here, by inserting an engineered llama immunoglobulin heavy chain (IgH) locus into IgH-deficient mice, we generate a transgenic mouse line, which we refer to as 'LamaMouse'. We demonstrate that LamaMice solely express llama IgH molecules without association to Igκ or λ light chains. Immunization of LamaMice with AAV8, the receptor-binding domain of the SARS-CoV-2 spike protein, IgE, IgG2c, and CLEC9A enabled us to readily select respective target-specific nanobodies using classical hybridoma and phage display technologies, single B cell screening, and direct cloning of the nanobody-repertoire into a mammalian expression vector. Our work shows that the LamaMouse represents a flexible and broadly applicable platform for a facilitated selection of target-specific nanobodies.
Assuntos
Camelídeos Americanos , Cadeias Pesadas de Imunoglobulinas , Camundongos Transgênicos , Anticorpos de Domínio Único , Glicoproteína da Espícula de Coronavírus , Animais , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Camelídeos Americanos/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Camundongos , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , Lectinas Tipo C/metabolismo , Lectinas Tipo C/imunologia , Lectinas Tipo C/genética , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Imunoglobulina E/imunologia , Humanos , Dependovirus/genética , Dependovirus/imunologia , Imunoglobulina G/imunologia , COVID-19/imunologia , Linfócitos B/imunologiaRESUMO
Tel1 (ATM in humans) is a large kinase that resides in the cell in an autoinhibited dimeric state and upon activation orchestrates the cellular response to DNA damage. We report the structure of an endogenous Tel1 dimer from Chaetomium thermophilum. Major parts are at 2.8 Å resolution, including the kinase active site with ATPγS bound, and two different N-terminal solenoid conformations are at 3.4 Å and 3.6 Å, providing a side-chain model for 90% of the Tel1 polypeptide. We show that the N-terminal solenoid has DNA binding activity, but that its movements are not coupled to kinase activation. Although ATPγS and catalytic residues are poised for catalysis, the kinase resides in an autoinhibited state. The PIKK regulatory domain acts as a pseudo-substrate, blocking direct access to the site of catalysis. The structure allows mapping of human cancer mutations and defines mechanisms of autoinhibition at near-atomic resolution.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Chaetomium/enzimologia , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Chaetomium/química , DNA/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Homeostase , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Multimerização ProteicaRESUMO
Cyanate hydratase (CynS) catalyzes the decomposition of cyanate and bicarbonate into ammonia and carbon dioxide. Here, the serendipitous crystallization of CynS from Serratia proteamaculans (SpCynS) is reported. SpCynS was crystallized as an impurity and its identity was determined using mass-spectrometric analysis. The crystals belonged to space group P1 and diffracted to 2.1â Å resolution. The overall structure of SpCynS is very similar to a previously determined structure of CynS from Escherichia coli. Density for a ligand bound to the SpCynS active site was observed, but could not be unambiguously identified. Additionally, glycerol molecules bound at the entry to the active site of the enzyme indicate conserved residues that might be important for the trafficking of substrates and products.
Assuntos
Carbono-Nitrogênio Liases/química , Serratia/enzimologia , Sequência de Aminoácidos , Carbono-Nitrogênio Liases/genética , Cristalização , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Serratia/genéticaRESUMO
DNA double-strand breaks are repaired by two major pathways, homologous recombination or nonhomologous end joining. The commitment to one or the other pathway proceeds via different steps of resection of the DNA ends, which is controlled and executed by a set of DNA double-strand break sensors, endo- and exonucleases, helicases, and DNA damage response factors. The molecular choreography of the underlying protein machinery is beginning to emerge. In this review, we discuss the early steps of genetic recombination and double-strand break sensing with an emphasis on structural and molecular studies.