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2.
Nat Commun ; 14(1): 3763, 2023 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-37353482

RESUMO

Altered protein phosphorylation in cancer cells often leads to surface presentation of phosphopeptide neoantigens. However, their role in cancer immunogenicity remains unclear. Here we describe a mechanism by which an HLA-B*0702-specific acute myeloid leukemia phosphoneoantigen, pMLL747-755 (EPR(pS)PSHSM), is recognized by a cognate T cell receptor named TCR27, a candidate for cancer immunotherapy. We show that the replacement of phosphoserine P4 with serine or phosphomimetics does not affect pMHC conformation or peptide-MHC affinity but abrogates TCR27-dependent T cell activation and weakens binding between TCR27 and pMHC. Here we describe the crystal structures for TCR27 and cognate pMHC, map of the interface produced by nuclear magnetic resonance, and a ternary complex generated using information-driven protein docking. Our data show that non-covalent interactions between the epitope phosphate group and TCR27 are crucial for TCR specificity. This study supports development of new treatment options for cancer patients through target expansion and TCR optimization.


Assuntos
Fosfopeptídeos , Receptores de Antígenos de Linfócitos T , Humanos , Fosfopeptídeos/metabolismo , Ligação Proteica
3.
Nat Commun ; 14(1): 2616, 2023 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-37147290

RESUMO

The TCR integrates forces in its triggering process upon interaction with pMHC. Force elicits TCR catch-slip bonds with strong pMHCs but slip-only bonds with weak pMHCs. We develop two models and apply them to analyze 55 datasets, demonstrating the models' ability to quantitatively integrate and classify a broad range of bond behaviors and biological activities. Comparing to a generic two-state model, our models can distinguish class I from class II MHCs and correlate their structural parameters with the TCR/pMHC's potency to trigger T cell activation. The models are tested by mutagenesis using an MHC and a TCR mutated to alter conformation changes. The extensive comparisons between theory and experiment provide model validation and testable hypothesis regarding specific conformational changes that control bond profiles, thereby suggesting structural mechanisms for the inner workings of the TCR mechanosensing machinery and plausible explanations of why and how force may amplify TCR signaling and antigen discrimination.


Assuntos
Receptores de Antígenos de Linfócitos T , Transdução de Sinais , Receptores de Antígenos de Linfócitos T/metabolismo , Ativação Linfocitária , Genes MHC da Classe II , Mutagênese , Ligação Proteica
4.
Nat Biotechnol ; 36(12): 1152-1154, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30520863
5.
Cell Rep ; 14(12): 2833-45, 2016 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-26997265

RESUMO

Antigen recognition of peptide-major histocompatibility complexes (pMHCs) by T cells, a key step in initiating adaptive immune responses, is performed by the T cell receptor (TCR) bound to CD3 heterodimers. However, the biophysical basis of the transmission of TCR-CD3 extracellular interaction into a productive intracellular signaling sequence remains incomplete. Here we used nuclear magnetic resonance (NMR) spectroscopy combined with mutational analysis and computational docking to derive a structural model of the extracellular TCR-CD3 assembly. In the inactivated state, CD3γε interacts with the helix 3 and helix 4-F strand regions of the TCR Cß subunit, whereas CD3δε interacts with the F and C strand regions of the TCR Cα subunit in this model, placing the CD3 subunits on opposing sides of the TCR. This work identifies the molecular contacts between the TCR and CD3 subunits, identifying a physical basis for transmitting an activating signal through the complex.


Assuntos
Complexo CD3/química , Modelos Moleculares , Complexo Receptor-CD3 de Antígeno de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/química , Complexo CD3/genética , Complexo CD3/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo
6.
Nucleic Acids Res ; 40(5): 2076-88, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22084199

RESUMO

The phosphoesterase (PE) domain of the bacterial DNA repair enzyme LigD possesses distinctive manganese-dependent 3'-phosphomonoesterase and 3'-phosphodiesterase activities. PE exemplifies a new family of DNA end-healing enzymes found in all phylogenetic domains. Here, we determined the structure of the PE domain of Pseudomonas aeruginosa LigD (PaePE) using solution NMR methodology. PaePE has a disordered N-terminus and a well-folded core that differs in instructive ways from the crystal structure of a PaePE•Mn(2+)• sulfate complex, especially at the active site that is found to be conformationally dynamic. Chemical shift perturbations in the presence of primer-template duplexes with 3'-deoxynucleotide, 3'-deoxynucleotide 3'-phosphate, or 3' ribonucleotide termini reveal the surface used by PaePE to bind substrate DNA and suggest a more efficient engagement in the presence of a 3'-ribonucleotide. Spectral perturbations measured in the presence of weakly catalytic (Cd(2+)) and inhibitory (Zn(2+)) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn(2+).


Assuntos
DNA Ligases/química , Proteínas de Ligação a DNA/química , DNA/química , Cristalografia por Raios X , Fluorescência , Metais/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/enzimologia
7.
Biomol NMR Assign ; 5(2): 151-5, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21213076

RESUMO

DNA ligase D (LigD), consisting of polymerase, ligase and phosphoesterase domains, is the essential catalyst of the bacterial non-homologous end-joining pathway of DNA double-strand break repair. The phosphoesterase (PE) module performs manganese-dependent 3'-phosphomonoesterase and 3'-ribonucleoside resection reactions that heal broken ends in preparation for sealing. LigD PE exemplifies a structurally and mechanistically unique class of DNA end-processing enzymes. Here, we present the resonance assignments of the PE domain of Pseudomonas aeruginosa LigD comprising the N-terminal 177 residues.


Assuntos
Proteínas de Bactérias/química , DNA Ligases/química , Ressonância Magnética Nuclear Biomolecular , Monoéster Fosfórico Hidrolases/química , Pseudomonas aeruginosa/enzimologia , Isótopos de Carbono , Reparo do DNA , Isótopos de Nitrogênio , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/química
8.
J Biol Chem ; 281(42): 31863-75, 2006 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-16905547

RESUMO

Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain (DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-kappaB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone 15N NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding alpha2-alpha3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the alpha2-alpha3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the alpha2-alpha3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas do Citoesqueleto/química , Inflamação/metabolismo , Ribonucleoproteínas/química , Sequência de Aminoácidos , Apoptose , Proteínas Reguladoras de Apoptose/fisiologia , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Modelos Estatísticos , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Pirina , Ribonucleoproteínas/fisiologia , Transdução de Sinais
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