RESUMO
Protein arginine methyltransferases (PRMTs) play important roles in several cellular processes, including signaling, gene regulation, and transport of proteins and nucleic acids, to impact growth, differentiation, proliferation, and development. PRMT5 symmetrically di-methylates the two-terminal ω-guanidino nitrogens of arginine residues on substrate proteins. PRMT5 acts as part of a multimeric complex in concert with a variety of partner proteins that regulate its function and specificity. A core component of these complexes is the WD40 protein MEP50/WDR77/p44, which mediates interactions with binding partners and substrates. We have determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The structure of the surprising hetero-octameric complex reveals the close interaction between the seven-bladed ß-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteína-Arginina N-Metiltransferases/química , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
Antibody therapeutics are one of the most important classes of drugs. Antibody structures have become an integral part of predicting the behavior of potential therapeutics, either directly or as the basis of modeling. Structures of Fab:antigen complexes have even greater value. While the crystallization and structure determination of Fabs is easy relative to many other protein classes, especially membrane proteins, broad screening and optimization of crystalline hits is still necessary. Through a comprehensive review of rabbit Fab crystal contacts and their incompatibility with human Fabs, we identified a small secondary structural element from the rabbit light chain constant domain potentially responsible for hindering the crystallization of human Fabs. Upon replacing the human kappa constant domain FG loop (HQGLSSP) with the two residue shorter rabbit loop (QGTTS), we dramatically improved the crystallization of human Fabs and Fab:antigen complexes. Our design, which we call "Crystal Kappa", enables rapid crystallization of human fabs and fab complexes in a broad range of conditions, with less material in smaller screens or from dilute solutions.
Assuntos
Fragmentos Fab das Imunoglobulinas/química , Cadeias kappa de Imunoglobulina/química , Sequência de Aminoácidos , Animais , Células CHO , Cricetulus , Cristalização , Cristalografia por Raios X , Humanos , Conformação Proteica em Folha beta , CoelhosRESUMO
CXCR1 and CXCR2 signaling play a critical role in neutrophil migration, angiogenesis, and tumorigenesis and are therefore an attractive signaling axis to target in a variety of indications. In human, a total of seven chemokines signal through these receptors and comprise the ELR+CXC chemokine family, so named because of the conserved ELRCXC N-terminal motif. To fully antagonize CXCR1 and CXCR2 signaling, an effective therapeutic should block either both receptors or all seven ligands, yet neither approach has been fully realized clinically. In this work, we describe the generation and characterization of LY3041658, a humanized monoclonal antibody that binds and neutralizes all seven human and cynomolgus monkey ELR+CXC chemokines and three of five mouse and rat ELR+CXC chemokines with high affinity. LY3041658 is able to block ELR+CXC chemokine-induced Ca2+ mobilization, CXCR2 internalization, and chemotaxis in vitro as well as neutrophil mobilization in vivo without affecting other neutrophil functions. In addition to the in vitro and in vivo activity, we characterized the epitope and structural basis for binding in detail through alanine scanning, crystallography, and mutagenesis. Together, these data provide a robust preclinical characterization of LY3041658 for which the efficacy and safety is being evaluated in human clinical trials for neutrophilic skin diseases.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8B/antagonistas & inibidores , Animais , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Afinidade de Anticorpos , Quimiotaxia de Leucócito/imunologia , Humanos , Macaca fascicularis , Camundongos , Neutrófilos/imunologia , RatosRESUMO
Recent studies have implicated a role of the epidermal growth factor receptor (EGFR) pathway in kidney disease. Skin toxicity associated with therapeutics which completely block the EGFR pathway precludes their use in chronic dosing. Therefore, we developed antibodies which specifically neutralize the EGFR ligands TGFα (transforming growth factor-alpha) and epiregulin but not EGF (epidermal growth factor), amphiregulin, betacellulin, HB-EGF (heparin-binding epidermal growth factor), or epigen. The epitope of one such neutralizing antibody, LY3016859, was characterized in detail to elucidate the structural basis for ligand specificity. Here we report a crystal structure of the LY3016859 Fab fragment in complex with soluble human TGFα. Our data demonstrate a conformational epitope located primarily within the C-terminal subdomain of the ligand. In addition, point mutagenesis experiments were used to highlight specific amino acids which are critical for both antigen binding and neutralization, most notably Ala41 , Glu44 , and His45 . These results illustrate the structural basis for the ligand specificity/selectivity of LY3016859 and could also provide insight into further engineering to alter specificity and/or affinity of LY3016859.
Assuntos
Anticorpos Neutralizantes/química , Especificidade de Anticorpos , Epirregulina/química , Epitopos/química , Fragmentos Fab das Imunoglobulinas/química , Fator de Crescimento Transformador alfa , Animais , Humanos , Camundongos , Fator de Crescimento Transformador alfa/antagonistas & inibidores , Fator de Crescimento Transformador alfa/químicaRESUMO
The enhancer-of-zeste homolog 2 (EZH2) gene product is an 87 kDa polycomb group (PcG) protein containing a C-terminal methyltransferase SET domain. EZH2, along with binding partners, i.e., EED and SUZ12, upon which it is dependent for activity forms the core of the polycomb repressive complex 2 (PRC2). PRC2 regulates gene silencing by catalyzing the methylation of histone H3 at lysine 27. Both overexpression and mutation of EZH2 are associated with the incidence and aggressiveness of various cancers. The novel crystal structure of the SET domain was determined in order to understand disease-associated EZH2 mutations and derive an explanation for its inactivity independent of complex formation. The 2.00 Å crystal structure reveals that, in its uncomplexed form, the EZH2 C-terminus folds back into the active site blocking engagement with substrate. Furthermore, the S-adenosyl-L-methionine (SAM) binding pocket observed in the crystal structure of homologous SET domains is notably absent. This suggests that a conformational change in the EZH2 SET domain, dependent upon complex formation, must take place for cofactor and substrate binding activities to be recapitulated. In addition, the data provide a structural context for clinically significant mutations found in the EZH2 SET domain.
Assuntos
Domínio Catalítico/genética , Doença/genética , Mutação , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/genética , Células Sf9 , SpodopteraRESUMO
Aminoacyl transfer RNA (tRNA) synthetases are intensely studied enzymes because of their importance in the establishment of the genetic code and their connection to disease and medicine. During the advancement of this field, several assays were developed. Despite many innovations, the sensitivity, simplicity, and reliability of the radiometric assays (which were among the first to be developed) have ensured their continued use. Four activities are measured by these assays: active site titration, amino acid activation, aminoacylation, and posttransfer editing (deacylation). In an effort to maintain the advantage of these assays while enhancing throughput, reducing waste, and improving data quality, a universal 96-well filter plate format was developed. This format facilitates the assays for all four of the widely studied activities.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Radiometria/métodos , Aminoacilação , Sítios de Ligação , Modelos Biológicos , Radiometria/instrumentação , Contagem de Cintilação , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Little is known about the role of specific base modifications of transfer RNAs. Wyosine bases are tRNA(Phe)-specific modifications that are distinguished by differentiated, lateral side chains and base methylations appended to the core ring structure of a universally conserved G37, adjacent to the anticodon of Phe tRNAs. Based on previous data, we hypothesized that this modification was needed for -1 frameshifting. Using a reporter system incorporating a SCV-LA yeast virus slippery site for detecting -1 frameshifts in vivo, yeast strains were created that enabled chemical-genetic dissection of the role of different functional groups of wyebutosine that are added in a three-step post-transcriptional set of reactions. With this system, hypomodification increased Phe-specific frameshifting, with incremental changes in frameshift efficiency after specific intermediates in the progression of wyebutosine synthesis. These data combined with investigations of wild-type and hypomodified tRNA binding to ribosomes suggest that frameshift efficiency is kinetically and not thermodynamically controlled. The progressive nature of frameshift efficiency with the stage of modification is consistent with a stepwise evolution and tuning of frameshift potential. The stepwise tuning of frameshift efficiency could explain why tRNA(Phe) in some eukaryotes is not fully modified but, rather, hypomodified to capture a specific frameshift potential.
Assuntos
Evolução Molecular , Mudança da Fase de Leitura do Gene Ribossômico , Processamento Pós-Transcricional do RNA/fisiologia , RNA Fúngico/metabolismo , RNA de Transferência de Fenilalanina/metabolismo , Saccharomyces cerevisiae/metabolismo , Mudança da Fase de Leitura do Gene Ribossômico/genética , Genes Reporter/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Fúngico/genética , RNA de Transferência de Fenilalanina/genética , Saccharomyces cerevisiae/genéticaRESUMO
With the recent availability of high-resolution structures of bacterial ribosomes, studies of ribosome-catalyzed protein biosynthesis are now focusing on the nature of conformational changes that occur as the ribosome exerts its complex catalytic function. Photocrosslinking can be relevant for this purpose by providing clues to ribosomal structural fluctuations and dynamics. Here we describe crosslinking experiments on 70S ribosomes using two photolabile anticodon stem-loop derivatives of Escherichia coli tRNAPhe carrying a 4-thiouridine in either position 33 or 37 and denoted Ph-ASLs. One or both of these Ph-ASLs bind to the tRNA A-, P-, and E-sites on the ribosome, with both binding to and photocrosslinking from the E-site showing strong dependence on the presence of a tRNA in the P-site. Both Ph-ASLs crosslink to the extreme 3'-end of 16S rRNA from both the P- and E-sites, providing direct confirmatory evidence in solution for the folding back of the 3'-end toward the decoding region. This suggests that the 3'-end of 16S rRNA may act as a switch in controlling mRNA access to the decoding center, a phenomenon of potential relevance for the translation of leaderless mRNA. E-site bound Ph-ASLs also form photocrosslinks to nucleotides 1395-1398, 1399-1400, and 1491-1494 at the top of helix 44 of 16S rRNA, indicating movement of the decoding center from a position between the A- and P-sites seen in the crystal structure to one neighboring the E-site.