RESUMO
Since the late 1980s, mutations in the RAS genes have been recognized as major oncogenes with a high occurrence rate in human cancers. Such mutations reduce the ability of the small GTPase RAS to hydrolyze GTP, keeping this molecular switch in a constitutively active GTP-bound form that drives, unchecked, oncogenic downstream signaling. One strategy to reduce the levels of active RAS is to target guanine nucleotide exchange factors, which allow RAS to cycle from the inactive GDP-bound state to the active GTP-bound form. Here, we describe the identification of potent and cell-active small-molecule inhibitors which efficiently disrupt the interaction between KRAS and its exchange factor SOS1, a mode of action confirmed by a series of biophysical techniques. The binding sites, mode of action, and selectivity were elucidated using crystal structures of KRASG12C-SOS1, SOS1, and SOS2. By preventing formation of the KRAS-SOS1 complex, these inhibitors block reloading of KRAS with GTP, leading to antiproliferative activity. The final compound 23 (BAY-293) selectively inhibits the KRAS-SOS1 interaction with an IC50 of 21 nM and is a valuable chemical probe for future investigations.
Assuntos
Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteína SOS1/antagonistas & inibidores , Linhagem Celular , Cristalografia por Raios X , Descoberta de Drogas , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína SOS1/química , Proteína SOS1/metabolismo , Transdução de SinaisRESUMO
TSG-6 is an inflammation-associated hyaluronan (HA)-binding protein that has anti-inflammatory and protective functions in arthritis and asthma as well as a critical role in mammalian ovulation. The interaction between TSG-6 and HA is pH-dependent, with a marked reduction in affinity on increasing the pH from 6.0 to 8.0. Here we have investigated the mechanism underlying this pH dependence using a combined approach of site-directed mutagenesis, NMR, isothermal titration calorimetry and microtiter plate assays. Analysis of single-site mutants of the TSG-6 Link module indicated that the loss in affinity above pH 6.0 is mediated by the change in ionization state of a histidine residue (His(4)) that is not within the HA-binding site. To understand this in molecular terms, the pH-dependent folding profile and the pK(a) values of charged residues within the Link module were determined using NMR. These data indicated that His(4) makes a salt bridge to one side-chain oxygen atom of a buried aspartate residue (Asp(89)), whereas the other oxygen is simultaneously hydrogen-bonded to a key HA-binding residue (Tyr(12)). This molecular network transmits the change in ionization state of His(4) to the HA-binding site, which explains the loss of affinity at high pH. In contrast, simulations of the pH affinity curves indicate that another histidine residue, His(45), is largely responsible for the gain in affinity for HA between pH 3.5 and 6.0. The pH-dependent interaction of TSG-6 with HA (and other ligands) provides a means of differentially regulating the functional activity of this protein in different tissue microenvironments.
Assuntos
Moléculas de Adesão Celular/química , Ácido Hialurônico/química , Dobramento de Proteína , Aminoácidos/química , Aminoácidos/metabolismo , Moléculas de Adesão Celular/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Concentração de Íons de Hidrogênio , Ligantes , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
The TipAL protein, a bacterial transcriptional regulator of the MerR family, is activated by numerous cyclic thiopeptide antibiotics. Its C-terminal drug-binding domain, TipAS, defines a subfamily of broadly distributed bacterial proteins including Mta, a central regulator of multidrug resistance in Bacillus subtilis. The structure of apo TipAS, solved by solution NMR [Brookhaven Protein Data Bank entry 1NY9], is composed of a globin-like alpha-helical fold with a deep surface cleft and an unfolded N-terminal region. Antibiotics bind within the cleft at a position that is close to the corresponding heme pocket in myo- and hemoglobin, and induce folding of the N-terminus. Thus the classical globin fold is well adapted not only for accommodating its canonical cofactors, heme and other tetrapyrroles, but also for the recognition of a variety of antibiotics where ligand binding leads to transcriptional activation and drug resistance.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Transativadores/química , Transativadores/metabolismo , Sequência de Aminoácidos , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Relação Estrutura-Atividade , Transativadores/genéticaRESUMO
The solution structure of the Link module from human TSG-6, a hyaladherin with important roles in inflammation and ovulation, has been determined in both its free and hyaluronan-bound conformations. This reveals a well defined hyaluronan-binding groove on one face of the Link module that is closed in the absence of ligand. The groove is lined with amino acids that have been implicated in mediating the interaction with hyaluronan, including two tyrosine residues that appear to form essential intermolecular hydrogen bonds and two basic residues capable of supporting ionic interactions. This is the first structure of a non-enzymic hyaladherin in its active state, and identifies a ligand-induced conformational change that is likely to be conserved across the Link module superfamily. NMR and isothermal titration calorimetry experiments with defined oligosaccharides have allowed us to infer the minimum length of hyaluronan that can be accommodated within the binding site and its polarity in the groove; these data have been used to generate a model of the complex formed between the Link module and a hyaluronan octasaccharide.
Assuntos
Moléculas de Adesão Celular/metabolismo , Ácido Hialurônico/metabolismo , Inflamação/fisiopatologia , Ovulação/fisiologia , Sequência de Aminoácidos , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/fisiologia , Escherichia coli/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
The N-terminal domain of the vaccinia virus protein E3L (Z alpha(E3L)) is essential for full viral pathogenicity in mice. It has sequence similarity to the high-affinity human Z-DNA-binding domains Z alpha(ADAR1) and Z alpha(DLM1). Here, we report the solution structure of Z alpha(E3L) and the chemical shift map of its interaction surface with Z-DNA. The global structure and the Z-DNA interaction surface of Z alpha(E3L) are very similar to the high-affinity Z-DNA-binding domains Z alpha(ADAR1) and Z alpha(DLM1). However, the key Z-DNA contacting residue Y48 of Z alpha(E3L) adopts a different side chain conformation in unbound Z alpha(E3L), which requires rearrangement for binding to Z-DNA. This difference suggests a molecular basis for the significantly lower in vitro affinity of Z alpha(E3L) to Z-DNA compared with its homologues.
Assuntos
DNA Forma Z/química , Proteínas de Ligação a RNA/química , Tirosina , Proteínas Virais/química , Sequência de Aminoácidos , Sítios de Ligação , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , SoluçõesRESUMO
Adhesive interactions involving CD44, the cell surface receptor for hyaluronan, underlie fundamental processes such as inflammatory leukocyte homing and tumor metastasis. Regulation of such events is critical and appears to be effected by changes in CD44 N-glycosylation that switch the receptor "on" or "off" under appropriate circumstances. How altered glycosylation influences binding of hyaluronan to the lectin-like Link module in CD44 is unclear, although evidence suggests additional flanking sequences peculiar to CD44 may be involved. Here we show using X-ray crystallography and NMR spectroscopy that these sequences form a lobular extension to the Link module, creating an enlarged HA binding domain and a formerly unidentified protein fold. Moreover, the disposition of key N-glycosylation sites reveals how specific sugar chains could alter both the affinity and avidity of CD44 HA binding. Our results provide the necessary structural framework for understanding the diverse functions of CD44 and developing novel therapeutic strategies.