RESUMO
The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.
Assuntos
Actinobacteria/genética , Antivirais/farmacologia , Genoma Bacteriano , Macrolídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/metabolismo , Actinobacteria/metabolismo , Sequência de Aminoácidos , Antivirais/química , Antivirais/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Evolução Molecular , Células HEK293 , Humanos , Macrolídeos/química , Macrolídeos/metabolismo , Modelos Moleculares , Conformação Proteica , Homologia de Sequência , Sirolimo/química , Sirolimo/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
IL-1 is a key inflammatory and immune mediator in many diseases, including dry-eye disease, and its inhibition is clinically efficacious in rheumatoid arthritis and cryopyrin-associated periodic syndromes. To treat ocular surface disease with a topical biotherapeutic, the uniqueness of the site necessitates consideration of the agent's size, target location, binding kinetics, and thermal stability. Here we chimerized two IL-1 receptor ligands, IL-1ß and IL-1Ra, to create an optimized receptor antagonist, EBI-005, for topical ocular administration. EBI-005 binds its target, IL-1R1, 85-fold more tightly than IL-1Ra, and this increase translates to an â¼100-fold increase in potency in vivo. EBI-005 preserves the affinity bias of IL-1Ra for IL-1R1 over the decoy receptor (IL-1R2), and, surprisingly, is also more thermally stable than either parental molecule. This rationally designed antagonist represents a unique approach to therapeutic design that can potentially be exploited for other ß-trefoil family proteins in the IL-1 and FGF families.
Assuntos
Citocinas/antagonistas & inibidores , Desenho de Fármacos , Administração Tópica , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Citocinas/química , Estabilidade de Medicamentos , Feminino , Humanos , Proteína Antagonista do Receptor de Interleucina 1/antagonistas & inibidores , Proteína Antagonista do Receptor de Interleucina 1/química , Proteína Antagonista do Receptor de Interleucina 1/genética , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/química , Interleucina-1beta/genética , Cinética , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Soluções Oftálmicas , Conformação Proteica , Receptores Tipo I de Interleucina-1/antagonistas & inibidores , Receptores Tipo I de Interleucina-1/química , Receptores Tipo I de Interleucina-1/genética , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaRESUMO
Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-ß superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-ß family ligands BMP9 and BMP10 as well as the three type II TGF-ß family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies.
Assuntos
Receptores de Activinas Tipo II/química , Proteínas Morfogenéticas Ósseas/química , Fatores de Diferenciação de Crescimento/química , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Inibidores da Angiogênese/química , Inibidores da Angiogênese/uso terapêutico , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Cristalografia por Raios X , Fator 2 de Diferenciação de Crescimento , Fatores de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/metabolismo , Células HEK293 , Humanos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Type IIL restriction enzymes have rejuvenated the search for user-specified DNA binding and cutting. By aligning and contrasting the highly comparable amino-acid sequences yet diverse recognition specificities across the family of enzymes, amino acids involved in DNA binding have been identified and mutated to produce alternative binding specificities. To date, the specificity of MmeI (a type IIL restriction enzyme) has successfully been altered at positions 3, 4 and 6 of the asymmetric TCCRAC (where R is a purine) DNA-recognition sequence. To further understand the structural basis of MmeI DNA-binding specificity, the enzyme has been crystallized in complex with its DNA substrate. The crystal belonged to space group P1, with unit-cell parameters a = 61.73, b = 94.96, c = 161.24 Å, α = 72.79, ß = 89.12, γ = 71.68°, and diffracted to 2.6 Å resolution when exposed to synchrotron radiation. The structure promises to reveal the basis of MmeI DNA-binding specificity and will complement efforts to create enzymes with novel specificities.
Assuntos
DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Cristalização , Cristalografia por Raios X , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Ligação ProteicaRESUMO
DNA recognition by proteins is essential for specific expression of genes in a living organism. En route to a target DNA site, a protein will often sample noncognate DNA sites through nonspecific protein-DNA interactions, resulting in a variety of conformationally different binding states. We present here the crystal structure of endonuclease BstYI bound to a noncognate DNA. Surprisingly, the structure reveals the enzyme in a "hemispecific" binding state on the pathway between nonspecific and specific recognition. A single base pair change in the DNA abolishes binding of only one monomer, with the second monomer bound specifically. We show that the enzyme binds essentially as a rigid body, and that one end of the DNA is accommodated loosely in the binding cleft while the other end is held tightly. Another intriguing feature of the structure is Ser172, which has a dual role in establishing nonspecific and specific contacts. Taken together, the structure provides a snapshot of an enzyme in a "paused" intermediate state that may be part of a more general mechanism of scanning DNA.
Assuntos
DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/fisiologia , Cristalografia por Raios X , DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Geobacillus stearothermophilus/enzimologia , Geobacillus stearothermophilus/genética , Ligação ProteicaRESUMO
K-Ras exists in two distinct structural conformations specific to binding of GDP and GTP nucleotides. The cycling between an inactive, GDP-bound state and an active, GTP-bound state is regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. The activated form of K-Ras regulates cell proliferation, differentiation and survival by controlling several downstream signaling pathways. Oncogenic mutations that attenuate the GTPase activity of K-Ras result in accumulation of this key signaling protein in its hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. Mutations at position 12 are the most prevalent in K-Ras associated cancers, hence K-RasG12C has become a recent focus of research for therapeutic intervention. Here we report 1HN, 15N, and 13C backbone and 1H, 13C side-chain resonance assignments for the 19.3 kDa (aa 1-169) human K-Ras protein harboring an oncogenic G12C mutation in the active GppNHp-bound form (K-RasG12C-GppNHp), using heteronuclear, multidimensional NMR spectroscopy at 298K. Triple-resonance data assisted the assignments of the backbone 1H, 15N, and 13C resonances of 126 out of 165 non-proline residues. The vast majority of unassigned residues are exchange-broadened beyond detection on the NMR time scale and belong to the P-loop and two flexible Switch regions.
Assuntos
Guanosina Trifosfato/metabolismo , Proteínas Mutantes/química , Ressonância Magnética Nuclear Biomolecular , Proteínas Proto-Oncogênicas p21(ras)/química , Isótopos de Carbono , Humanos , Isótopos de Nitrogênio , Ligação Proteica , Estrutura Secundária de Proteína , PrótonsRESUMO
K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here 1HN, 15N, and 13C resonance assignments for the 19.3 kDa (aa 1-169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RASG12C-GDP), using heteronuclear, multidimensional NMR spectroscopy. Backbone 1H-15N correlations have been assigned for all non-proline residues, except for the first methionine residue.
Assuntos
Guanosina Difosfato/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Ressonância Magnética Nuclear Biomolecular , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Humanos , Proteínas Mutantes/genética , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
The type II restriction endonuclease BstYI recognizes the degenerate sequence 5'-RGATCY-3' (where R = A/G and Y = C/T), which overlaps with both BamHI (GGATCC) and BglII (AGATCT), and thus raises the question of whether BstYI DNA recognition will be more BamHI-like or BglII-like. We present here the structure of BstYI bound to a cognate DNA sequence (AGATCT). We find the complex to be more BglII-like with similarities mapping to DNA conformation, domain organization, and residues involved in catalysis. However, BstYI is unique in containing an extended arm subdomain, and the mechanism of DNA capture has both BglII-like and BamHI-like elements. Further, DNA recognition is more minimal than BglII and BamHI, where only two residues mediate recognition of the entire core sequence. Taken together, the structure reveals a mechanism of degenerate DNA recognition and offers insights into the possibilities and limitations in altering specificities of closely related restriction enzymes.
Assuntos
Proteínas de Bactérias/química , DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação/genética , Cristalização , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Mutação , Conformação de Ácido Nucleico , Conformação Proteica , Especificidade por SubstratoRESUMO
We report here the structure of BstYI, an "intermediate" type II restriction endonuclease with overlapping sequence specificities to BamHI and BglII. BstYI, a thermophilic endonuclease, recognizes and cleaves the degenerate hexanucleotide sequence 5'-RGATCY-3' (where R=A or G and Y=C or T), cleaving DNA after the 5'-R on each strand to produce four-base (5') staggered ends. The crystal structure of free BstYI was solved at 1.85A resolution by multi-wavelength anomalous dispersion (MAD) phasing. Comparison with BamHI and BglII reveals a strong structural consensus between all three enzymes mapping to the alpha/beta core domain and residues involved in catalysis. Unexpectedly, BstYI also contains an additional "arm" substructure outside of the core protein, which enables the enzyme to adopt a more compact, intertwined dimer structure compared with BamHI and BglII. This arm substructure may underlie the thermostability of BstYI. We identify putative DNA recognition residues and speculate as to how this enzyme achieves a "relaxed" DNA specificity.
Assuntos
Proteínas de Bactérias/química , Desoxirribonuclease BamHI/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desoxirribonuclease BamHI/genética , Desoxirribonuclease BamHI/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Dimerização , Modelos Moleculares , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
The long circulating half-life of serum albumin, the most abundant protein in mammalian plasma, derives from pH-dependent endosomal salvage from degradation, mediated by the neonatal Fc receptor (FcRn). Using yeast display, we identified human serum albumin (HSA) variants with increased affinity for human FcRn at endosomal pH, enabling us to solve the crystal structure of a variant HSA/FcRn complex. We find an extensive, primarily hydrophobic interface stabilized by hydrogen-bonding networks involving protonated histidines internal to each protein. The interface features two key FcRn tryptophan side chains inserting into deep hydrophobic pockets on HSA that overlap albumin ligand binding sites. We find that fatty acids (FAs) compete with FcRn, revealing a clash between ligand binding and recycling, and that our high-affinity HSA variants have significantly increased circulating half-lives in mice and monkeys. These observations open the way for the creation of biotherapeutics with significantly improved pharmacokinetics.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Receptores Fc/química , Albumina Sérica/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Ligação Competitiva , Feminino , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Cinética , Ligantes , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Ratos , Homologia de Sequência , Albumina Sérica/genética , Microglobulina beta-2/químicaRESUMO
Lipid kinases are central players in lipid signaling pathways involved in inflammation, tumorigenesis, and metabolic syndrome. A number of these kinase targets have proven difficult to investigate in higher throughput cell-free assay systems. This challenge is partially due to specific substrate interaction requirements for several of the lipid kinase family members and the resulting incompatibility of these substrates with most established, homogeneous assay formats. Traditional, cell-free in vitro investigational methods for members of the lipid kinase family typically involve substrate incorporation of [gamma-32P] and resolution of signal by thin-layer chromatography (TLC) and autoradiograph densitometry. This approach, although highly sensitive, does not lend itself to high-throughput testing of large numbers of small molecules (100 s to 1 MM+). The authors present the development and implementation of a fully synthetic, liposome-based assay for assessing in vitro activity of phosphatidylinositol-5-phosphate-4-kinase isoforms (PIP4KIIbeta and alpha) in 2 commonly used homogeneous technologies. They have validated these assays through compound testing in both traditional TLC and radioactive filterplate approaches as well as binding validation using isothermic calorimetry. A directed library representing known kinase pharmacophores was screened against type IIbeta phosphatidylinositol-phosphate kinase (PIPK) to identify small-molecule inhibitors. This assay system can be applied to other types and isoforms of PIPKs as well as a variety of other lipid kinase targets.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Metabolismo dos Lipídeos , Lipossomos/metabolismo , Fosfotransferases/metabolismo , Trifosfato de Adenosina/metabolismo , Calorimetria , Sistema Livre de Células , Cromatografia em Camada Fina , Cristalografia por Raios X , Densitometria , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Concentração Inibidora 50 , Cinética , Metabolismo dos Lipídeos/efeitos dos fármacos , Radioisótopos de Fósforo , Fosfotransferases/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Reprodutibilidade dos Testes , Especificidade por Substrato/efeitos dos fármacosRESUMO
Human DNA polymerase kappa (Pol kappa) is a proficient extender of mispaired primer termini on undamaged DNAs and is implicated in the extension step of lesion bypass. We present here the structure of Pol kappa catalytic core in ternary complex with DNA and an incoming nucleotide. The structure reveals encirclement of the DNA by a unique "N-clasp" at the N terminus of Pol kappa, which augments the conventional right-handed grip on the DNA by the palm, fingers, and thumb domains and the PAD and provides additional thermodynamic stability. The structure also reveals an active-site cleft that is constrained by the close apposition of the N-clasp and the fingers domain, and therefore can accommodate only a single Watson-Crick base pair. Together, DNA encirclement and other structural features help explain Pol kappa's ability to extend mismatches and to promote replication through various minor groove DNA lesions, by extending from the nucleotide incorporated opposite the lesion by another polymerase.
Assuntos
Pareamento Incorreto de Bases , Dano ao DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Sequência de Aminoácidos , Pareamento de Bases , Sítios de Ligação , Cristalografia por Raios X , DNA/biossíntese , DNA/química , Adutos de DNA/química , Primers do DNA/metabolismo , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Dímeros de Pirimidina/químicaRESUMO
WW domains mediate protein recognition, usually though binding to proline-rich sequences. In many proteins, WW domains occur in tandem arrays. Whether or how individual domains within such arrays cooperate to recognize biological partners is, as yet, poorly characterized. An important question is whether functional diversity of different WW domain proteins is reflected in the structural organization and ligand interaction mechanisms of their multiple domains. We have determined the solution structure and dynamics of a pair of WW domains (WW3-4) from a Drosophila Nedd4 family protein called Suppressor of deltex (Su(dx)), a regulator of Notch receptor signaling. We find that the binding of a type 1 PPPY ligand to WW3 stabilizes the structure with effects propagating to the WW4 domain, a domain that is not active for ligand binding. Both WW domains adopt the characteristic triple-stranded beta-sheet structure, and significantly, this is the first example of a WW domain structure to include a domain (WW4) lacking the second conserved Trp (replaced by Phe). The domains are connected by a flexible linker, which allows a hinge-like motion of domains that may be important for the recognition of functionally relevant targets. Our results contrast markedly with those of the only previously determined three-dimensional structure of tandem WW domains, that of the rigidly oriented WW domain pair from the RNA-splicing factor Prp40. Our data illustrate that arrays of WW domains can exhibit a variety of higher order structures and ligand interaction mechanisms.