RESUMO
Ticks produce chemokine-binding proteins, known as evasins, in their saliva to subvert the host's immune response. Evasins bind to chemokines and thereby inhibit the activation of their cognate chemokine receptors, thus suppressing leukocyte recruitment and inflammation. We recently described subclass A3 evasins, which, like other class A evasins, exclusively target CC chemokines but appear to use a different binding site architecture to control target selectivity among CC chemokines. We now describe the structural basis of chemokine recognition by the class A3 evasin EVA-ACA1001. EVA-ACA1001 binds to almost all human CC chemokines and inhibits receptor activation. Truncation mutants of EVA-ACA1001 showed that, unlike class A1 evasins, both the N- and C-termini of EVA-ACA1001 play minimal roles in chemokine binding. To understand the structural basis of its broad chemokine recognition, we determined the crystal structure of EVA-ACA1001 in complex with the human chemokine CCL16. EVA-ACA1001 forms backbone-backbone interactions with the CC motif of CCL16, a conserved feature of all class A evasin-chemokine complexes. A hydrophobic pocket in EVA-ACA1001, formed by several aromatic side chains and the unique disulfide bond of class A3 evasins, accommodates the residue immediately following the CC motif (the "CC + 1 residue") of CCL16. This interaction is shared with EVA-AAM1001, the only other class A3 evasins characterized to date, suggesting it may represent a common mechanism that accounts for the broad recognition of CC chemokines by class A3 evasins.
Assuntos
Modelos Moleculares , Humanos , Animais , Carrapatos/química , Carrapatos/metabolismo , Cristalografia por Raios X , Sítios de Ligação , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Proteínas de Artrópodes/genética , Ligação Proteica , Quimiocinas/química , Quimiocinas/metabolismo , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/metabolismoRESUMO
Chemokines are key regulators of leukocyte trafficking and attractive targets for anti-inflammatory therapy. Evasins are chemokine-binding proteins from tick saliva, whose application as anti-inflammatory therapeutics will require manipulation of their chemokine target selectivity. Here we describe subclass A3 evasins, which are unique to the tick genus Amblyomma and distinguished from "classical" class A1 evasins by an additional disulfide bond near the chemokine recognition interface. The A3 evasin EVA-AAM1001 (EVA-A) bound to CC chemokines and inhibited their receptor activation. Unlike A1 evasins, EVA-A was not highly dependent on N- and C-terminal regions to differentiate chemokine targets. Structures of chemokine-bound EVA-A revealed a deep hydrophobic pocket, unique to A3 evasins, that interacts with the residue immediately following the CC motif of the chemokine. Mutations to this pocket altered the chemokine selectivity of EVA-A. Thus, class A3 evasins provide a suitable platform for engineering proteins with applications in research, diagnosis or anti-inflammatory therapy.
Assuntos
Carrapatos , Animais , Carrapatos/metabolismo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Quimiocinas/metabolismo , Quimiocinas CC/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/metabolismoRESUMO
Cytoplasmic detection of DNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) is an essential component of antiviral responses. Upon synthesis, cGAMP binds to the stimulator of interferon (IFN) genes (STING) in infected and adjacent cells through intercellular transfer by connexins forming gap-junctions, eliciting a strong IFN-ß-driven antiviral response. We demonstrate here that Genistein, a flavonoid compound naturally occurring in soy-based foods, inhibits cGAS-STING antiviral signaling at two levels. First, Genistein pretreatment of cGAMP-producing cells inhibited gap-junction intercellular communication, resulting in reduced STING responses in adjacent cells. In addition, Genistein directly blocked STING activation by the murine agonist DMXAA, by decreasing the interaction of STING with TBK1 and IKKε. As a result, Genistein attenuated STING signaling in human and mouse cells, dampening antiviral activity against Semliki Forest Virus infection. Collectively, our findings identify a previously unrecognized proviral activity of Genistein mediated via its inhibitory effects at two levels of cGAS-STING signaling. IMPORTANCE Several reports suggest that Genistein exhibits antiviral activities against DNA viruses. Our work uncovers a previously unrecognized proviral effect of Genistein, through inhibition of the cGAS-STING pathway at the level of cGAMP transfer and its sensing by STING. This suggests that the use of Genistein as an antiviral should be taken with caution as it may reduce the protective antiviral effects elicited by host STING activation.
Assuntos
Genisteína , Proteínas de Membrana , Animais , Antivirais/farmacologia , Genisteína/farmacologia , Humanos , Imunidade Inata/genética , Interferon beta/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Nucleotidiltransferases/genéticaRESUMO
The development of peptide inhibitors against intracellular targets depends upon the dual challenge of achieving a high affinity and specificity for the target and maintaining cellular permeability for biological activity. Previous efforts to develop bicyclic peptides targeted to the Grb7 signalling protein implicated in HER2+ve cancer progression have resulted in improved affinity. However, these same peptides demonstrated a lowered activity due to their decreased ability to penetrate cell membranes. Here, we report the testing of a new series of bicyclic G7 peptides designed to possess improved bioactivity. We discovered that the incorporation of two amino acids (Phe-Pro, Phe-Trp or Phe-Arg) within the bicyclic peptide framework maintains an enhanced binding affinity for the Grb7-SH2 domain compared to that of the first-generation monocyclic peptide G7-18NATE. Structure determination using X-ray crystallography revealed that the mode of binding by the expanded bicyclic G7 peptide is analogous to that of G7-18NATE. Interestingly, while the bicyclic peptide containing Phe-Trp did not display the highest affinity for Grb7-SH2 in the series, it was the most potent inhibitor of HER2+ve SKBR3 breast cancer cell migration when coupled to Penetratin. Together, this demonstrates that peptide flexibility as well as the amino acid tryptophan can play important roles in the uptake of peptides into the cell.
RESUMO
As natural chemokine inhibitors, evasin proteins produced in tick saliva are potential therapeutic agents for numerous inflammatory diseases. Engineering evasins to block the desired chemokines and avoid off-target side effects requires structural understanding of their target selectivity. Structures of the class A evasin EVA-P974 bound to human CC chemokine ligands 7 and 17 (CCL7 and CCL17) and to a CCL8-CCL7 chimera reveal that the specificity of class A evasins for chemokines of the CC subfamily is defined by conserved, rigid backbone-backbone interactions, whereas the preference for a subset of CC chemokines is controlled by side-chain interactions at four hotspots in flexible structural elements. Hotspot mutations alter target preference, enabling inhibition of selected chemokines. The structure of an engineered EVA-P974 bound to CCL2 reveals an underlying molecular mechanism of EVA-P974 target preference. These results provide a structure-based framework for engineering evasins as targeted antiinflammatory therapeutics.
Assuntos
Proteínas de Artrópodes/química , Quimiocinas/metabolismo , Inflamação/metabolismo , Engenharia de Proteínas , Carrapatos/metabolismo , Animais , Proteínas de Artrópodes/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Quimiocinas/metabolismoRESUMO
TIA-1 is an RNA-binding protein that sequesters target RNA into stress granules under conditions of cellular stress. Promotion of stress granule formation by TIA-1 depends upon self-association of its prion-like domain that facilitates liquid-liquid phase separation and is thought to be enhanced via RNA binding. However, the mechanisms underlying the influence of RNA on TIA-1 self-association have not been previously demonstrated. Here we have investigated the self-associating properties of full-length TIA-1 in the presence of designed and native TIA-1 nucleic acid binding sites in vitro, monitoring phase separation, fibril formation and shape. We show that single stranded RNA and DNA induce liquid-liquid phase separation of TIA-1 in a multisite, sequence-specific manner and also efficiently promote formation of amyloid-like fibrils. Although RNA binding to a single site induces a small conformational change in TIA-1, this alone does not enhance phase separation of TIA-1. Tandem binding sites are required to enhance phase separation of TIA-1 and this is finely tuned by the protein:binding site stoichiometry rather than nucleic acid length. Native tandem TIA-1 binding sites within the 3' UTR of p53 mRNA also efficiently enhance phase separation of TIA-1 and thus may potentially act as potent nucleation sites for stress granule assembly.
Assuntos
RNA/metabolismo , Antígeno-1 Intracelular de Células T/química , Regiões 3' não Traduzidas , Amiloide/ultraestrutura , Sítios de Ligação , DNA/química , DNA/metabolismo , Humanos , Modelos Moleculares , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Conformação Proteica , RNA/química , Antígeno-1 Intracelular de Células T/metabolismo , Antígeno-1 Intracelular de Células T/ultraestrutura , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Ovarian-derived inhibin A and inhibin B (heterodimers of common α- and differing ß-subunits) are secreted throughout the menstrual cycle in a discordant pattern, with smaller follicles producing inhibin B, whereas the dominant follicle and corpus luteum produce inhibin A. The classical function for endocrine inhibins is to block signalling by activins (homodimers of ß-subunits) in gonadotrope cells of the anterior pituitary and, thereby, inhibit the synthesis of FSH. Whether inhibin A and inhibin B have additional physiological functions is unknown, primarily because producing sufficient quantities of purified inhibins, in the absence of contaminating activins, for preclinical studies has proven extremely difficult. Here, we describe novel methodology to enhance inhibin A and inhibin B activity and to produce these ligands free of contaminating activins. Using computational modeling and targeted mutagenesis, we identified a point mutation in the activin ßâA-subunit, A347H, which completely disrupted activin dimerization and activity. Importantly, this ßâA-subunit mutation had minimal effect on inhibin A bioactivity. Mutation of the corresponding residue in the inhibin ßâB-subunit, G329E, similarly disrupted activin B synthesis/activity without affecting inhibin B production. Subsequently, we enhanced inhibin A potency by modifying the binding site for its co-receptor, betaglycan. Introducing a point mutation into the α-subunit (S344I) increased inhibin A potency ~12-fold. This study has identified a means to eliminate activin A/B interference during inhibin A/B production, and has facilitated the generation of potent inhibin A and inhibin B agonists for physiological exploration.
Assuntos
Inibinas , Engenharia de Proteínas/métodos , Feminino , Células HEK293 , Humanos , Inibinas/genética , Inibinas/isolamento & purificação , Inibinas/metabolismo , Inibinas/farmacologia , Proteínas de Membrana , Modelos Moleculares , Mutagênese/fisiologia , Ovário/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Multimerização Proteica/genética , Estrutura Quaternária de Proteína/genética , Estrutura Terciária de Proteína/genética , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Subunidades Proteicas/farmacologia , Proteínas de Saccharomyces cerevisiae , TransfecçãoRESUMO
The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem-loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral proteinase 3CD, translation initiation ceases allowing the next stage of replication to commence. Here, we investigate the interaction of PCBP2 with the apical region of stem-loop IV (SLIVm) of poliovirus RNA in its full-length and truncated form. CryoEM structure reconstruction of the full-length PCBP2 in complex with SLIVm solved to 6.1 Å resolution reveals a compact globular complex of PCBP2 interacting with the cruciform RNA via KH domains and featuring a prominent GNRA tetraloop. SEC-SAXS, SHAPE and hydroxyl-radical cleavage establish that PCBP2 stabilizes the SLIVm structure, but upon cleavage in the linker domain the complex becomes more flexible and base accessible. Limited proteolysis and REMSA demonstrate the accessibility of the linker region in the PCBP2/SLIVm complex and consequent loss of affinity of PCBP2 for the SLIVm upon cleavage. Together this study sheds light on the structural features of the PCBP2/SLIV complex vital for ribosomal docking, and the way in which this key functional interaction is regulated following translation of the poliovirus genome.
Assuntos
Iniciação Traducional da Cadeia Peptídica , Poliovirus/genética , RNA Viral/química , Proteínas de Ligação a RNA/química , Microscopia Crioeletrônica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Here, we report the design, synthesis, and evaluation of a series of inhibitors of Staphylococcus aureus BPL (SaBPL), where the central acyl phosphate of the natural intermediate biotinyl-5'-AMP (1) is replaced by a sulfonamide isostere. Acylsulfamide (6) and amino sulfonylurea (7) showed potent in vitro inhibitory activity (Ki = 0.007 ± 0.003 and 0.065 ± 0.03 µM, respectively) and antibacterial activity against S. aureus ATCC49775 with minimum inhibitory concentrations of 0.25 and 4 µg/mL, respectively. Additionally, the bimolecular interactions between the BPL and inhibitors 6 and 7 were defined by X-ray crystallography and molecular dynamics simulations. The high acidity of the sulfonamide linkers of 6 and 7 likely contributes to the enhanced in vitro inhibitory activities by promoting interaction with SaBPL Lys187. Analogues with alkylsulfamide (8), ß-ketosulfonamide (9), and ß-hydroxysulfonamide (10) isosteres were devoid of significant activity. Binding free energy estimation using computational methods suggests deprotonated 6 and 7 to be the best binders, which is consistent with enzyme assay results. Compound 6 was unstable in whole blood, leading to poor pharmacokinetics. Importantly, 7 has a vastly improved pharmacokinetic profile compared to that of 6 presumably due to the enhanced metabolic stability of the sulfonamide linker moiety.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Carbono-Nitrogênio Ligases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Sulfonamidas/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/farmacocinética , Proteínas de Bactérias/química , Carbono-Nitrogênio Ligases/química , Cristalografia por Raios X , Desenho de Fármacos , Estabilidade de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Camundongos , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Ratos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Sulfonamidas/síntese química , Sulfonamidas/farmacocinéticaRESUMO
Macrophage migration inhibitory factor (MIF) exerts multiple effects on immune cells, as well as having functions outside the immune system. MIF can promote inflammation through the induction of other cytokines, including TNF, IL-6, and IL-1 family cytokines. Here, we show that inhibition of MIF regulates the release of IL-1α, IL-1ß, and IL-18, not by affecting transcription or translation of these cytokines, but via activation of the NLRP3 inflammasome. MIF is required for the interaction between NLRP3 and the intermediate filament protein vimentin, which is critical for NLRP3 activation. Further, we demonstrate that MIF interacts with NLRP3, indicating a role for MIF in inflammasome activation independent of its role as a cytokine. These data advance our understanding of how MIF regulates inflammation and identify it as a factor critical for NLRP3 inflammasome activation.
Assuntos
Inflamassomos/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Células Cultivadas , Citocinas/metabolismo , Feminino , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Fatores Inibidores da Migração de Macrófagos/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Células THP-1RESUMO
RIG-I (retinoic acid inducible gene-I) is a cytosolic innate immune protein that senses viral dsRNA with a 5'-triphosphate overhang. Upon interaction with dsRNA a de-repression of the RIG-I CARD domains takes place that ultimately leads to the production of type I interferons and pro-inflammatory cytokines. Here we investigate the RIG-I conformational rearrangement upon interaction with an activating 5'-triphosphate-10-base pair dsRNA hairpin loop (10bp) compared with a less active 5'-triphosphate-8-base pair dsRNA hairpin loop (8bp). We use size-exclusion chromatography-coupled small-angle X-ray scattering (SAXS) and limited tryptic digest experiments to show that that upon binding to 10 bp, but not 8 bp, RIG-I becomes extended and shows greater flexibility, reflecting the release of its CARDs. We also examined the effect of different ATP analogues on the conformational changes of RIG-I/dsRNA complexes. Of the analogues tested, the addition of ATP transition state analogue ADP-AlFx further assisted in the complete activation of RIG-I in complex with 10bp and also to some extent RIG-I bound to 8bp. Together these data provide solution-based evidence for the molecular mechanism of innate immune signaling by RIG-I as stimulated by short hairpin RNA and ATP.
Assuntos
Trifosfato de Adenosina/química , Proteína DEAD-box 58/química , Domínios Proteicos , RNA de Cadeia Dupla/química , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Cromatografia em Gel , Proteína DEAD-box 58/metabolismo , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Receptores Imunológicos , Espalhamento a Baixo Ângulo , Transdução de Sinais , Soluções/química , Difração de Raios XRESUMO
We report the synthesis and evaluation of 5-halogenated-1,2,3-triazoles as inhibitors of biotin protein ligase from Staphylococcus aureus. The halogenated compounds exhibit significantly improved antibacterial activity over their nonhalogenated counterparts. Importantly, the 5-fluoro-1,2,3-triazole compound 4c displays antibacterial activity against S. aureus ATCC49775 with a minimum inhibitory concentration (MIC) of 8 µg/mL.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Ligases/antagonistas & inibidores , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Antibacterianos/química , Sítios de Ligação , Biotina/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Halogenação , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação ProteicaRESUMO
Grb7 is a signaling protein with critical roles in tumor cell proliferation and migration and an established cancer therapeutic target. Here we explore chemical space to develop a new bicyclic peptide inhibitor, incorporating thioether and lactam linkers that binds with affinity (KD = 1.1 µM) and specificity to the Grb7-SH2 domain. Structural analysis of the Grb7-SH2/peptide complex revealed an unexpected binding orientation underlying the binding selectivity by this new scaffold. We further incorporated carboxymethylphenylalanine and carboxyphenylalanine phosphotyrosine mimetics and arrived at an optimized inhibitor that potently binds Grb7-SH2 (KD = 0.13 µM) under physiological conditions. X-ray crystal structures of these Grb7-SH2/peptide complexes reveal the structural basis for the most potent and specific inhibitors of Grb7 developed to date. Finally, we demonstrate that cell permeable versions of these peptides successfully block Grb7 mediated interactions in a breast cancer cell line, establishing the potential of these peptides in the development of novel therapeutics targeted to Grb7.
Assuntos
Proteína Adaptadora GRB7/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Descoberta de Drogas , Quinase 1 de Adesão Focal/metabolismo , Proteína Adaptadora GRB7/química , Proteína Adaptadora GRB7/metabolismo , Humanos , Lactamas/síntese química , Lactamas/química , Lactamas/farmacologia , Ligantes , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Fosfatos/química , Conformação Proteica , Receptor ErbB-2/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Domínios de Homologia de srcRESUMO
TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5Î-UUUUUACUCC-3Î). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression.
Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Ligação a Poli(A)/química , Ribonucleosídeo Difosfato Redutase/química , Cristalografia por Raios X , DNA/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Humanos , Oligonucleotídeos/química , Proteínas de Ligação a Poli(A)/genética , Ligação Proteica/genética , Mapas de Interação de Proteínas/genética , Motivo de Reconhecimento de RNA/genética , Ribonucleosídeo Difosfato Redutase/genética , Antígeno-1 Intracelular de Células TRESUMO
The design of potent and specific peptide inhibitors to therapeutic targets is of enormous utility for both proof-of-concept studies and for the development of potential new therapeutics. Grb7 is a key signaling molecule in the progression of HER2 positive and triple negative breast cancers. Here we report the crystal structure of a stapled bicyclic peptide inhibitor G7-B1 in complex with the Grb7-SH2 domain. This revealed an unexpected binding mode of the peptide, in which the staple forms an alternative contact with the surface of the target protein. Based on this structural information, we designed a new series of bicyclic G7 peptides that progressively constrain the starting peptide, to arrive at the G7-B4 peptide that binds with an approximately 2-fold enhanced affinity to the Grb7-SH2 domain (KD = 0.83 µM) compared to G7-B1 and shows low affinity binding to Grb2-, Grb10- and Grb14-SH2 domains (KD > 100 µM). Furthermore, we determined the structure of the G7-B4 bicyclic peptide in complex with the Grb7-SH2 domain, both before and after ring closing metathesis to show that the closed staple is essential to the target interaction. The G7-B4 peptide represents an advance in the development of Grb7 inhibitors and is a classical example of structure aided inhibitor development.
Assuntos
Proteína Adaptadora GRB7/química , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Cristalografia por Raios X , Desenho de Fármacos , Proteína Adaptadora GRB7/antagonistas & inibidores , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de ProteínaRESUMO
The Grb7 adaptor protein is a therapeutic target for both TNBC and HER2+ breast cancer. A nonphosphorylated cyclic peptide 1 (known as G7-18NATE) inhibits Grb7 via targeting the Grb7-SH2 domain, but requires the presence of phosphate ions for both affinity and specificity. Here we report the discovery of malonate bound in the phosphotyrosine binding pocket of the apo-Grb7-SH2 structure. Based on this, we carried out the rational design and synthesis of two analogues of peptide 1 that incorporate carboxymethylphenylalanine (cmF) and carboxyphenylalanine (cF) as mimics of phosphotyrosine (pY). Binding studies using SPR confirmed that affinity for Grb7-SH2 domain is improved up to 9-fold over peptide 1 under physiological phosphate conditions (KD = 2.1-5.7 µM) and that binding is specific for Grb7-SH2 over closely related domains (low or no detectable binding to Grb2-SH2 and Grb10-SH2). X-ray crystallographic structural analysis of the analogue bearing a cmF moiety in complex with Grb7-SH2 has identified the precise contacts conferred by the pY mimic that underpin this improved molecular interaction. Together this study identifies and characterizes the tightest specific inhibitor of Grb7 to date, representing a significant development toward a new Grb7-targeted therapeutic.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Proteína Adaptadora GRB7/antagonistas & inibidores , Peptídeos Cíclicos/química , Fosfotirosina/química , Antineoplásicos/síntese química , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Cristalografia por Raios X , Feminino , Proteína Adaptadora GRB7/metabolismo , Humanos , Malonatos/química , Terapia de Alvo Molecular , Peptídeos Cíclicos/síntese química , Peptidomiméticos , Fosfatos/química , Fosfatos/metabolismo , Conformação Proteica , Domínios de Homologia de srcRESUMO
The biogenesis of membranes from constituent proteins and lipids is a fundamental aspect of cell biology. In the case of proteins assembled into bacterial outer membranes, an overarching question concerns how the energy required for protein insertion and folding is accessed at this remote location of the cell. The translocation and assembly module (TAM) is a nanomachine that functions in outer membrane biogenesis and virulence in diverse bacterial pathogens. Here we demonstrate the interactions through which TamA and TamB subunits dock to bridge the periplasm, and unite the outer membrane aspects to the inner membrane of the bacterial cell. We show that specific functional features in TamA have been conserved through evolution, including residues surrounding the lateral gate and an extensive surface of the POTRA domains. Analysis by nuclear magnetic resonance spectroscopy and small angle X-ray scattering document the characteristic structural features of these POTRA domains and demonstrate rigidity in solution. Quartz crystal microbalance measurements pinpoint which POTRA domain specifically docks the TamB subunit of the nanomachine. We speculate that the POTRA domain of TamA functions as a lever arm in order to drive the activity of the TAM, assembling proteins into bacterial outer membranes.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/fisiologia , Sequência Conservada , Evolução Molecular , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
Both RNA-binding proteins (RBP) and miRNA play important roles in the regulation of mRNA expression, often acting together to regulate a target mRNA. In some cases the RBP and miRNA have been reported to act competitively, but in other instances they function cooperatively. Here, we investigated HuR function as an enhancer of let-7-mediated translational repression of c-Myc despite the separation of their binding sites. Using an in vitro system, we determined that a let-7 mimic, consisting of single-stranded (ss)DNA complementary to the let-7 binding site, enhanced the affinity of HuR for a 122-nt MYC RNA encompassing both binding sites. This finding supports the biophysical principle of cooperative binding by an RBP and miRNA purely through interactions at distal mRNA binding sites.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , MicroRNAs/metabolismo , Mimetismo Molecular/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Semelhante a ELAV 1/química , Humanos , MicroRNAs/química , Ligação Proteica/fisiologia , Proteínas Proto-Oncogênicas c-myc/químicaRESUMO
Phanta is a reversibly photoswitching chromoprotein (ΦF, 0.003), useful for pcFRET, that was isolated from a mutagenesis screen of the bright green fluorescent eCGP123 (ΦF, 0.8). We have investigated the contribution of substitutions at positions His193, Thr69 and Gln62, individually and in combination, to the optical properties of Phanta. Single amino acid substitutions at position 193 resulted in proteins with very low ΦF, indicating the importance of this position in controlling the fluorescence efficiency of the variant proteins. The substitution Thr69Val in Phanta was important for supressing the formation of a protonated chromophore species observed in some His193 substituted variants, whereas the substitution Gln62Met did not significantly contribute to the useful optical properties of Phanta. X-ray crystal structures for Phanta (2.3 Å), eCGP123T69V (2.0 Å) and eCGP123H193Q (2.2 Å) in their non-photoswitched state were determined, revealing the presence of a cis-coplanar chromophore. We conclude that changes in the hydrogen-bonding network supporting the cis-chromophore, and its contacts with the surrounding protein matrix, are responsible for the low fluorescence emission of eCGP123 variants containing a His193 substitution.
Assuntos
Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Substituição de Aminoácidos , Cristalografia por Raios X , Escherichia coli/genética , Fluorescência , Corantes Fluorescentes/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Processos Fotoquímicos , Conformação ProteicaRESUMO
The engagement of natural killer cell immunoglobulin-like receptors (KIRs) with their target ligands, human leukocyte antigen (HLA) molecules, is a critical component of innate immunity. Structurally, KIRs typically have either two (D1-D2) or three (D0-D1-D2) extracellular immunoglobulin domains, with the D1 and D2 domain recognizing the α1 and α2 helices of HLA, respectively, whereas the D0 domain of the KIR3DLs binds a loop region flanking the α1 helix of the HLA molecule. KIR2DL4 is distinct from other KIRs (except KIR2DL5) in that it does not contain a D1 domain and instead has a D0-D2 arrangement. Functionally, KIR2DL4 is also atypical in that, unlike all other KIRs, KIR2DL4 has both activating and inhibitory signaling domains. Here, we determined the 2.8 Å crystal structure of the extracellular domains of KIR2DL4. Structurally, KIR2DL4 is reminiscent of other KIR2DL receptors, with the D0 and D2 adopting the C2-type immunoglobulin fold arranged with an acute elbow angle. However, KIR2DL4 self-associated via the D0 domain in a concentration-dependent manner and was observed as a tetramer in the crystal lattice by size exclusion chromatography, dynamic light scattering, analytical ultracentrifugation, and small angle x-ray scattering experiments. The assignment of residues in the D0 domain to forming the KIR2DL4 tetramer precludes an interaction with HLA akin to that observed for KIR3DL1. Accordingly, no interaction was observed to HLA by direct binding studies. Our data suggest that the unique functional properties of KIR2DL4 may be mediated by self-association of the receptor.