RESUMO
Antibodies that target the glycan cap epitope on the ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization are not well understood. Here, we present cryoelectron microscopy (cryo-EM) structures of diverse glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLDs) to the glycan cap, which we call the MLD anchor and cradle. Antibodies that bind to the MLD cradle share common features, including use of IGHV1-69 and IGHJ6 germline genes, which exploit hydrophobic residues and form ß-hairpin structures to mimic the MLD anchor, disrupt MLD attachment, destabilize GP quaternary structure, and block cleavage events required for receptor binding. Our results provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/tratamento farmacológico , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Especificidade de Anticorpos , Sítios de Ligação , Microscopia Crioeletrônica , Ebolavirus/crescimento & desenvolvimento , Ebolavirus/imunologia , Ebolavirus/patogenicidade , Epitopos/química , Epitopos/imunologia , Feminino , Células HEK293 , Células HeLa , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/patologia , Doença pelo Vírus Ebola/virologia , Humanos , Células Jurkat , Camundongos , Modelos Moleculares , Polissacarídeos/química , Polissacarídeos/imunologia , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIß is structurally unique in that the type IIß holoenzyme is much more compact than the free RIIß homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIß C-terminal deletion mutant (RIIß(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIß holoenzyme and the RIIß domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIß holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIß homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIß isoform of PKA.
Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/química , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/química , AMP Cíclico/química , Holoenzimas/química , Animais , Domínio Catalítico , AMP Cíclico/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Holoenzimas/genética , Holoenzimas/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Modelos Moleculares , Mutação , Difração de Nêutrons , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The gastrointestinal peptide hormone gastrin is responsible for initiating the release of gastric acid in the stomach in response to the presence of food and/or humoral factors such as gastrin releasing peptide. However, it has a role in the growth and maintenance of the gastric epithelium, and has been implicated in the formation and growth of gastric cancers. Hypergastrinemia resulting from atrophic gastritis and pernicious anemia leads to hyperplasia and carcinoid formation in rats, and contributes to tumor formation in humans. Additionally, gastrin has been suspected to play a role in the formation and growth of cancers of the colon, but recent studies have instead implicated gastrin processing intermediates, such as gastrin-17-Gly, acting upon a putative, non-cholecystokinin receptor. This review summarizes the production and chemical structures of gastrin and of the processing intermediate gastrin-17-Gly, as well as their activities in the gastrointestinal tract, particularly the promotion of colon cancers.
Assuntos
Gastrinas/biossíntese , Neoplasias Gastrointestinais/metabolismo , Neoplasias Gastrointestinais/patologia , Animais , Colecistocinina/metabolismo , Ácido Gástrico/metabolismo , Gastrite Atrófica/metabolismo , Gastrite Atrófica/patologia , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/patologia , Humanos , Ratos , Receptor de Colecistocinina B/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Gastrin-17-Gly (G17-Gly) has been shown to bind to non-CCK nanomolar and micromolar affinity sites on DLD-1 and HT-29 human colonic carcinoma cells and to stimulate cellular proliferation. However, in previous studies, we showed that C-terminal truncation of the gastrin-17 (G17) to the G17 analog G17(1-12) and then to G17(1-6)-NH(2) did not remove the ability to bind to DLD-1 cells or to activate proliferation. This implies that residues and/or structural motifs required for bioactivity at these receptors rest in the N-terminal region of G17. In this work, radioligand binding studies conducted with further C-terminally truncated analogs revealed that sequences as short as G17(1-4) still bind to a single receptor with micromolar affinity. Additionally, cell proliferation assays showed that G17(1-12) stimulates proliferation of DLD-1 cells, as of HT-29 cells, but the sequences shorter than G17(1-6)-NH(2), including non-amidated G17(1-6), were incapable of stimulating proliferation. These observations indicate that the tetrapeptide pGlu-Gly-Pro-Trp is the minimum N-terminal sequence for binding to the probable growth-promoting site on DLD-1 cells. Since analogs shorter than G17(1-6) are able to bind the receptor, these peptides may be of use for developing selective antagonists.
Assuntos
Gastrinas/química , Peptídeos/química , Peptídeos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Peptídeos/síntese química , Relação Estrutura-AtividadeRESUMO
Gastrin-17 (G17) processing intermediates bind to non-CCK receptors which mediate growth of the colonic mucosa but also the formation and development of colonic cancers. In previous studies, we removed the C-terminal region of G17 to form G17(1-12) and considerably shorter C-terminally amidated and non-amidated analogs. Peptides as short as G17(1-4) continued to bind to a single site on DLD-1 human colonic carcinoma cells, while only the G17(1-6)-NH(2) and G17(1-12) peptides retained the ability to activate the receptor and stimulate cell proliferation in vitro. In this report, we studied the structure of these analogs, using a combination of ECD and VCD spectroscopy and replica exchange molecular dynamics (REMD) simulations in water, TFE, and membrane-mimicking environments, in order to determine preferred conformations that may have importance in promoting the biological activities. Mostly random meander structures, punctuated by a beta-turn at residues 1-4, were found in most peptides by REMD simulations. G17(1-3)-NH(2), which cannot form a beta-turn, failed to bind the non-CCK receptor, suggesting the importance of this feature for binding. Additionally, the beta-turn appeared more frequently in longer sequences, possibly explaining the higher affinity of the non-CCK receptor for these peptides seen previously. Finally, C-terminally amidated peptides generally showed greater formation of turn structure than their non-amidated counterparts as shown by ECD spectra, suggesting the importance of peptide length in stabilizing turn structure in N-terminal sequences, and perhaps explaining the ability of G17(1-6)-NH(2) to activate the non-CCK receptor where as the non-amidated G17(1-6) and shorter peptides do not.
Assuntos
Gastrinas/química , Simulação de Dinâmica Molecular , Peptídeos/química , Linhagem Celular Tumoral , Dicroísmo Circular , Humanos , Peptídeos/síntese químicaRESUMO
TC5b is a 20 residue polypeptide notable for its compact tertiary structure, a rarity for a short peptide. This structure is due to the "Trp-cage" motif, an association of aromatic, Pro, and Gly residues. The structure of TC5b has been fully characterized by NMR and electronic circular dichroism (ECD) studies, but has never been studied with vibrational circular dichroism (VCD) spectroscopy, which may reveal finer structure. In this study, we examine the VCD spectra of TC5b to characterize the spectroscopic signature of the peptide and its comprising structural elements. TC5b exhibited a negative-positive-negative triplet which is associated with alpha-helical structure in deuterated solvents but also signs of a polyproline II (PPII) helix in the amide I' region. Detection of this element was complicated by the aforementioned triplet form, as well as by an upfrequency shift in PPII helical elements due to the use of the deuterated organic solvents DMSO-d(6) and TFE-d(1). Nevertheless, while ECD spectra showed only alpha-helical structure for TC5b, VCD spectroscopy revealed a more complex structure which was in agreement with NMR results. VCD spectroscopy also showed a rapid conformational change of the peptide at temperatures above 35 degrees C in D(2)O and in aqueous solvent with greater than 75% DMSO-d(6) content. Molecular dynamics (MD) simulations to investigate this latter effect of DMSO-d(6) on TC5b were conducted in DMSO and 50% (v/v) DMSO in H(2)O. In DMSO unfolding of the peptide was rapid while in 50% (v/v) DMSO in H(2)O the unfolding was more gradual.
Assuntos
Peptídeos/química , Proteínas Recombinantes/química , Dicroísmo Circular , Peptídeos/síntese química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/síntese química , TermodinâmicaRESUMO
Our previous studies have shown that stimulation of proliferation of DLD-1 and HT29 human colonic cancer cells by nanomolar gastrin (G17) and carboxymethyl gastrin (G17Gly) and reversal of growth by micromolar G17 and G17Gly involves binding sites which can neither be CCK1 nor CCK2 receptors; the N terminal fragment, G17(1-12), is sufficient to increase the number of HT-29 cells by binding the higher affinity binding site but is without a suppressing effect through the lower affinity site. In this study with DLD-1 cells, competitive binding using 125I-G17(1-12) showed that G17(1-12) binds both high and low affinity sites, as do G17 and G17Gly. G17(1-6)-NH2, even without the central-to-C-terminal portion of G17, was still able to bind a single site and to promote a dose-dependent increase in cell number at nanomolar concentrations. The results indicate the presence of a non-CCK receptor on human colonic cancer cells which could mediate the tumor-promoting activity of the N-terminal-to-central portion of G17Gly which, unlike G17, is produced by such cells.
Assuntos
Carcinógenos/farmacologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Gastrinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/etiologia , Humanos , Fragmentos de Peptídeos/farmacologia , Ensaio Radioligante , Receptores da Colecistocinina/metabolismoRESUMO
An equimolar mixture of avian pancreatic polypeptide (aPP) fragments aPP(1-11)-NH2 and Ac-aPP(12-36) had an electronic circular dichroism (ECD) spectrum that was similar to that of whole aPP in H2O and even more so in 30% (v/v) trifluoroethanol (TFE) in 15 mM Na2HPO4, but was different from the sum of the spectra of the individual fragments. The vibrational circular dichroism (VCD) spectrum of the combined fragments in 30% (v/v) TFE in 15 mM Na2HPO4 in D2O was also similar to that of the intact aPP and unlike the sum of the VCD spectra of the fragments. The interaction of these fragments is thus sufficient to support the conformation of whole aPP. This study demonstrates that VCD, in combination with ECD, is useful for the study of protein-protein interactions.