RESUMO
Voltage-gated sodium (NaV) and calcium channels (CaV) form targets for calmodulin (CaM), which affects channel inactivation properties. A major interaction site for CaM resides in the C-terminal (CT) region, consisting of an IQ domain downstream of an EF-hand domain. We present a crystal structure of fully Ca2+-occupied CaM, bound to the CT of NaV1.5. The structure shows that the C-terminal lobe binds to a site â¼90° rotated relative to a previous site reported for an apoCaM complex with the NaV1.5 CT and for ternary complexes containing fibroblast growth factor homologous factors (FHF). We show that the binding of FHFs forces the EF-hand domain in a conformation that does not allow binding of the Ca2+-occupied C-lobe of CaM. These observations highlight the central role of the EF-hand domain in modulating the binding mode of CaM. The binding sites for Ca2+-free and Ca2+-occupied CaM contain targets for mutations linked to long-QT syndrome, a type of inherited arrhythmia. The related NaV1.4 channel has been shown to undergo Ca2+-dependent inactivation (CDI) akin to CaVs. We present a crystal structure of Ca2+/CaM bound to the NaV1.4 IQ domain, which shows a binding mode that would clash with the EF-hand domain. We postulate the relative reorientation of the EF-hand domain and the IQ domain as a possible conformational switch that underlies CDI.
Assuntos
Cálcio/química , Calmodulina/química , Canal de Sódio Disparado por Voltagem NAV1.5/química , Sítios de Ligação , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Cristalografia , Motivos EF Hand , Humanos , Síndrome do QT Longo , Modelos Moleculares , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação ProteicaRESUMO
The allosteric interplay between distant functional sites present in a single protein provides for one of the most important regulatory mechanisms in biological systems. While the design of ligand-binding sites into proteins remains challenging, this holds even truer for the coupling of a newly engineered binding site to an allosteric mechanism that regulates the ligand affinity. Here it is shown how computational design algorithms enabled the introduction of doxycycline- and doxorubicin-binding sites into the serine proteinase inhibitor (serpin) family member α1-antichymotrypsin. Further engineering allowed exploitation of the proteinase-triggered serpin-typical S-to-R transition to modulate the ligand affinities. These design variants follow strategies observed in naturally occurring plasma globulins that allow for the targeted delivery of hormones in the blood. By analogy, we propose that the variants described in the present study could be further developed to allow for the delivery of the antibiotic doxycycline and the anticancer compound doxorubicin to tissues/locations that express specific proteinases, such as bacterial infection sites or tumor cells secreting matrix metalloproteinases.
Assuntos
Doxorrubicina/metabolismo , Doxiciclina/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes , Sítio Alostérico/genética , Doxorrubicina/química , Doxiciclina/química , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Humanos , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , alfa 1-Antiquimotripsina/química , alfa 1-Antiquimotripsina/genética , alfa 1-Antiquimotripsina/metabolismoRESUMO
The human gut microbiota, which underpins nutrition and systemic health, is compositionally sensitive to the availability of complex carbohydrates in the diet. The Bacteroidetes comprise a dominant phylum in the human gut microbiota whose members thrive on dietary and endogenous glycans by employing a diversity of highly specific, multi-gene polysaccharide utilization loci (PUL), which encode a variety of carbohydrases, transporters, and sensor/regulators. PULs invariably also encode surface glycan-binding proteins (SGBPs) that play a central role in saccharide capture at the outer membrane. Here, we present combined biophysical, structural, and in vivo characterization of the two SGBPs encoded by the Bacteroides ovatus mixed-linkage ß-glucan utilization locus (MLGUL), thereby elucidating their key roles in the metabolism of this ubiquitous dietary cereal polysaccharide. In particular, molecular insight gained through several crystallographic complexes of SGBP-A and SGBP-B with oligosaccharides reveals that unique shape complementarity of binding platforms underpins specificity for the kinked MLG backbone vis-à-vis linear ß-glucans. Reverse-genetic analysis revealed that both the presence and binding ability of the SusD homolog BoSGBPMLG-A are essential for growth on MLG, whereas the divergent, multi-domain BoSGBPMLG-B is dispensable but may assist in oligosaccharide scavenging from the environment. The synthesis of these data illuminates the critical role SGBPs play in concert with other MLGUL components, reveals new structure-function relationships among SGBPs, and provides fundamental knowledge to inform future (meta)genomic, biochemical, and microbiological analyses of the human gut microbiota.
Assuntos
Bacteroides/fisiologia , Grão Comestível/metabolismo , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Proteínas de Membrana/fisiologia , Polissacarídeos/metabolismo , beta-Glucanas/metabolismo , Bacteroides/genética , Bacteroides/metabolismo , Metabolismo dos Carboidratos/fisiologia , Sequência de Carboidratos , Fibras na Dieta/metabolismo , Microbioma Gastrointestinal/fisiologia , Regulação Bacteriana da Expressão Gênica , Loci Gênicos , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Humanos , Proteínas de Membrana/metabolismoRESUMO
The introduction of ligand-binding sites into proteins and the engineering of molecular allosteric coupling pathways are topical issues in protein design. Here, we show that these issues can be addressed concurrently, using the serpin human α1-antichymotrypsin (ACT) as a model. We have introduced up to 15 amino acid substitutions into ACT, converting it into a surrogate corticosteroid-binding globulin (CBG), thereby creating a new binding globulin (NewBG). Human CBG and ACT share 46% sequence identity, and CBG served as the blue-print for our design, which was guided by side-chain-packing calculations, ITC measurements and crystal structure determinations. Upon transfer of ligand-interacting residues from CBG to ACT and mutation of specific second shell residues, a NewBG variant was obtained, which binds cortisol with 1.5⯵M affinity. This novel serpin (NewBG-III) binds cortisol with a 33-fold lower affinity than CBG, but shares a similar ligand-binding profile and binding mode when probed with different steroid ligands and site-directed mutagenesis. An additional substitution, i.e. A349R, created NewBG-III-allo, which introduced an allosteric coupling between ligand binding and the serpin-like S-to-R transition in ACT. In NewBG-III-allo, the proteinase-triggered S-to-R transition leads to a greater than 200-fold reduction in ligand affinity, and crystal structures suggest that this is mediated by the L55V and A349R substitutions. This reduction significantly exceeds the 10-fold reduction in binding affinity observed in human CBG.
Assuntos
Complexos Multiproteicos/química , Engenharia de Proteínas , Transcortina/química , alfa 1-Antiquimotripsina/química , Substituição de Aminoácidos/genética , Sítios de Ligação/genética , Cristalografia por Raios X , Humanos , Hidrocortisona/química , Ligantes , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Mutação/genética , Ligação Proteica/genética , Conformação Proteica , Homologia de Sequência de Aminoácidos , Transcortina/genética , Transcortina/ultraestrutura , alfa 1-Antiquimotripsina/genética , alfa 1-Antiquimotripsina/ultraestruturaRESUMO
The anti-inflammatory actions of interleukin-10 (IL10) are thought to be mediated primarily by the STAT3 transcription factor, but pro-inflammatory cytokines such as interleukin-6 (IL6) also act through STAT3. We now report that IL10, but not IL6 signaling, induces formation of a complex between STAT3 and the inositol polyphosphate-5-phosphatase SHIP1 in macrophages. Both SHIP1 and STAT3 translocate to the nucleus in macrophages. Remarkably, sesquiterpenes of the Pelorol family, which we previously described as allosteric activators of SHIP1 phosphatase activity, could induce SHIP1/STAT3 complex formation in cells and mimic the anti-inflammatory action of IL10 in a mouse model of colitis. Using crystallography and docking studies we identified a drug-binding pocket in SHIP1. Our studies reveal new mechanisms of action for both STAT3 and SHIP1 and provide a rationale for use of allosteric SHIP1-activating compounds, which mimic the beneficial anti-inflammatory actions of IL10. VIDEO ABSTRACT.
RESUMO
Voltage-gated sodium channels (NaV) are responsible for the rapid depolarization of many excitable cells. They readily inactivate, a process where currents diminish after milliseconds of channel opening. They are also targets for a multitude of disease-causing mutations, many of which have been shown to affect inactivation. A cluster of disease mutations, linked to Long-QT and Brugada syndromes, is located in a C-terminal EF-hand like domain of NaV1.5, the predominant cardiac sodium channel isoform. Previous studies have suggested interactions with the III-IV linker, a cytosolic element directly involved in inactivation. Here we validate and map the interaction interface using isothermal titration calorimetry (ITC) and NMR spectroscopy. We investigated the impact of various disease mutations on the stability of the domain, and found that mutations that cause misfolding of the EF-hand domain result in hyperpolarizing shifts in the steady-state inactivation curve. Conversely, mutations in the III-IV linker that disrupt the interaction with the EF-hand domain also result in large hyperpolarization shifts, supporting the interaction between both elements in intact channels. Disrupting the interaction also causes large late currents, pointing to a dual role of the interaction in reducing the population of channels entering inactivation and in stabilizing the inactivated state.
Assuntos
Suscetibilidade a Doenças , Motivos EF Hand , Mutação , Canais de Sódio Disparados por Voltagem/genética , Canais de Sódio Disparados por Voltagem/metabolismo , Sítios de Ligação , Humanos , Ativação do Canal Iônico , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Relação Estrutura-Atividade , Canais de Sódio Disparados por Voltagem/químicaRESUMO
Corticosteroid-binding globulin (CBG) transports glucocorticoids and progesterone in the blood and thereby modulates the tissue availability of these hormones. As a member of the serine protease inhibitor (SERPIN) family, CBG displays a reactive center loop (RCL) that is targeted by proteinases. Cleavage of the RCL is thought to trigger a SERPIN-typical stressed-to-relaxed (S-to-R) transition that leads to marked structural rearrangements and a reduced steroid-binding affinity. To characterize structure-function relationships in CBG we studied various conformational states of E. coli-produced rat and human CBG. In the 2.5 Å crystal structure of human CBG in complex with progesterone, the RCL is cleaved at a novel site that differs from the known human neutrophil elastase recognition site. Although the cleaved RCL segment is five residues longer than anticipated, it becomes an integral part of ß-sheet A as a result of the S-to-R transition. The atomic interactions observed between progesterone and CBG explain the lower affinity of progesterone in comparison to corticosteroids. Surprisingly, CD measurements in combination with thermal unfolding experiments show that rat CBG fails to undergo an S-to-R transition upon proteolytic cleavage of the RCL hinting that the S-to-R transition observed in human CBG is not a prerequisite for CBG function in rat. This observation cautions against drawing general conclusions about molecular mechanisms by comparing and merging structural data from different species.
Assuntos
Transcortina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Progesterona/química , Ligação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteólise , Ratos , Especificidade da Espécie , Homologia Estrutural de ProteínaRESUMO
CONTEXT: Cortisol is transported by corticosteroid-binding globulin (CBG) in blood. Single nucleotide polymorphisms (SNP) in the human CBG (SERPINA6) gene that disrupt CBG production or steroid binding are considered rare. OBJECTIVE: The objective of the study was to identify and determine the frequency of SNP in SERPINA6 that influence the production or cortisol-binding properties of CBG in Chinese subjects. PARTICIPANTS AND DESIGN: Blood samples from 2287 anonymous Chinese workers undergoing routine health tests were screened for the SERPINA6 coding sequence polymorphisms. MAIN OUTCOME MEASURES AND RESULTS: In a pilot study of 108 Chinese women, two nonsynonymous SNP were identified within SERPINA6 exon 2 encoding CBG A51V (n = 3) and CBG E102G (n = 1) variants. Sequence analysis of SERPINA6 exon 2 in a further 137 Chinese women revealed two other individuals with nonsynonymous SNP encoding CBGs R64Q and R64W as well as another CBG A51V carrier. The surprisingly high frequency of heterozygous CBG A51V carriers was confirmed in 1011 Chinese men (1:35) and 1031 other women (1:37). Individuals homozygous for these SNP were not identified. When expressed in Chinese hamster ovary cells, CBG A51V bound steroid normally, but its production/secretion was severely impaired; CBG E102G was produced normally, but its cortisol-binding capacity was abnormally low, whereas CBG R64Q and R64W were produced and bound cortisol normally. CONCLUSIONS: Defects in CBG A51V production explain why plasma CBG levels in individuals heterozygous for this variant are approximately 50% lower than normal. The high frequency of CBG A51V will allow clinical consequences of CBG deficiencies to be assessed for the first time in large patient populations.
Assuntos
Hidrocortisona/metabolismo , Polimorfismo de Nucleotídeo Único , Transcortina/análise , Adulto , Substituição de Aminoácidos , Animais , Células CHO , China , Estudos de Coortes , Cricetinae , Cricetulus , Éxons , Feminino , Estudos de Associação Genética , Heterozigoto , Humanos , Masculino , Modelos Moleculares , Projetos Piloto , Conformação Proteica , Proteínas Recombinantes/metabolismo , Transcortina/química , Transcortina/genética , Transcortina/metabolismoRESUMO
Corticosteroid-binding globulin (CBG) is a non-inhibitory serine proteinase inhibitor (serpin) that transports cortisol and progesterone in blood. Crystal structures of rat CBG and a thrombin-cleaved human CBG:anti-trypsin (Pittsburgh) chimera show how structural transitions after proteolytic cleavage of the CBG reactive center loop (RCL) could disrupt steroid binding. This ligand release mechanism is assumed to involve insertion of the cleaved RCL into the beta-sheet A of the serpin structure. We have, therefore, examined how amino acid substitutions in the human CBG RCL influence steroid binding before and after its cleavage by neutrophil elastase. Elastase-cleaved wild-type CBG or variants with substitutions at P15 and/or P16 (E334G/G335N or E334A) lost steroid binding completely, whereas deletion of Glu-334 resulted in no loss of steroid binding after RCL cleavage, presumably because this prevents its insertion into beta-sheet A. Similarly, the steroid binding properties of CBG variants with substitutions at P15 (G335P), P14 (V336R), or P12 (T338P) in the RCL hinge were largely unaffected after elastase cleavage, most likely because the re-orientation and/or insertion of the cleaved RCL was blocked. Substitutions at P10 (G340P, G340S) or P8 (T342P, T342N) resulted in a partial loss of steroid binding after proteolysis which we attribute to incomplete insertion of the cleaved RCL. Remarkably, several substitutions (E334A, V336R, G340S, and T342P) increased the steroid binding affinities of human CBG even before elastase cleavage, consistent with the concept that CBG normally toggles between a high affinity ligand binding state where the RCL is fully exposed and a lower affinity state in which the RCL is partly inserted into beta-sheet A.