RESUMO
De novo design of complex protein folds using solely computational means remains a substantial challenge1. Here we use a robust deep learning pipeline to design complex folds and soluble analogues of integral membrane proteins. Unique membrane topologies, such as those from G-protein-coupled receptors2, are not found in the soluble proteome, and we demonstrate that their structural features can be recapitulated in solution. Biophysical analyses demonstrate the high thermal stability of the designs, and experimental structures show remarkable design accuracy. The soluble analogues were functionalized with native structural motifs, as a proof of concept for bringing membrane protein functions to the soluble proteome, potentially enabling new approaches in drug discovery. In summary, we have designed complex protein topologies and enriched them with functionalities from membrane proteins, with high experimental success rates, leading to a de facto expansion of the functional soluble fold space.
Assuntos
Desenho Assistido por Computador , Aprendizado Profundo , Proteínas de Membrana , Dobramento de Proteína , Solubilidade , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Estabilidade Proteica , Proteoma/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos , Estudo de Prova de ConceitoRESUMO
The bacterium Clostridium perfringens causes severe, sometimes lethal gastrointestinal disorders in humans, including enteritis and enterotoxemia. Type F strains produce an enterotoxin (CpE) that causes the third most common foodborne illness in the United States. CpE induces gut breakdown by disrupting barriers at cell-cell contacts called tight junctions (TJs), which are formed and maintained by claudins. Targeted binding of CpE to specific claudins, encoded by its C-terminal domain (cCpE), loosens TJ barriers to trigger molecular leaks between cells. Cytotoxicity results from claudin-bound CpE complexes forming pores in cell membranes. In mammalian tissues, â¼24 claudins govern TJ barriers-but the basis for CpE's selective targeting of claudins in the gut was undetermined. We report the structure of human claudin-4 in complex with cCpE, which reveals that enterotoxin targets a motif conserved in receptive claudins and how the motif imparts high-affinity CpE binding to these but not other subtypes. The structural basis of CpE targeting is supported by binding affinities, kinetics, and half-lives of claudin-enterotoxin complexes and by the cytotoxic effects of CpE on claudin-expressing cells. By correlating the binding residence times of claudin-CpE complexes we determined to claudin expression patterns in the gut, we uncover that the primary CpE receptors differ in mice and humans due to sequence changes in the target motif. These findings provide the molecular and structural element CpE employs for subtype-specific targeting of claudins during pathogenicity of C. perfringens in the gut and a framework for new strategies to treat CpE-based illnesses in domesticated mammals and humans.
Assuntos
Claudina-4/química , Enterotoxinas/química , Junções Íntimas/efeitos dos fármacos , Animais , Sítios de Ligação , Claudina-4/metabolismo , Clostridium perfringens , Enterotoxinas/toxicidade , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Células Sf9 , Spodoptera , Junções Íntimas/metabolismoRESUMO
Strains of Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflicts humans and domesticated animals, causing prevalent gastrointestinal illnesses. CpE's C-terminal domain (cCpE) binds cell surface receptors, followed by a restructuring of its N-terminal domain to form a membrane-penetrating ß-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are known receptors for CpE and also control the architecture and function of cell-cell contacts (tight junctions) that create barriers to intercellular molecular transport. CpE binding and assembly disables claudin barrier function and induces cytotoxicity via ß-pore formation, disrupting gut homeostasis; however, a structural basis of this process and strategies to inhibit the claudin-CpE interactions that trigger it are both lacking. Here, we used a synthetic antigen-binding fragment (sFab) library to discover two sFabs that bind claudin-4 and cCpE complexes. We established these sFabs' mode of molecular recognition and binding properties and determined structures of each sFab bound to claudin-4-cCpE complexes using cryo-EM. The structures reveal that the sFabs bind a shared epitope, but conform distinctly, which explains their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in binding changes, validating the structures, and uncovering the sFab's targeting mechanism. From these insights, we generated a model for CpE's claudin-bound ß-pore that predicted sFabs would not prevent cytotoxicity, which we then verified in vivo. Taken together, this work demonstrates the development and mechanism of claudin/cCpE-binding sFabs that provide a framework and strategy for obstructing claudin/CpE assembly to treat CpE-linked gastrointestinal diseases.
Assuntos
Claudinas , Enterotoxinas , Animais , Claudina-3/genética , Claudina-3/metabolismo , Claudina-4/genética , Claudina-4/metabolismo , Claudinas/metabolismo , Clostridium perfringens , Enterotoxinas/metabolismo , Epitopos/metabolismo , Humanos , Ligação ProteicaRESUMO
Biological membranes define the boundaries of cells and compartmentalize the chemical and physical processes required for life. Many biological processes are carried out by proteins embedded in or associated with such membranes. Determination of membrane protein (MP) structures at atomic or near-atomic resolution plays a vital role in elucidating their structural and functional impact in biology. This endeavor has determined 1198 unique MP structures as of early 2021. The value of these structures is expanded greatly by deposition of their three-dimensional (3D) coordinates into the Protein Data Bank (PDB) after the first atomic MP structure was elucidated in 1985. Since then, free access to MP structures facilitates broader and deeper understanding of MPs, which provides crucial new insights into their biological functions. Here we highlight the structural and functional biology of representative MPs and landmarks in the evolution of new technologies, with insights into key developments influenced by the PDB in magnifying their impact.
Assuntos
Bases de Dados de Proteínas , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Bases de Dados de Proteínas/história , História do Século XX , História do Século XXI , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The human pathogenic bacterium Clostridium perfringens secretes an enterotoxin (CpE) that targets claudins through its C-terminal receptor-binding domain (cCpE). Isoform-specific binding by CpE causes dissociation of claudins and tight junctions (TJs), resulting in cytotoxicity and breakdown of the gut epithelial barrier. Here, we present crystal structures of human claudin-9 (hCLDN-9) in complex with cCpE at 3.2 and 3.3 Å. We show that hCLDN-9 is a high-affinity CpE receptor and that hCLDN-9-expressing cells undergo cell death when treated with CpE but not cCpE, which lacks its cytotoxic domain. Structures reveal cCpE-induced alterations to 2 epitopes known to enable claudin self-assembly and expose high-affinity interactions between hCLDN-9 and cCpE that explain isoform-specific recognition. These findings elucidate the molecular bases for hCLDN-9 selective ion permeability and binding by CpE, and provide mechanisms for how CpE disrupts gut homeostasis by dissociating claudins and TJs to affect epithelial adhesion and intercellular transport.
Assuntos
Claudinas/química , Claudinas/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Toxinas Biológicas/química , Toxinas Biológicas/toxicidade , Animais , Sítios de Ligação , Enterotoxinas/química , Enterotoxinas/metabolismo , Enterotoxinas/toxicidade , Humanos , Mucosa Intestinal/patologia , Camundongos , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Toxinas Biológicas/metabolismoRESUMO
Aspirin and other nonsteroidal anti-inflammatory drugs target the cyclooxygenase enzymes (COX-1 and COX-2) to block the formation of prostaglandins. Aspirin is unique in that it covalently modifies each enzyme by acetylating Ser-530 within the cyclooxygenase active site. Acetylation of COX-1 leads to complete loss of activity, while acetylation of COX-2 results in the generation of the monooxygenated product 15(R)-hydroxyeicosatetraenoic acid (15R-HETE). Ser-530 has also been shown to influence the stereochemistry for the addition of oxygen to the prostaglandin product. We determined the crystal structures of S530T murine (mu) COX-2, aspirin-acetylated human (hu) COX-2, and huCOX-2 in complex with salicylate to 1.9, 2.0, and 2.4 Å, respectively. The structures reveal that (1) the acetylated Ser-530 completely blocks access to the hydrophobic groove, (2) the observed binding pose of salicylate is reflective of the enzyme-inhibitor complex prior to acetylation, and (3) the observed Thr-530 rotamer in the S530T muCOX-2 crystal structure does not impede access to the hydrophobic groove. On the basis of these structural observations, along with functional analysis of the S530T/G533V double mutant, we propose a working hypothesis for the generation of 15R-HETE by aspirin-acetylated COX-2. We also observe differential acetylation of COX-2 purified in various detergent systems and nanodiscs, indicating that detergent and lipid binding within the membrane-binding domain of the enzyme alters the rate of the acetylation reaction in vitro.
Assuntos
Acetilação/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/química , Aspirina/química , Ciclo-Oxigenase 2/química , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Cristalografia por Raios X , Ciclo-Oxigenase 2/metabolismo , Humanos , Camundongos , Simulação de Acoplamento Molecular , Conformação ProteicaRESUMO
Pathogenic strains of Clostridium perfringens secrete an enterotoxin (CpE) that causes prevalent, severe, and sometimes deadly gastrointestinal disorders in humans and domesticated animals. CpE binds selectively to membrane protein receptors called claudins on the apical surfaces of small intestinal epithelium. Claudins normally construct tight junctions that regulate epithelial paracellular transport but are hijacked from doing so by CpE and are instead led to form claudin/CpE small complexes. Small complexes are building blocks for assembling oligomeric ß-barrel pores that penetrate the plasma membrane and induce gut cytotoxicity. Here we present structures of CpE in complexes with its native claudin receptor in humans, claudin-4, at 4.0 and 2.8 Å using cryogenic electron microscopy. The structures reveal the overall architecture of the small complex, that the small complex can be kinetically trapped, and resolve its key features; like the residues used in claudin/CpE complex binding, the orientation of CpE relative to the membrane, and CpE-induced structural changes to claudin-4. Further, the structures allude to the biophysical procession from small complex to cytotoxic ß-barrel pore used by CpE during pathogenesis and the role of trypsin in this process. In full, this work elucidates the structure and mechanism of claudin-bound CpE pore assembly and provides strategies to obstruct its formation to treat CpE-induced gastrointestinal diseases.
RESUMO
Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties-these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes using cryogenic electron microscopy. Structural and biophysical results reveal this sFabs mechanism of select binding to human claudin-4 over other homologous claudins and establish the ability of sFabs to bind hard-to-target claudins to probe tight junction structure and function. The findings provide a framework for tight junction modulation by sFabs for tissue-selective therapies.
Assuntos
Claudina-4 , Claudina-4/metabolismo , Humanos , Junções Íntimas/metabolismo , Microscopia Crioeletrônica , Enterotoxinas/metabolismo , Enterotoxinas/química , Enterotoxinas/imunologia , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/metabolismo , Ligação Proteica , Modelos MolecularesRESUMO
De novo design of complex protein folds using solely computational means remains a significant challenge. Here, we use a robust deep learning pipeline to design complex folds and soluble analogues of integral membrane proteins. Unique membrane topologies, such as those from GPCRs, are not found in the soluble proteome and we demonstrate that their structural features can be recapitulated in solution. Biophysical analyses reveal high thermal stability of the designs and experimental structures show remarkable design accuracy. The soluble analogues were functionalized with native structural motifs, standing as a proof-of-concept for bringing membrane protein functions to the soluble proteome, potentially enabling new approaches in drug discovery. In summary, we designed complex protein topologies and enriched them with functionalities from membrane proteins, with high experimental success rates, leading to a de facto expansion of the functional soluble fold space.
RESUMO
The cyclooxygenases (COX-1 and COX-2) generate prostaglandin H(2) from arachidonic acid (AA). In its catalytically productive conformation, AA binds within the cyclooxygenase channel with its carboxylate near Arg-120 and Tyr-355 and ω-end located within a hydrophobic groove above Ser-530. Although AA is the preferred substrate for both isoforms, COX-2 can oxygenate a broad spectrum of substrates. Mutational analyses have established that an interaction of the carboxylate of AA with Arg-120 is required for high affinity binding by COX-1 but not COX-2, suggesting that hydrophobic interactions between the ω-end of substrates and cyclooxygenase channel residues play a significant role in COX-2-mediated oxygenation. We used structure-function analyses to investigate the role that Arg-120 and residues lining the hydrophobic groove play in the binding and oxygenation of substrates by murine (mu) COX-2. Mutations to individual amino acids within the hydrophobic groove exhibited decreased rates of oxygenation toward AA with little effect on binding. R120A muCOX-2 oxygenated 18-carbon ω-6 and ω-3 substrates albeit at reduced rates, indicating that an interaction with Arg-120 is not required for catalysis. Structural determinations of Co(3+)-protoporphyrin IX-reconstituted muCOX-2 with α-linolenic acid and G533V muCOX-2 with AA indicate that proper bisallylic carbon alignment is the major determinant for efficient substrate oxygenation by COX-2. Overall, these findings implicate Arg-120 and hydrophobic groove residues as determinants that govern proper alignment of the bisallylic carbon below Tyr-385 for catalysis in COX-2 and confirm nuances between COX isoforms that explain substrate promiscuity.
Assuntos
Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/metabolismo , Animais , Ácido Araquidônico/metabolismo , Arginina/química , Linhagem Celular , Cristalografia por Raios X , Ciclo-Oxigenase 1/química , Ciclo-Oxigenase 1/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Especificidade por Substrato , Ácido alfa-Linolênico/metabolismoRESUMO
Clostridium perfringens enterotoxin (CpE) is a ß-pore forming toxin that disrupts gastrointestinal homeostasis in mammals by binding membrane protein receptors called claudins. Although structures of CpE fragments bound to claudins have been determined, the mechanisms that trigger CpE activation and oligomerization that lead to the formation of cytotoxic ß-pores remain undetermined. Proteolysis of CpE in the gut by trypsin has been shown to play a role in this and subsequent cytotoxicity processes. Here, we report solution structures of full-length and trypsinized CpE using small-angle X-ray scattering (SAXS) and crystal structures of trypsinized CpE and its C-terminal claudin-binding domain (cCpE) using X-ray crystallography. Mass spectrometry and SAXS uncover that removal of the CpE N-terminus by trypsin alters the CpE structure to expose areas that are normally unexposed. Crystal structures of trypsinized CpE and cCpE reveal unique dimer interfaces that could serve as oligomerization sites. Moreover, comparisons of these structures to existing ones predict the functional implications of oligomerization in the contexts of cell receptor binding and ß-pore formation. This study sheds light on trypsin's role in altering CpE structure to activate its function via inducing oligomerization on its path toward cytotoxic ß-pore formation. Its findings can incite new approaches to inhibit CpE-based cytotoxicity with oligomer-disrupting therapeutics.
Assuntos
Clostridium perfringens , Enterotoxinas , Animais , Tripsina , Espalhamento a Baixo Ângulo , Clostridium perfringens/metabolismo , Difração de Raios X , Enterotoxinas/metabolismo , Claudinas/metabolismo , Mamíferos/metabolismoRESUMO
Claudins are a family of â¼25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. Humans have 27 subtypes, which homo- and hetero-oligomerize to impart distinct properties and physiological functions to tissues and organs. As the structural and functional backbone of tight junctions, claudins are attractive targets for therapeutics capable of modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties-these traits also make therapy development a challenge. We have developed a synthetic antibody fragment (sFab) that binds human claudin-4 and used it to resolve structures of its complex with Clostridium perfringens enterotoxin (CpE) using cryogenic electron microscopy (cryo-EM). The resolution of the structures reveals the architectures of 22 kDa claudin-4, the 14 kDa C-terminal domain of CpE, and the mechanism by which this sFab binds claudins. Further, we elucidate the biochemical and biophysical bases of sFab binding and demonstrate that this molecule exhibits subtype-selectivity by assaying homologous claudins. Our results provide a framework for developing sFabs against hard-to-target claudins and establishes the utility of sFabs as fiducial markers for determining cryo-EM structures of this small membrane protein family at resolutions that surpass X-ray crystallography. Taken together, this work highlights the ability of sFabs to elucidate claudin structure and function and posits their potential as therapeutics for modulating tight junctions by targeting specific claudin subtypes.
RESUMO
Therapy resistance is the primary problem in treating late-stage colorectal cancer (CRC). Claudins are frequently dysregulated in cancer, and several are being investigated as novel therapeutic targets and biomarkers. We have previously demonstrated that Claudin-1 (CLDN1) expression in CRC promotes epithelial-mesenchymal transition, metastasis, and resistance to anoikis. Here, we hypothesize that CLDN1 promotes cancer stemness and chemoresistance in CRC. We found that high CLDN1 expression in CRC is associated with cancer stemness and chemoresistance signaling pathways in patient datasets, and it promotes chemoresistance both in vitro and in vivo. Using functional stemness assays, proteomics, biophysical binding assays, and patient-derived organoids, we found that CLDN1 promotes properties of cancer stemness including CD44 expression, tumor-initiating potential, and chemoresistance through a direct interaction with ephrin type-A receptor 2 (EPHA2) tyrosine kinase. This interaction is dependent on the CLDN1 PDZ-binding motif, increases EPHA2 protein expression by inhibiting its degradation, and enhances downstream AKT signaling and CD44 expression to promote stemness and chemoresistance. These results suggest CLDN1 is a viable target for pharmacological intervention and/or biomarker development.
Assuntos
Neoplasias Colorretais , Humanos , Linhagem Celular Tumoral , Claudina-1/genética , Claudina-1/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Transdução de SinaisRESUMO
The cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid (AA) in the committed step of prostaglandin biogenesis. Substitutions of I434V, H513R, and I523V constitute the only differences in residues lining the cyclooxygenase channel between COX-1 and COX-2. These changes create a hydrophobic pocket in COX-2, with Arg-513 located at the base of the pocket, which has been exploited in the design of COX-2-selective inhibitors. Previous studies have shown that COX-2, but not COX-1, can oxygenate endocannabinoid substrates, including 2-arachidonoyl glycerol (2-AG). To investigate the isoform-specific structural basis of endocannabinoid binding to COX-2, we determined the crystal structure of the 2-AG isomer 1-arachidonoyl glycerol (1-AG) in complex with wild type and R513H murine (mu) COX-2 to 2.2 and 2.35 Å, respectively, and R513H muCOX-2 in complex with AA to 2.45 Å resolution. The 2,3-dihydroxypropyl moiety of 1-AG binds near the opening of the cyclooxygenase channel in the space vacated by the movement of the Leu-531 side chain, validating our previous hypothesis implicating the flexibility of the Leu-531 side chain as a determinant for the ability of COX-2 to oxygenate endocannabinoid substrates. Functional analyses carried out to compliment our structural findings indicated that Y355F and R513H muCOX-2 constructs had no effect on the oxygenation of 1-AG and 2-AG, whereas substitutions that resulted in a shortened side chain for Leu-531 had only modest effects. Both AA and 1-AG bind to R513H muCOX-2 in conformations similar to those observed in the co-crystal structures of these substrates with wild type enzyme.
Assuntos
Ácidos Araquidônicos/química , Moduladores de Receptores de Canabinoides/química , Ciclo-Oxigenase 2/química , Endocanabinoides , Glicerídeos/química , Substituição de Aminoácidos , Animais , Ácidos Araquidônicos/metabolismo , Moduladores de Receptores de Canabinoides/metabolismo , Cristalografia por Raios X , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Glicerídeos/metabolismo , Camundongos , Mutação de Sentido Incorreto , Oxirredução , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Prostaglandin endoperoxide H synthases 1 and 2, also known as cyclooxygenases (COXs) 1 and 2, convert arachidonic acid (AA) to prostaglandin endoperoxide H(2). Prostaglandin endoperoxide H synthases are targets of nonspecific nonsteroidal anti-inflammatory drugs and COX-2-specific inhibitors called coxibs. PGHS-2 is a sequence homodimer. Each monomer has a peroxidase and a COX active site. We find that human PGHS-2 functions as a conformational heterodimer having a catalytic monomer (E(cat)) and an allosteric monomer (E(allo)). Heme binds tightly only to the peroxidase site of E(cat), whereas substrates, as well as certain inhibitors (e.g. celecoxib), bind the COX site of E(cat). E(cat) is regulated by E(allo) in a manner dependent on what ligand is bound to E(allo). Substrate and nonsubstrate fatty acids (FAs) and some COX inhibitors (e.g. naproxen) preferentially bind to the COX site of E(allo). AA can bind to E(cat) and E(allo), but the affinity of AA for E(allo) is 25 times that for E(cat). Palmitic acid, an efficacious stimulator of human PGHS-2, binds only E(allo) in palmitic acid/murine PGHS-2 co-crystals. Nonsubstrate FAs can potentiate or attenuate actions of COX inhibitors depending on the FA and whether the inhibitor binds E(cat) or E(allo). Our studies suggest that the concentration and composition of the free FA pool in the environment in which PGHS-2 functions in cells, the FA tone, is a key factor regulating PGHS-2 activity and its responses to COX inhibitors. We suggest that differences in FA tone occurring with different diets will likely affect both base-line prostanoid synthesis and responses to COX inhibitors.
Assuntos
Ciclo-Oxigenase 2/química , Multimerização Proteica/fisiologia , Animais , Domínio Catalítico , Linhagem Celular , Cristalografia por Raios X , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/química , Heme/química , Heme/genética , Heme/metabolismo , Humanos , Camundongos , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Claudins are a family of integral membrane proteins that enable epithelial cell/cell interactions by localizing to and driving the formation of tight junctions. Via claudin self-assembly within the membranes of adjoining cells, their extracellular domains interact, forming barriers to the paracellular transport of small molecules and ions. The bacterium Clostridium perfringens causes prevalent gastrointestinal disorders in mammals by employing an enterotoxin (CpE) that targets claudins. CpE binds to claudins at or near tight junctions in the gut and disrupts their barrier function, potentially by disabling their assembly or via cell signaling means-the mechanism(s) remain unclear. CpE ultimately destroys claudin-expressing cells through the formation of a cytotoxic membrane-penetrating ß-barrel pore. Structures obtained by X-ray crystallography of CpE, claudins, and claudins in complex with CpE fragments have provided the structural bases of claudin and CpE functions, revealing potential mechanisms for the CpE-mediated disruption of claudin-made tight junctions. This review highlights current progress in this space-what has been discovered and what remains unknown-toward efforts to elucidate the molecular mechanism of CpE disruption of tight junction barriers. It further underscores the key insights obtained through structure that are being applied to develop CpE-based therapeutics that combat claudin-overexpressing cancers or modulate tight junction barriers.
Assuntos
Claudinas , Junções Íntimas , Animais , Biologia , Claudinas/metabolismo , Enterotoxinas/toxicidade , Mamíferos/metabolismo , Junções Íntimas/metabolismoRESUMO
The cyclooxygenases (COX-1 and COX-2) are membrane-associated, heme-containing homodimers that generate prostaglandin H(2) from arachidonic acid (AA) in the committed step of prostaglandin biogenesis and are the targets for nonsteroidal anti-inflammatory drugs (NSAIDs). N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) was the first in a series of isoform-selective drugs designed to preferentially inhibit COX-2, with the aim of ameliorating many of the toxic gastrointestinal side effects caused by conventional NSAID inhibition. We determined the X-ray crystal structure of murine COX-2 in complex with NS-398 utilizing synchrotron radiation to 3.0A resolution. NS-398 binds in the cyclooxygenase channel in a conformation that is different than that observed for other COX-2-selective inhibitors, such as celecoxib, with no discernible penetration into the side pocket formed in COX-2 by the isoform-specific substitutions of I434V, H513R, and I523V. Instead, the methanesulfonamide moiety of NS-398 interacts with the side chain of Arg-120 at the opening of the cyclooxygenase channel, similar to that observed for acidic, nonselective NSAIDs such as indomethacin and flurbiprofen. Our structure validates inhibitor studies that identified Arg-120 as a molecular determinant for time-dependent inhibition of COX-2 by NS-398.
Assuntos
Inibidores de Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/química , Nitrobenzenos/química , Sulfonamidas/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Ligação de Hidrogênio , Camundongos , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
The cyclooxygenases (COX-1 and COX-2) are membrane-associated heme-containing homodimers that generate prostaglandin H(2) from arachidonic acid (AA). Although AA is the preferred substrate, other fatty acids are oxygenated by these enzymes with varying efficiencies. We determined the crystal structures of AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) bound to Co(3+)-protoporphyrin IX-reconstituted murine COX-2 to 2.1, 2.4, and 2.65 A, respectively. AA, EPA, and docosahexaenoic acid bind in different conformations in each monomer constituting the homodimer in their respective structures such that one monomer exhibits nonproductive binding and the other productive binding of the substrate in the cyclooxygenase channel. The interactions identified between protein and substrate when bound to COX-1 are conserved in our COX-2 structures, with the only notable difference being the lack of interaction of the carboxylate of AA and EPA with the side chain of Arg-120. Leu-531 exhibits a different side chain conformation when the nonproductive and productive binding modes of AA are compared. Unlike COX-1, mutating this residue to Ala, Phe, Pro, or Thr did not result in a significant loss of activity or substrate binding affinity. Determination of the L531F:AA crystal structure resulted in AA binding in the same global conformation in each monomer. We speculate that the mobility of the Leu-531 side chain increases the volume available at the opening of the cyclooxygenase channel and contributes to the observed ability of COX-2 to oxygenate a broad spectrum of fatty acid and fatty ester substrates.
Assuntos
Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Substituição de Aminoácidos/genética , Animais , Ácido Araquidônico/química , Ácido Araquidônico/metabolismo , Cristalografia por Raios X , Ácidos Docosa-Hexaenoicos/química , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/química , Ácido Eicosapentaenoico/metabolismo , Leucina/metabolismo , Camundongos , Modelos Moleculares , Conformação Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxigênio/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Structural biology has revealed predicting heterologous expression levels, homogeneity, and stability of a protein from its primary structure are exceedingly difficult. Membrane proteins, in particular, present numerous challenges that make obtaining milligram quantities of quality samples problematic. For structural and functional investigation of these molecules, however, this is what is required. Fluorescence size-exclusion chromatography (F-SEC), a technique where a protein of biological interest is fused to green fluorescent protein (GFP) and monitored, circumvents many bottlenecks inherent to membrane protein structural biology. In vivo expression yields, as well as in vitro homogeneity and stability, can be rapidly evaluated utilizing nanogram quantities of unpurified protein. In this chapter we describe our most current protocols for expression screening and biochemical characterization of membrane proteins using F-SEC, as it pertains to a high-throughput (HTP) crystallographic pipeline. Therein, the methods and workflow were designed and optimized for structure-function elucidation of eukaryotic integral membrane proteins, but may be applied to prokaryotic or water-soluble proteins with minor modifications, thus making it a useful general approach.