RESUMEN
Among coupled exchangers, CLCs uniquely catalyze the exchange of oppositely charged ions (Cl- for H+). Transport-cycle models to describe and explain this unusual mechanism have been proposed based on known CLC structures. While the proposed models harmonize with many experimental findings, gaps and inconsistencies in our understanding have remained. One limitation has been that global conformational change - which occurs in all conventional transporter mechanisms - has not been observed in any high-resolution structure. Here, we describe the 2.6 Å structure of a CLC mutant designed to mimic the fully H+-loaded transporter. This structure reveals a global conformational change to improve accessibility for the Cl- substrate from the extracellular side and new conformations for two key glutamate residues. Together with DEER measurements, MD simulations, and functional studies, this new structure provides evidence for a unified model of H+/Cl- transport that reconciles existing data on all CLC-type proteins.
Cells are shielded from harmful molecules and other threats by a thin, flexible layer called the membrane. However, this barrier also prevents chloride, sodium, protons and other ions from moving in or out of the cell. Channels and transporters are two types of membrane proteins that form passageways for these charged particles. Channels let ions flow freely from one side of the membrane to the other. To do so, these proteins change their three-dimensional shape to open or close as needed. On the other hand, transporters actively pump ions across the membrane to allow the charged particles to accumulate on one side. The shape changes needed for that type of movement are different: the transporters have to open a passageway on one side of the membrane while closing it on the other side, alternating openings to one side or the other. In general, channels and transporters are not related to each other, but one exception is a group called CLCs proteins. Present in many organisms, this family contains a mixture of channels and transporters. For example, humans have nine CLC proteins: four are channels that allow chloride ions in and out, and five are 'exchange transporters' that make protons and chloride ions cross the membrane in opposite directions. These proteins let one type of charged particle move freely across the membrane, which generates energy that the transporter then uses to actively pump the other ion in the direction needed by the cell. Yet, the exact three-dimensional changes required for CLC transporters and channels to perform their roles are still unknown. To investigate this question, Chavan, Cheng et al. harnessed a technique called X-ray crystallography, which allows scientists to look at biological molecules at the level of the atom. This was paired with other methods to examine a CLC mutant that adopts the shape of a normal CLC transporter when it is loaded with a proton. The experiments revealed how various elements in the transporter move relative to each other to adopt a structure that allows protons and chloride ions to enter the protein from opposite sides of the membrane, using separate pathways. While obtained on a bacterial CLC, these results can be applied to other CLC channels and transporters (including those in humans), shedding light on how this family transports charged particles across membranes. From bone diseases to certain types of seizures, many human conditions are associated with poorly functioning CLCs. Understanding the way these structures change their shapes to perform their roles could help to design new therapies for these health problems.
Asunto(s)
Antiportadores/química , Cloruros/metabolismo , Proteínas de Escherichia coli/química , Antiportadores/genética , Cristalografía por Rayos X , Proteínas de Escherichia coli/genética , Transporte Iónico , Mutación , Conformación Proteica , Bombas de Protones/fisiología , Protones , Análisis EspectralRESUMEN
CLC proteins are a ubiquitously expressed family of chloride-selective ion channels and transporters. A dearth of pharmacological tools for modulating CLC gating and ion conduction limits investigations aimed at understanding CLC structure/function and physiology. Herein, we describe the design, synthesis, and evaluation of a collection of N-arylated benzimidazole derivatives (BIMs), one of which (BIM1) shows unparalleled (>20-fold) selectivity for CLC-Ka over CLC-Kb, the two most closely related human CLC homologs. Computational docking to a CLC-Ka homology model has identified a BIM1 binding site on the extracellular face of the protein near the chloride permeation pathway in a region previously identified as a binding site for other less selective inhibitors. Results from site-directed mutagenesis experiments are consistent with predictions of this docking model. The residue at position 68 is 1 of only â¼20 extracellular residues that differ between CLC-Ka and CLC-Kb. Mutation of this residue in CLC-Ka and CLC-Kb (N68D and D68N, respectively) reverses the preference of BIM1 for CLC-Ka over CLC-Kb, thus showing the critical role of residue 68 in establishing BIM1 selectivity. Molecular docking studies together with results from structure-activity relationship studies with 19 BIM derivatives give insight into the increased selectivity of BIM1 compared with other inhibitors and identify strategies for further developing this class of compounds.
Asunto(s)
Canales de Cloruro/antagonistas & inhibidores , Cloruros/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Sitios de Unión , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Electrofisiología , Humanos , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Relación Estructura-Actividad , Xenopus laevisRESUMEN
Is nucleotide exchange sufficient to activate K-Ras4B? To signal, oncogenic rat sarcoma (Ras) anchors in the membrane and recruits effectors by exposing its effector lobe. With the use of NMR and molecular dynamics (MD) simulations, we observed that in solution, farnesylated guanosine 5'-diphosphate (GDP)-bound K-Ras4B is predominantly autoinhibited by its hypervariable region (HVR), whereas the GTP-bound state favors an activated, HVR-released state. On the anionic membrane, the catalytic domain adopts multiple orientations, including parallel (â¼180°) and perpendicular (â¼90°) alignments of the allosteric helices, with respect to the membrane surface direction. In the autoinhibited state, the HVR is sandwiched between the effector lobe and the membrane; in the active state, with membrane-anchored farnesyl and unrestrained HVR, the catalytic domain fluctuates reinlessly, exposing its effector-binding site. Dimerization and clustering can reduce the fluctuations. This achieves preorganized, productive conformations. Notably, we also observe HVR-autoinhibited K-Ras4B-GTP states, with GDP-bound-like orientations of the helices. Thus, we propose that the GDP/GTP exchange may not be sufficient for activation; instead, our results suggest that the GDP/GTP exchange, HVR sequestration, farnesyl insertion, and orientation/localization of the catalytic domain at the membrane conjointly determine the active or inactive state of K-Ras4B. Importantly, K-Ras4B-GTP can exist in active and inactive states; on its own, GTP binding may not compel K-Ras4B activation.-Jang, H., Banerjee, A., Chavan, T. S, Lu, S., Zhang, J., Gaponenko, V., Nussinov, R. The higher level of complexity of K-Ras4B activation at the membrane.
Asunto(s)
Membrana Celular/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Dominio Catalítico , Guanosina Difosfato/química , Guanosina Trifosfato/química , Espectroscopía de Resonancia Magnética , Ratones , Simulación de Dinámica Molecular , Estructura Molecular , Células 3T3 NIH , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas p21(ras)/químicaRESUMEN
CLC secondary active transporters exchange Cl(-) for H(+). Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Using (19)F NMR, we show that as [H(+)] is increased to protonate Gluex and enrich the outward-facing state, a residue ~20 Å away from Gluex, near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function.
Asunto(s)
Canales de Cloruro/química , Canales de Cloruro/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible.
Asunto(s)
Dominio Catalítico , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas ras/química , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Biocatálisis , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión ProteicaRESUMEN
Ras proteins recruit and activate effectors, including Raf, that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, activation of Raf requires its dimerization. It has been suspected that dimeric Ras may promote dimerization and activation of Raf. Here, we show that the GTP-bound catalytic domain of K-Ras4B, a highly oncogenic splice variant of the K-Ras isoform, forms stable homodimers. We observe two major dimer interfaces. The first, highly populated ß-sheet dimer interface is at the Switch I and effector binding regions, overlapping the binding surfaces of Raf, PI3K, RalGDS, and additional effectors. This interface has to be inhibitory to such effectors. The second, helical interface also overlaps the binding sites of some effectors. This interface may promote activation of Raf. Our data reveal how Ras self-association can regulate effector binding and activity, and suggest that disruption of the helical dimer interface by drugs may abate Raf signaling in cancer.
Asunto(s)
Guanosina Trifosfato/química , Proteínas Proto-Oncogénicas p21(ras)/química , Dominio Catalítico , Humanos , Cinética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de ProteínaRESUMEN
Recent evidence suggests that chemokine (C-X-C motif) receptor 4 (CXCR4) contributes to the regulation of blood pressure through interactions with α1-adrenergic receptors (ARs) in vascular smooth muscle. The underlying molecular mechanisms, however, are unknown. Using proximity ligation assays to visualize single-molecule interactions, we detected that α1A/B-ARs associate with CXCR4 on the cell surface of rat and human vascular smooth muscle cells (VSMC). Furthermore, α1A/B-AR could be coimmunoprecipitated with CXCR4 in a HeLa expression system and in human VSMC. A peptide derived from the second transmembrane helix of CXCR4 induced chemical shift changes in the NMR spectrum of CXCR4 in membranes, disturbed the association between α1A/B-AR and CXCR4, and inhibited Ca(2+) mobilization, myosin light chain (MLC) 2 phosphorylation, and contraction of VSMC upon α1-AR activation. CXCR4 silencing reduced α1A/B-AR:CXCR4 heteromeric complexes in VSMC and abolished phenylephrine-induced Ca(2+) fluxes and MLC2 phosphorylation. Treatment of rats with CXCR4 agonists (CXCL12, ubiquitin) reduced the EC50 of the phenylephrine-induced blood pressure response three- to fourfold. These observations suggest that disruption of the quaternary structure of α1A/B-AR:CXCR4 heteromeric complexes by targeting transmembrane helix 2 of CXCR4 and depletion of the heteromeric receptor complexes by CXCR4 knockdown inhibit α1-AR-mediated function in VSMC and that activation of CXCR4 enhances the potency of α1-AR agonists. Our findings extend the current understanding of the molecular mechanisms regulating α1-AR and provide an example of the importance of G protein-coupled receptor (GPCR) heteromerization for GPCR function. Compounds targeting the α1A/B-AR:CXCR4 interaction could provide an alternative pharmacological approach to modulate blood pressure.
Asunto(s)
Receptores Adrenérgicos alfa 1/metabolismo , Receptores CXCR4/metabolismo , Secuencias de Aminoácidos , Animales , Bencilaminas , Presión Sanguínea/efectos de los fármacos , Membrana Celular , Quimiocina CXCL12/metabolismo , Ciclamas , Dimerización , Células HeLa , Compuestos Heterocíclicos/química , Humanos , Masculino , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Fenilefrina/química , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling.
Asunto(s)
Membrana Celular/metabolismo , GTP Fosfohidrolasas/metabolismo , Péptidos/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Membrana Celular/química , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Fluidez de la Membrana , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Microscopía Confocal , Modelos Químicos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Fosfolípidos/química , Fosfolípidos/metabolismo , Fosforilación , Unión Proteica , Multimerización de Proteína , Prenilación de Proteína , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Serina/química , Serina/genética , Serina/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Post-translational modifications in proteins play a major functional role. Post-translational modifications affect the way proteins interact with each other, bind nucleotides, and localize in cellular compartments. Given the importance of post-translational modifications in protein biology, development of methods to produce post-translationally modified proteins for biochemical and biophysical studies is timely and significant. At the same time, obtaining post-translationally modified proteins in bacterial expression systems is often problematic. Here, we describe a novel recombinant approach to prepare human K-Ras 4B, a protein that is post-translationally farnesylated, proteolytically cleaved, and methylated in its C-terminus. K-Ras 4B is a member of the Ras subfamily of small GTPases and is of interest because it is frequently mutated in human cancer. The method relies on separate production of two structural domains-the N-terminal catalytic domain and the C-terminal peptide chemically modified with S-farnesyl-L-cysteine methyl ester. After the two domains are prepared, they are ligated together using the transpeptidase enzyme, sortase. Our procedure starts with the use of the plasmid of K-Ras 4B catalytic domain containing the sortase recognition sequence. After this, we describe the bacterial expression and purification steps used to purify K-Ras 4B and the preparation of the conjugated C-terminal peptide. The procedure ends with the sortase-mediated ligation technique. The produced post-translationally modified K-Ras 4B is active in a number of assays, including a GTP hydrolysis assay, Raf-1 binding assay, and surface plasmon resonance-based phospholipid binding assay.
Asunto(s)
Ingeniería Genética/métodos , Proteínas Proto-Oncogénicas p21(ras)/biosíntesis , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis , Prenilación de Proteína , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/aislamiento & purificación , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
NAC (N-acetyl-L-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H2O2. However, only NAC, and not catalase or another ROS scavenger Trolox, was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine, leading to its description as a ROS inducer. In contrast, our own experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1 (Forkhead box protein M1), stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox, interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Taken together, the findings of the present study suggest that, as a result of the dual nature of NAC, data interpretation might not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis.
Asunto(s)
Acetilcisteína/farmacología , Depuradores de Radicales Libres/farmacología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Acetilcisteína/metabolismo , Antineoplásicos Fitogénicos/antagonistas & inhibidores , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Catalasa/genética , Catalasa/metabolismo , Línea Celular Tumoral , Cromanos/antagonistas & inhibidores , Cromanos/metabolismo , Cromanos/farmacología , Citomegalovirus/enzimología , Dioxolanos/antagonistas & inhibidores , Dioxolanos/farmacología , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/antagonistas & inhibidores , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Depuradores de Radicales Libres/metabolismo , Humanos , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/farmacología , Oxidantes/antagonistas & inhibidores , Oxidantes/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/metabolismo , Estabilidad Proteica/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Proteínas Ubiquitinadas/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
NMR is commonly used to investigate macromolecular interactions. However, sensitivity problems hamper its use for studying such interactions at low physiologically relevant concentrations. At high concentrations, proteins or peptides tend to aggregate. In order to overcome this problem, we make use of reductive ¹³C-methylation to study protein interactions at low micromolar concentrations. Methyl groups in dimethyl lysines are degenerate with one ¹³CH3 signal arising from two carbons and six protons, as compared to one carbon and three protons in aliphatic amino acids. The improved sensitivity allows us to study protein-protein or protein-peptide interactions at very low micromolar concentrations. We demonstrate the utility of this method by studying the interaction between the post-translationally lipidated hypervariable region of a human proto-oncogenic GTPase K-Ras and a calcium sensor protein calmodulin. Calmodulin specifically binds K-Ras and modulates its downstream signaling. This binding specificity is attributed to the unique lipidated hypervariable region of K-Ras. At low micromolar concentrations, the post-translationally modified hypervariable region of K-Ras aggregates and binds calmodulin in a non-specific manner, hence conventional NMR techniques cannot be used for studying this interaction, however, upon reductively methylating the lysines of calmodulin, we detected signals of the lipidated hypervariable region of K-Ras at physiologically relevant nanomolar concentrations. Thus, we utilize ¹³C-reductive methylation of lysines to enhance the sensitivity of conventional NMR methods for studying protein interactions at low concentrations.