RESUMEN

Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), GtCCR4 has higher light sensitivity than typical channelrhodopsins. Furthermore, GtCCR4 shows superior properties as an optogenetic tool, such as minimal desensitization. Our structural analyses of GtCCR2 and GtCCR4 revealed that GtCCR4 has an outwardly bent transmembrane helix, resembling the conformation of activated G-protein-coupled receptors. Spectroscopic and electrophysiological comparisons suggested that this helix bend in GtCCR4 omits channel recovery time and contributes to high light sensitivity. An electrophysiological comparison of GtCCR4 and the well-characterized optogenetic tool ChRmine demonstrated that GtCCR4 has superior current continuity and action-potential spike generation with less invasiveness in neurons. We also identified highly active mutants of GtCCR4. These results shed light on the diverse structures and dynamics of microbial rhodopsins and demonstrate the strong optogenetic potential of GtCCR4.

RESUMEN

The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets complementary to an RNA guide, and is widely used as a powerful genome-editing tool. Here, we report the crystal structure of Brevibacillus laterosporus Cas9 (BlCas9, also known as BlatCas9), in complex with a guide RNA and its target DNA at 2.4-Å resolution. The structure reveals that the BlCas9 guide RNA adopts an unexpected architecture containing a triple-helix, which is specifically recognized by BlCas9, and that BlCas9 recognizes a unique N4CNDN protospacer adjacent motif through base-specific interactions on both the target and non-target DNA strands. Based on the structure, we rationally engineered a BlCas9 variant that exhibits enhanced genome- and base-editing activities with an expanded target scope in human cells. This approach may further improve the performance of the enhanced BlCas9 variant to generate useful genome-editing tools that require only a single C PAM nucleotide and can be packaged into a single AAV vector for in vivo gene therapy.

Asunto(s)

Brevibacillus , Proteína 9 Asociada a CRISPR , Edición Génica , Brevibacillus/genética , Brevibacillus/metabolismo , Brevibacillus/enzimología , Edición Génica/métodos , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/química , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Humanos , Sistemas CRISPR-Cas , Ingeniería de Proteínas/métodos

RESUMEN

The endothelin receptor type B (ETB) exhibits promiscuous coupling with various heterotrimeric G protein subtypes including Gs, Gi/o, Gq/11, and G12/13. Recent fluorescence and structural studies have raised questions regarding the coupling efficiencies and determinants of these G protein subtypes. Herein, by utilizing an integrative approach, combining hydrogen/deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cellular systems, we investigated conformational changes of Gs, Gi, and Gq triggered by ETB activation. ETB coupled to Gi and Gq but not with Gs. We underscored the critical roles of specific regions, including the C terminus of Gα and intracellular loop 2 (ICL2) of ETB in ETB-Gi1 or ETB-Gq coupling. Although The C terminus of Gα is essential for ETB-Gi1 and ETB-Gq coupling, ETB ICL2 influences Gq-coupling but not Gi1-coupling. Our results suggest a differential coupling efficiency of ETB with Gs, Gi1, and Gq, accompanied by distinct conformational changes in G proteins upon ETB-induced activation.

RESUMEN

Vitamin C plays important roles as a cofactor in many enzymatic reactions and as an antioxidant against oxidative stress. As some mammals including humans cannot synthesize vitamin C de novo from glucose, its uptake from dietary sources is essential, and is mediated by the sodium-dependent vitamin C transporter 1 (SVCT1). Despite its physiological significance in maintaining vitamin C homeostasis, the structural basis of the substrate transport mechanism remained unclear. Here, we report the cryo-EM structures of human SVCT1 in different states at 2.5-3.5 Å resolutions. The binding manner of vitamin C together with two sodium ions reveals the counter ion-dependent substrate recognition mechanism. Furthermore, comparisons of the inward-open and occluded structures support a transport mechanism combining elevator and distinct rotational motions. Our results demonstrate the molecular mechanism of vitamin C transport with its underlying conformational cycle, potentially leading to future industrial and medical applications.

Asunto(s)

Ácido Ascórbico , Microscopía por Crioelectrón , Transportadores de Sodio Acoplados a la Vitamina C , Humanos , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/química , Transportadores de Sodio Acoplados a la Vitamina C/genética , Ácido Ascórbico/metabolismo , Ácido Ascórbico/química , Transporte Biológico , Sodio/metabolismo , Modelos Moleculares , Multimerización de Proteína , Unión Proteica , Células HEK293 , Conformación Proteica

RESUMEN

The immunoproteasome subunit LMP7 (ß5i)/LMP2 (ß1i) dual blockade has been reported to suppress B cell differentiation and activation, suggesting that the dual inhibition of LMP7/LMP2 is a promising approach for treating autoimmune diseases. In contrast, the inhibition of the constitutive proteasome subunit ß5c correlates with cytotoxicity against non-immune cells. Therefore, LMP7/LMP2 dual inhibitors with high selectivity over ß5c may be desirable for treating autoimmune diseases. In this study, we present the optimization and discovery of α-amido boronic acids using cryo-electron microscopy (cryo-EM). The exploitation of structural differences between the proteasome subunits led to the identification of a highly selective LMP7/LMP2 dual inhibitor 19. Molecular dynamics simulation based on cryo-EM structures of the proteasome subunits complexed with 19 explained the inhibitory activity profile. In mice immunized with 4-hydroxy-3-nitrophenylacetyl conjugated to ovalbumin, results indicate that 19 is orally bioavailable and shows promise as potential treatment for autoimmune diseases.

Asunto(s)

Ácidos Borónicos , Microscopía por Crioelectrón , Complejo de la Endopetidasa Proteasomal , Inhibidores de Proteasoma , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Animales , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/síntesis química , Ratones , Ácidos Borónicos/química , Ácidos Borónicos/farmacología , Ácidos Borónicos/síntesis química , Humanos , Relación Estructura-Actividad , Cisteína Endopeptidasas/metabolismo , Estructura Molecular , Simulación de Dinámica Molecular , Descubrimiento de Drogas

RESUMEN

Pyroglutamylated RF-amide peptide (QRFP) is a peptide hormone with a C-terminal RF-amide motif. QRFP selectively activates a class A G-protein-coupled receptor (GPCR) GPR103 to exert various physiological functions such as energy metabolism and appetite regulation. Here, we report the cryo-electron microscopy structure of the QRFP26-GPR103-Gq complex at 3.19 Å resolution. QRFP26 adopts an extended structure bearing no secondary structure, with its N-terminal and C-terminal sides recognized by extracellular and transmembrane domains of GPR103 respectively. This movement, reminiscent of class B1 GPCRs except for orientation and structure of the ligand, is critical for the high-affinity binding and receptor specificity of QRFP26. Mutagenesis experiments validate the functional importance of the binding mode of QRFP26 by GPR103. Structural comparisons with closely related receptors, including RY-amide peptide-recognizing GPCRs, revealed conserved and diversified peptide recognition mechanisms, providing profound insights into the biological significance of RF-amide peptides. Collectively, this study not only advances our understanding of GPCR-ligand interactions, but also paves the way for the development of novel therapeutics targeting metabolic and appetite disorders and emergency medical care.

Asunto(s)

Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Humanos , Células HEK293 , Unión Proteica , Ligandos , Péptidos y Proteínas de Señalización Intercelular

RESUMEN

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.

Asunto(s)

Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Francisella , Edición Génica , Humanos , Edición Génica/métodos , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Francisella/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Amaurosis Congénita de Leber/genética , Streptococcus pyogenes/genética , Células HEK293 , Mutación , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Ingeniería de Proteínas/métodos , Genoma Humano

RESUMEN

The integrin family is a transmembrane receptor that plays critical roles in the cell-cell and cell-extracellular matrix adhesion, signal transduction such as cell cycle regulation, organization of the intracellular cytoskeleton, and immune responses. Consequently, dysfunction of integrins is associated with a wide range of human diseases, including cancer and immune diseases, which makes integrins therapeutic targets for drug discovery. Here we report the cryo-EM structure of the human α-I domain-containing full-length integrin αEß7, which is expressed in the leukocytes of the immune system and a drug target for inflammatory bowel disease (IBD). The structure reveals the half-bent conformation, an intermediate between the close and the open conformation, while the α-I domain responsible for the ligand binding covers the headpiece domain by a unique spatial arrangement. Our results provide the structural information for the drug design targeting IBD.

Asunto(s)

Microscopía por Crioelectrón , Modelos Moleculares , Dominios Proteicos , Humanos , Integrinas/metabolismo , Integrinas/química , Integrinas/ultraestructura , Conformación Proteica

RESUMEN

The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing guide RNA (pegRNA) to facilitate a wide variety of precise genome edits in living cells1. However, owing to a lack of structural information, the molecular mechanism of pegRNA-guided reverse transcription by the prime editor remains poorly understood. Here we present cryo-electron microscopy structures of the SpCas9-M-MLV RTΔRNaseH-pegRNA-target DNA complex in multiple states. The termination structure, along with our functional analysis, reveals that M-MLV RT extends reverse transcription beyond the expected site, resulting in scaffold-derived incorporations that cause undesired edits at the target loci. Furthermore, structural comparisons among the pre-initiation, initiation and elongation states show that M-MLV RT remains in a consistent position relative to SpCas9 during reverse transcription, whereas the pegRNA-synthesized DNA heteroduplex builds up along the surface of SpCas9. On the basis of our structural insights, we rationally engineered pegRNA variants and prime-editor variants in which M-MLV RT is fused within SpCas9. Collectively, our findings provide structural insights into the stepwise mechanism of prime editing, and will pave the way for the development of a versatile prime editing toolbox.

Asunto(s)

Proteína 9 Asociada a CRISPR , Edición Génica , Virus de la Leucemia Murina de Moloney , ARN Guía de Sistemas CRISPR-Cas , ADN Polimerasa Dirigida por ARN , Transcripción Reversa , Streptococcus pyogenes , Humanos , Proteína 9 Asociada a CRISPR/química , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/ultraestructura , Microscopía por Crioelectrón , ADN/química , ADN/metabolismo , ADN/genética , ADN/ultraestructura , Modelos Moleculares , Virus de la Leucemia Murina de Moloney/enzimología , Virus de la Leucemia Murina de Moloney/genética , Ribonucleasa H/deficiencia , Ribonucleasa H/genética , ARN Guía de Sistemas CRISPR-Cas/química , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/ultraestructura , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/metabolismo , ADN Polimerasa Dirigida por ARN/ultraestructura , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/genética , Proteínas Virales/química , Proteínas Virales/metabolismo , Proteínas Virales/ultraestructura , Proteínas Virales/genética , Células HEK293

RESUMEN

Melatonin (5-methoxy-N-acetyltryptamine) binds with high affinity and specificity to membrane receptors. Several receptor subtypes exist in different species, of which the mammalian MT1 and MT2 receptors are the best-characterized. They are members of the G protein-coupled receptor superfamily, preferentially coupling to Gi/o proteins but also to other G proteins in a cell-context-depending manner. In this review, experts on melatonin receptors will summarize the current state of the field. We briefly report on the discovery and classification of melatonin receptors, then focus on the molecular structure of human MT1 and MT2 receptors and highlight the importance of molecular simulations to identify new ligands and to understand the structural dynamics of these receptors. We then describe the state-of-the-art of the intracellular signaling pathways activated by melatonin receptors and their complexes. Brief statements on the molecular toolbox available for melatonin receptor studies and future perspectives will round-up this review.

Asunto(s)

Melatonina , Receptor de Melatonina MT1 , Animales , Humanos , Receptores de Melatonina , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT2/metabolismo , Melatonina/metabolismo , Transducción de Señal , Receptores Acoplados a Proteínas G , Mamíferos/metabolismo

RESUMEN

G-protein-coupled receptors (GPCRs) constitute a prominent superfamily in humans and are categorized into six classes (A-F) that play indispensable roles in cellular communication and therapeutics. Nonetheless, their structural comprehension has been limited by challenges in high-resolution data acquisition. This review highlights the transformative impact of cryogenic electron microscopy (cryo-EM) on the structural determinations of GPCR-G-protein complexes. Specific technologies, such as nanobodies and mini-G-proteins, stabilize complexes and facilitate structural determination. We discuss the structural alterations upon receptor activation in different GPCR classes, revealing their diverse mechanisms. This review highlights the robust foundation for comprehending GPCR function and pave the way for future breakthroughs in drug discovery and therapeutic targeting.

Asunto(s)

Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G , Microscopía por Crioelectrón/métodos , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Conformación Proteica , Animales , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/inmunología , Modelos Moleculares

RESUMEN

ERGIC-53 transports certain subsets of newly synthesized secretory proteins and membrane proteins from the endoplasmic reticulum to the Golgi apparatus. Despite numerous structural and functional studies since its identification, the overall architecture and mechanism of action of ERGIC-53 remain unclear. Here we present cryo-EM structures of full-length ERGIC-53 in complex with its functional partner MCFD2. These structures reveal that ERGIC-53 exists as a homotetramer, not a homohexamer as previously suggested, and comprises a four-leaf clover-like head and a long stalk composed of three sets of four-helix coiled-coil followed by a transmembrane domain. 3D variability analysis visualizes the flexible motion of the long stalk and local plasticity of the head region. Notably, MCFD2 is shown to possess a Zn2+-binding site in its N-terminal lid, which appears to modulate cargo binding. Altogether, distinct mechanisms of cargo capture and release by ERGIC- 53 via the stalk bending and metal binding are proposed.

Asunto(s)

Proteínas de la Membrana , Proteínas de Transporte Vesicular , Proteínas de Transporte Vesicular/metabolismo , Unión Proteica , Proteínas de la Membrana/metabolismo , Sitios de Unión , Aparato de Golgi/metabolismo , Lectinas de Unión a Manosa/metabolismo

RESUMEN

Histone H2B monoubiquitination (at Lys120 in humans) regulates transcription elongation and DNA repair. In humans, H2B monoubiquitination is catalyzed by the heterodimeric Bre1 complex composed of Bre1A/RNF20 and Bre1B/RNF40. The Bre1 proteins generally function as tumor suppressors, while in certain cancers, they facilitate cancer cell proliferation. To obtain structural insights of H2BK120 ubiquitination and its regulation, we report the cryo-electron microscopy structure of the human Bre1 complex bound to the nucleosome. The two RING domains of Bre1A and Bre1B recognize the acidic patch and the nucleosomal DNA phosphates around SHL 6.0-6.5, which are ideally located to recruit the E2 enzyme and ubiquitin for H2BK120-specific ubiquitination. Mutational experiments suggest that the two RING domains bind in two orientations and that ubiquitination occurs when Bre1A binds to the acidic patch. Our results provide insights into the H2BK120-specific ubiquitination by the Bre1 proteins and suggest that H2B monoubiquitination can be regulated by nuclesomal DNA flexibility.

Asunto(s)

Neoplasias , Nucleosomas , Humanos , Microscopía por Crioelectrón , ADN/metabolismo , Histonas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación

RESUMEN

GPR34 is a recently identified G-protein coupled receptor, which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative ligand. Here, we report cryo-electron microscopy structures of human GPR34-Gi complex bound with one of two ligands bound: either the LysoPS analogue S3E-LysoPS, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. The ligand-binding pocket is laterally open toward the membrane, allowing lateral entry of lipidic agonists into the cavity. The amine and carboxylate groups of the serine moiety are recognized by the charged residue cluster. The acyl chain of S3E-LysoPS is bent and fits into the L-shaped hydrophobic pocket in TM4-5 gap, and the aromatic fatty acid surrogate of M1 fits more appropriately. Molecular dynamics simulations further account for the LysoPS-regioselectivity of GPR34. Thus, using a series of structural and physiological experiments, we provide evidence that chemically unstable 2-acyl LysoPS is the physiological ligand for GPR34. Overall, we anticipate the present structures will pave the way for development of novel anticancer drugs that specifically target GPR34.

Asunto(s)

Ácidos Grasos , Lisofosfolípidos , Humanos , Microscopía por Crioelectrón , Ácidos Grasos/metabolismo , Ligandos , Lisofosfolípidos/metabolismo , Receptores Lisofosfolípidos/agonistas , Receptores Lisofosfolípidos/metabolismo

RESUMEN

Homoeostatic regulation of the acid-base balance is essential for cellular functional integrity. However, little is known about the molecular mechanism through which the acid-base balance regulates cellular responses. Here, we report that bicarbonate ions activate a G protein-coupled receptor (GPCR), i.e., GPR30, which leads to Gq-coupled calcium responses. Gpr30-Venus knock-in mice reveal predominant expression of GPR30 in brain mural cells. Primary culture and fresh isolation of brain mural cells demonstrate bicarbonate-induced, GPR30-dependent calcium responses. GPR30-deficient male mice are protected against ischemia-reperfusion injury by a rapid blood flow recovery. Collectively, we identify a bicarbonate-sensing GPCR in brain mural cells that regulates blood flow and ischemia-reperfusion injury. Our results provide a perspective on the modulation of GPR30 signalling in the development of innovative therapies for ischaemic stroke. Moreover, our findings provide perspectives on acid/base sensing GPCRs, concomitantly modulating cellular responses depending on fluctuating ion concentrations under the acid-base homoeostasis.

Asunto(s)

Isquemia Encefálica , Daño por Reperfusión , Accidente Cerebrovascular , Masculino , Ratones , Animales , Bicarbonatos , Calcio/metabolismo , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo

RESUMEN

ß-arrestins (ßarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo-electron microscopy structures of ßarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the ßarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of ßarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of ßarr2 from a ß strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-ßarr complexes with direct implications for exploring novel therapeutic avenues.

Asunto(s)

Dominios y Motivos de Interacción de Proteínas , Receptores Acoplados a Proteínas G , beta-Arrestinas , beta-Arrestinas/química , Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G/química , Transducción de Señal , Conformación Proteica en Lámina beta , Conformación Proteica en Hélice alfa , Humanos

RESUMEN

Sodium-glucose cotransporter 2 (SGLT2) is imporant in glucose reabsorption. SGLT2 inhibitors suppress renal glucose reabsorption, therefore reducing blood glucose levels in patients with type 2 diabetes. We and others have developed several SGLT2 inhibitors starting from phlorizin, a natural product. Using cryo-electron microscopy, we present the structures of human (h)SGLT2-MAP17 complexed with five natural or synthetic inhibitors. The four synthetic inhibitors (including canagliflozin) bind the transporter in the outward conformations, while phlorizin binds it in the inward conformation. The phlorizin-hSGLT2 interaction exhibits biphasic kinetics, suggesting that phlorizin alternately binds to the extracellular and intracellular sides. The Na+-bound outward-facing and unbound inward-open structures of hSGLT2-MAP17 suggest that the MAP17-associated bundle domain functions as a scaffold, with the hash domain rotating around the Na+-binding site. Thus, Na+ binding stabilizes the outward-facing conformation, and its release promotes state transition to inward-open conformation, exhibiting a role of Na+ in symport mechanism. These results provide structural evidence for the Na+-coupled alternating-access mechanism proposed for the transporter family.

Asunto(s)

Diabetes Mellitus Tipo 2 , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Humanos , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Transportador 2 de Sodio-Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa , Florizina/farmacología , Florizina/química , Florizina/metabolismo , Microscopía por Crioelectrón , Glucosa/metabolismo

RESUMEN

Cryo-EM single particle analysis has recently facilitated the high-resolution structural determination of numerous GPCR-G complexes. Diverse methodologies have been devised with this trend, and in the case of GPCR-Gi complexes, scFv16, an antibody that recognizes the intricate interface of the complex, has been mainly implemented to stabilize the complex. However, owing to their flexibility and heterogeneity, structural determinations of GPCR-Gi complexes remain both challenging and resource-intensive. By employing eGαt, which exhibits binding affinity to modified nanobody Nb35, the cryo-EM structure of Rhodopsin-eGαt complex was previously reported. Using this modified G protein, we determined the structure of the ETB-eGt complex bound to the modified Nb35. The determined structure of ETB receptor was the same as the previously reported ETB-Gi complex, and the resulting dataset demonstrated significantly improved anisotropy. This modified G protein will be utilized for the structural determination of other GPCR-Gi complexes.

Asunto(s)

Proteínas de Unión al GTP , Rodopsina , Microscopía por Crioelectrón , Proteínas de Unión al GTP/metabolismo , Rodopsina/metabolismo , Anticuerpos/metabolismo

RESUMEN

Cytochrome c oxidase (CcO) is part of the respiratory chain and contributes to the electrochemical membrane gradient in mitochondria as well as in many bacteria, as it uses the energy released in the reduction of oxygen to pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography to study the structural response of the active site upon flash photolysis of carbon monoxide (CO) from the reduced heme a3 of ba3-type CcO. In contrast with the aa3-type enzyme, our data show how CO is stabilized on CuB through interactions with a transiently ordered water molecule. These results offer a structural explanation for the extended lifetime of the CuB-CO complex in ba3-type CcO and, by extension, the extremely high oxygen affinity of the enzyme.

Asunto(s)

Monóxido de Carbono , Complejo IV de Transporte de Electrones , Complejo IV de Transporte de Electrones/metabolismo , Dominio Catalítico , Monóxido de Carbono/química , Cristalografía , Oxidación-Reducción , Oxígeno/metabolismo

RESUMEN

Modulation of large conductance intracellular ligand-activated potassium (BK) channel family (Slo1-3) by auxiliary subunits allows diverse physiological functions in excitable and non-excitable cells. Cryoelectron microscopy (cryo-EM) structures of voltage-gated potassium (Kv) channel complexes have provided insights into how voltage sensitivity is modulated by auxiliary subunits. However, the modulation mechanisms of BK channels, particularly as ligand-activated ion channels, remain unknown. Slo1 is a Ca2+-activated and voltage-gated BK channel and is expressed in neurons, muscle cells, and epithelial cells. Using cryo-EM and electrophysiology, we show that the LRRC26-γ1 subunit modulates not only voltage but also Ca2+ sensitivity of Homo sapiens Slo1. LRRC26 stabilizes the active conformation of voltage-senor domains of Slo1 by an extracellularly S4-locking mechanism. Furthermore, it also stabilizes the active conformation of Ca2+-sensor domains of Slo1 intracellularly, which is functionally equivalent to intracellular Ca2+ in the activation of Slo1. Such a dual allosteric modulatory mechanism may be general in regulating the intracellular ligand-activated BK channel complexes.

Asunto(s)

Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Humanos , Calcio/metabolismo , Microscopía por Crioelectrón , Activación del Canal Iónico/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Ligandos , Potasio , Regulación Alostérica