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1.
Methods Mol Biol ; 2449: 95-147, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35507260

RESUMEN

In the last two decades it has become increasingly evident that a large number of proteins adopt either a fully or a partially disordered conformation. Intrinsically disordered proteins are ubiquitous proteins that fulfill essential biological functions while lacking a stable 3D structure. Their conformational heterogeneity is encoded by the amino acid sequence, thereby allowing intrinsically disordered proteins or regions to be recognized based on their sequence properties. The identification of disordered regions facilitates the functional annotation of proteins and is instrumental for delineating boundaries of protein domains amenable to crystallization. This chapter focuses on the methods currently employed for predicting protein disorder and identifying intrinsically disordered binding sites.


Asunto(s)
Proteínas Intrínsecamente Desordenadas , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Intrínsecamente Desordenadas/química , Unión Proteica , Conformación Proteica , Dominios Proteicos
2.
Acta Crystallogr D Struct Biol ; 78(Pt 5): 553-559, 2022 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-35503204

RESUMEN

Crystallographers have an array of search-model options for structure solution by molecular replacement (MR). The well established options of homologous experimental structures and regular secondary-structure elements or motifs are increasingly supplemented by computational modelling. Such modelling may be carried out locally or may use pre-calculated predictions retrieved from databases such as the EBI AlphaFold database. MrParse is a new pipeline to help to streamline the decision process in MR by consolidating bioinformatic predictions in one place. When reflection data are provided, MrParse can rank any experimental homologues found using eLLG, which indicates the likelihood that a given search model will work in MR. Inbuilt displays of predicted secondary structure, coiled-coil and transmembrane regions further inform the choice of MR protocol. MrParse can also identify and rank homologues in the EBI AlphaFold database, a function that will also interest other structural biologists and bioinformaticians.


Asunto(s)
Proteínas , Bases de Datos de Proteínas , Modelos Moleculares , Dominios Proteicos , Estructura Secundaria de Proteína , Proteínas/química
3.
Front Immunol ; 13: 863529, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35514997

RESUMEN

Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) play important roles in the generation of antigenic peptides presented by Major Histocompatibility Class I (MHCI) molecules and indirectly regulate adaptive immune responses. Although the discrete function of these enzymes has been extensively characterized, recent reports have suggested that they can also form heterodimers with functional consequences. However, lack of structural characterization of a putative ERAP1/ERAP2 dimer has limited our understanding of its biological role and significance. To address this, we employed computational molecular dynamics calculations to explore the topology of interactions between these two, based on experimentally determined homo-dimerization interfaces observed in crystal structures of ERAP2 or homologous enzymes. Our analysis of 8 possible dimerization models, suggested that the most likely ERAP1/ERAP2 heterodimerization topology involves the exon 10 loop, a non-conserved loop previously implicated in interactions between ERAP1 and the disulfide-bond shuffling chaperone ERp44. This dimerization topology allows access to the active site of both enzymes and is consistent with a previously reported construct in which ERAP1 and ERAP2 were linked by Fos/Jun zipper tags. The proposed model constitutes a tentative structural template to help understand the physiological role and significance of ERAP1/ERAP2 molecular interactions.


Asunto(s)
Aminopeptidasas , Péptidos , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Antígenos , Antígenos de Histocompatibilidad Menor/genética , Dominios Proteicos
4.
J Gen Virol ; 103(5)2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35506985

RESUMEN

CD163, a macrophage-specific membrane scavenger receptor, serves as a cellular entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). The removal of scavenger receptor cysteine-rich (SRCR) domain 5 (SRCR5) of CD163 is sufficient to make transfected cells or genetically modified pigs resistant to PRRSV-1 and PRRSV-2 genotypes, and substitution of SRCR5 with SRCR8 from human CD163-like protein (hCD163L1) confers resistance to PRRSV-1 but not PRRSV-2 isolates. However, the specific regions within the SRCR5 polypeptide involved in PRRSV infection remain largely unknown. In this report, we performed mutational studies in order to identify which regions or amino acid sequences in the SRCR5 domain are critical for PRRSV infection. The approach used in this study was to make proline-arginine (PR) insertions along the SRCR5 polypeptide. Constructs were transfected into HEK293T cells, and then evaluated for infection with PRRSV-2 or PRRSV-1. For PRRSV-2, four PR insertions located after amino acids 8 (PR-9), 47 (PR-48), 54 (PR-55), and 99 (PR-100) had the greatest impact on infection. For PRRSV-1, insertions after amino acids 57 (PR-58) and 99 (PR-100) were critical. Computer simulations based on the crystal structure of SRCR5 showed that the mutations that affected infection localized to a similar region on the surface of the 3-D structure. Specifically, we found two surface patches that are essential for PRRSV infection. PR-58 and PR-55, which were separated by only three amino acids, had reciprocal effects on PRRSV-1 and PRRSV-2. Substitution of Glu-58 with Lys-58 reduced PRRSV-1 infection without affecting PRRSV-2, which partially explains the resistance to PRRSV-1 caused by the SRCR5 replacement with the homolog human SRCR8 previously observed. Finally, resistance to infection was observed following the disruption of any of the four conserved disulfide bonds within SRCR5. In summary, the results confirm that there are distinct differences between PRRSV-1 and PRRSV-2 on recognition of CD163; however, all mutations that affect infection locate on a similar region on the same face of SRCR5.


Asunto(s)
Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Animales , Antígenos CD , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Cisteína/genética , Células HEK293 , Humanos , Mutación , Síndrome Respiratorio y de la Reproducción Porcina/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , Dominios Proteicos , Receptores de Superficie Celular , Receptores Depuradores/genética , Porcinos
5.
Commun Biol ; 5(1): 421, 2022 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-35513706

RESUMEN

The Wnt signaling pathway plays a critical role in the developmental and physiological processes of metazoans. We previously reported that the Frizzled4 (FZD4) linker domain plays an important role in Norrin binding and signaling. However, the question remains whether the FZD linker contributes to Wnt signaling in general. Here, we show that the FZD linker is involved in Wnt binding and affects downstream Wnt signaling. A FZD4 chimera, in which the linker was swapped with that of the non-canonical receptor FZD6, impairs the binding with WNT3A and suppresses the recruitment of LRP6 and Disheveled, resulting in reduced canonical signaling. A similar effect was observed for non-canonical signaling. A FZD6 chimera containing the FZD1 linker showed reduced WNT5A binding and impaired signaling in ERK, JNK, and AKT mediated pathways. Altogether, our results suggest that the FZD linker plays an important role in specific Wnt binding and intracellular Wnt signaling.


Asunto(s)
Receptores Frizzled , Vía de Señalización Wnt , Proteínas Portadoras/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Dominios Proteicos , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
6.
Adv Protein Chem Struct Biol ; 130: 161-188, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35534107

RESUMEN

Within the modular protein domains there are five families that recognize proline-rich sequences: SH3, WW, EVH1, GYF and UEV domains. This chapter reviews the main strategies developed for the design of ligands for these families, including peptides, peptidomimetics and drugs. We also describe some studies aimed to understand the molecular reasons responsible for the intrinsic affinity and specificity of these domains.


Asunto(s)
Péptidos , Prolina , Sitios de Unión , Humanos , Ligandos , Péptidos/química , Prolina/química , Prolina/metabolismo , Unión Proteica , Dominios Proteicos
7.
Mol Cell ; 82(10): 1878-1893.e10, 2022 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-35537448

RESUMEN

Transcription factors (TFs) consist of a DNA-binding domain and an activation domain (AD) that are frequently considered to be independent and exchangeable modules. However, recent studies report that the physicochemical properties of the AD can control TF assembly at chromatin by driving phase separation into transcriptional condensates. Here, we dissected transcription activation by comparing different synthetic TFs at a reporter gene array with real-time single-cell fluorescence microscopy. In these experiments, binding site occupancy, residence time, and coactivator recruitment in relation to multivalent TF interactions were compared. While phase separation propensity and activation strength of the AD were linked, the actual formation of liquid-like TF droplets had a neutral or inhibitory effect on transcription activation. We conclude that multivalent AD-mediated interactions enhance the transcription activation capacity of a TF by increasing its residence time in the chromatin-bound state and facilitating the recruitment of coactivators independent of phase separation.


Asunto(s)
Cromatina , Factores de Transcripción , Sitios de Unión , Cromatina/genética , Dominios Proteicos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional
8.
Elife ; 112022 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-35506657

RESUMEN

De novo-designed receptor transmembrane domains (TMDs) present opportunities for precise control of cellular receptor functions. We developed a de novo design strategy for generating programmed membrane proteins (proMPs): single-pass α-helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces. We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor (CAR) that we expressed in mouse primary T cells and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD, from monomers up to tetramers. All programmed CARs stimulated substantially lower T cell cytokine release relative to the commonly used CD28 TMD, which we show elevated cytokine release through lateral recruitment of the endogenous T cell costimulatory receptor CD28. Precise design using orthogonal and modular TMDs thus provides a new way to program receptor structure and predictably tune activity for basic or applied synthetic biology.


Asunto(s)
Antígenos CD28 , Receptores Quiméricos de Antígenos , Animales , Antígenos CD28/metabolismo , Citocinas/metabolismo , Ratones , Dominios Proteicos , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T , Ensayos Antitumor por Modelo de Xenoinjerto
9.
Nature ; 605(7908): 172-178, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35444281

RESUMEN

Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that open their pores in response to binding of the agonist glutamate1-3. An ionic current through a single iGluR channel shows up to four discrete conductance levels (O1-O4)4-6. Higher conductance levels have been associated with an increased number of agonist molecules bound to four individual ligand-binding domains (LBDs)6-10. Here we determine structures of a synaptic complex of AMPA-subtype iGluR and the auxiliary subunit γ2 in non-desensitizing conditions with various occupancy of the LBDs by glutamate. We show that glutamate binds to LBDs of subunits B and D only after it is already bound to at least the same number of LBDs that belong to subunits A and C. Our structures combined with single-channel recordings, molecular dynamics simulations and machine-learning analysis suggest that channel opening requires agonist binding to at least two LBDs. Conversely, agonist binding to all four LBDs does not guarantee maximal channel conductance and favours subconductance states O1 and O2, with O3 and O4 being rare and not captured structurally. The lack of subunit independence and low efficiency coupling of glutamate binding to channel opening underlie the gating of synaptic complexes to submaximal conductance levels, which provide a potential for upregulation of synaptic activity.


Asunto(s)
Receptores de Glutamato , Receptores Ionotrópicos de Glutamato , Ácido Glutámico/metabolismo , Simulación de Dinámica Molecular , Dominios Proteicos , Receptores de Glutamato/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo
10.
Oncogene ; 41(21): 3000-3010, 2022 May.
Artículo en Inglés | MEDLINE | ID: mdl-35459779

RESUMEN

Members of the Inhibitor of Apoptosis Protein (IAP) family are essential for cell survival and appear to neutralize the cell death machinery by binding pro-apoptotic caspases. dcaf12 was recently identified as an apoptosis regulator in Drosophila. However, the underlying molecular mechanisms are unknown. Here we revealed that human DCAF12 homolog binds multiple IAPs, including XIAP, cIAP1, cIAP2, and BRUCE, through recognition of BIR domains in IAPs. The pro-apoptotic function of DCAF12 is dependent on its capacity to bind IAPs. In response to apoptotic stimuli, DCAF12 translocates from the nucleus to the cytoplasm, where it blocks the interaction between XIAP and pro-apoptotic caspases to facilitate caspase activation and apoptosis execution. Similarly, DCAF12 suppresses NF-κB activation in an IAP binding-dependent manner. Moreover, DCAF12 acts as a tumor suppressor to restrict the malignant phenotypes of cancer cells. Together, our results suggest that DCAF12 is an evolutionarily conserved IAP antagonist.


Asunto(s)
Proteínas Inhibidoras de la Apoptosis , FN-kappa B , Apoptosis , Caspasas/metabolismo , Supervivencia Celular , Humanos , Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/genética , FN-kappa B/metabolismo , Dominios Proteicos , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo
11.
Proc Natl Acad Sci U S A ; 119(15): e2119076119, 2022 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-35377810

RESUMEN

SignificanceOne of the key unresolved questions in the field of molecular chaperones is how they can actively unfold proteins. In this study, we discovered that the Hsp70/Hsp40 chaperone system completely unfolds a native soluble substrate protein, the ligand-binding domain of the glucocorticoid receptor, in a concerted action. Our high-resolution optical tweezers data show in real time how the substrate is attacked by the chaperone machinery. As soon as the hormone has left the binding pocket, up to five Hsp70/Hsp40 complexes bind and unfold the protein in a stepwise manner. This finding constitutes direct evidence that the chaperone machinery can bind to the folded core of the receptor, thus providing a mechanism for Hsp70-induced protein unfolding.


Asunto(s)
Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico , Receptores de Glucocorticoides , Adenosina Trifosfato/química , Proteínas del Choque Térmico HSP40/química , Proteínas HSP70 de Choque Térmico/química , Hidrólisis , Pinzas Ópticas , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Receptores de Glucocorticoides/química , Imagen Individual de Molécula
12.
Proc Natl Acad Sci U S A ; 119(15): e2116790119, 2022 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-35377815

RESUMEN

SignificanceExtracellular proteins with mechanical functions often require specialized assembly processes to form covalent oligomers. Progress in tissue bioengineering and repair will benefit from an understanding of how to harness and manipulate these processes. Here, we show that a particular supramolecular assembly mode was pre-encoded in the ancient domain organization common to gel-forming mucins and von Willebrand factor, glycoproteins that are deceptively different due to their divergence for distinct mechanical tasks. This finding highlights symmetry principles and building blocks retooled in nature to construct polymers with wide-ranging properties. These building blocks and knowledge of their self-assembly can be used to design new polymeric structures.


Asunto(s)
Evolución Molecular , Mucina-1 , Factor de von Willebrand , Mucina-1/química , Dominios Proteicos , Estructura Secundaria de Proteína , Factor de von Willebrand/química
13.
J Vis Exp ; (181)2022 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-35377353

RESUMEN

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily relative to traditional homologous recombination. The CRISPR-Cas9 system is particularly attractive when a single-point mutation is desired. The gap junction protein, Connexin 43 (Cx43), is encoded by gene Gja1, which has a single coding exon and cannot be spliced. However, Gja1 produces not only full-length Cx43 protein but up to six N-terminus truncated isoforms by a process known as internal translation, the result of ribosomal translation initiation at internal AUG (Methionine) start sites. GJA1-20k is the most commonly generated truncated isoform of Cx43 initiated at the AUG codon at position 213 of Gja1 mRNA. Because residue 213 occurs at the end of the last transmembrane domain of Cx43, GJA1-20k is effectively the 20 kDa C-terminus tail of Cx43 as an independent protein. Previous investigators identified, in cells, that a critical role of GJA1-20k is to facilitate trafficking of full-length Cx43 gap junction hemichannels to the plasma membrane. To examine this phenomenon in vivo, a mutant mouse with a Gja1 point-mutation was generated that replaces the ATG (Methionine) at residue 213 with TTA (Leucine, M213L mutation). The result of M213L is that Gja1 mRNA and full-length Cx43 are still generated, yet the translation of Gja1-20k is significantly reduced. This report focuses on choosing the restriction enzyme site to develop a one amino acid mutated (Gja1M213L/M213L) mouse model. This protocol describes genetically modified mice by the CRISPR-Cas9 system and rapid genotyping by combining PCR and restriction enzyme treatments.


Asunto(s)
Uniones Comunicantes , Animales , Modelos Animales de Enfermedad , Uniones Comunicantes/metabolismo , Ratones , Dominios Proteicos , Isoformas de Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo
14.
Bioorg Chem ; 123: 105768, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35378372

RESUMEN

Cat eye syndrome chromosome region candidate 2 (CECR2) bromodomain is a module of CECR2-containing remodeling factor (CERF), which is a chromatin remodeling complex correlating with transcriptional control and adjustment of chromatin architecture. Potent chemical probes would be beneficial to gain insights into the biochemical and pharmacological functions of CECR2 BRD. Herein, we report the discovery of a series of CECR2 BRD inhibitors with 7H-pyrrolo[2,3-d] pyrimidine scaffold based on molecular docking model of TP-248 and CECR2 BRD. The most potent inhibitor of this series, DC-CBi-22 with IC50 of 8.0 ± 1.4 nM against CECR2 BRD and selectivity over BPTF BRD up to 24.9-fold. The SARs were detailed according to molecular docking. DC-CBi-22 would serve as a useful chemical probe for the study of CECR2.


Asunto(s)
Pirimidinas , Factores de Transcripción , Simulación del Acoplamiento Molecular , Dominios Proteicos , Pirimidinas/farmacología , Relación Estructura-Actividad , Factores de Transcripción/química
15.
PLoS One ; 17(4): e0266937, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35417490

RESUMEN

Species-specific diversities are particular features of mammalian chloride channel regulator, calcium activated (CLCA) genes. In contrast to four complex gene clusters in mammals, only two CLCA genes appear to exist in chickens. CLCA2 is conserved in both, while only the galline CLCA1 (gCLCA1) displays close genetic distance to mammalian clusters 1, 3 and 4. In this study, sequence analyses and biochemical characterizations revealed that gCLCA1 as a putative avian prototype shares common protein domains and processing features with all mammalian CLCA homologues. It has a transmembrane (TM) domain in the carboxy terminal region and its mRNA and protein were detected in the alimentary canal, where the protein was localized in the apical membrane of enterocytes, similar to CLCA4. Both mammals and birds seem to have at least one TM domain containing CLCA protein with complex glycosylation in the apical membrane of enterocytes. However, some characteristic features of mammalian CLCA1 and 3 including entire protein secretion and expression in cell types other than enterocytes seem to be dispensable for chicken. Phylogenetic analyses including twelve bird species revealed that avian CLCA1 and mammalian CLCA3 form clades separate from a major branch containing mammalian CLCA1 and 4. Overall, our data suggest that gCLCA1 and mammalian CLCA clusters 1, 3 and 4 stem from a common ancestor which underwent complex gene diversification in mammals but not in birds.


Asunto(s)
Pollos , Canales de Cloruro , Animales , Membrana Celular/metabolismo , Pollos/genética , Pollos/metabolismo , Canales de Cloruro/metabolismo , Enterocitos/metabolismo , Mamíferos/genética , Mamíferos/metabolismo , Filogenia , Dominios Proteicos
16.
Int J Mol Sci ; 23(7)2022 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-35409101

RESUMEN

N-Glycosylation (NG) and disulfide bonds (DBs) are two prevalent co/post-translational modifications (PTMs) that are often conserved and coexist in membrane and secreted proteins involved in a large number of diseases. Both in the past and in recent times, the enzymes and chaperones regulating these PTMs have been constantly discovered to directly interact with each other or colocalize in the ER. However, beyond a few model proteins, how such cooperation affects N-glycan modification and disulfide bonding at selective sites in individual proteins is largely unknown. Here, we reviewed the literature to discover the current status in understanding the relationships between NG and DBs in individual proteins. Our results showed that more than 2700 human proteins carry both PTMs, and fewer than 2% of them have been investigated in the associations between NG and DBs. We summarized both these proteins with the reported relationships in the two PTMs and the tools used to discover the relationships. We hope that, by exposing this largely understudied field, more investigations can be encouraged to unveil the hidden relationships of NG and DBs in the majority of membranes and secreted proteins for pathophysiological understanding and biotherapeutic development.


Asunto(s)
Chaperonas Moleculares , Procesamiento Proteico-Postraduccional , Disulfuros/química , Glicosilación , Humanos , Chaperonas Moleculares/metabolismo , Dominios Proteicos
17.
Int J Mol Sci ; 23(7)2022 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-35409252

RESUMEN

YEATS (YAF9, ENL, AF9, TAF14, SAS5) family proteins recognize acylated histones and in turn regulate chromatin structure, gene transcription, and stress signaling. The chromosomal translocations of ENL and mixed lineage leukemia are considered oncogenic drivers in acute myeloid leukemia and acute lymphoid leukemia. However, known ENL YEATS domain inhibitors have failed to suppress the proliferation of 60 tested cancer cell lines. Herein, we identified four hits from the NMR fragment-based screening against the AF9 YEATS domain. Ten inhibitors of new chemotypes were then designed and synthesized guided by two complex structures and affinity assays. The complex structures revealed that these inhibitors formed an extra hydrogen bond to AF9, with respect to known ENL inhibitors. Furthermore, these inhibitors demonstrated antiproliferation activities in AF9-sensitive HGC-27 cells, which recapitulated the phenotype of the CRISPR studies against AF9. Our work will provide the basis for further structured-based optimization and reignite the campaign for potent AF9 YEATS inhibitors as a precise treatment for AF9-sensitive cancers.


Asunto(s)
Histonas , Leucemia Mieloide Aguda , Histonas/metabolismo , Humanos , Oncogenes , Dominios Proteicos
18.
Int J Mol Sci ; 23(7)2022 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-35409257

RESUMEN

Human vitamin K epoxide reductase (hVKORC1) enzymatic activity requires an initial activation by a specific redox protein, a less studied step in the hVKORC1 vital cycle. Significant steric conditions must be met by enzymes, being that to adapt their configurations is mandatory for hVKORC1 activation. We studied, by molecular dynamics (MD) simulations, the folding and conformational plasticity of hVKORC1 in its inactive (fully oxidised) state using available structures, crystallographic and from de novo modelling. According to the obtained results, hVKORC1 is a modular protein composed of the stable transmembrane domain (TMD) and intrinsically disordered luminal (L) loop, possessing the great plasticity/adaptability required to perform various steps of the activation process. The docking (HADDOCK) of Protein Disulfide Isomerase (PDI) onto different hVKORC1 conformations clearly indicated that the most interpretable solutions were found on the target closed L-loop form, a prevalent conformation of hVKORC1's oxidised state. We also suggest that the cleaved L-loop is an appropriate entity to study hVKORC1 recognition/activation by its redox protein. Additionally, the application of hVKORC1 (membrane protein) in aqueous solution is likely to prove to be very useful in practice in either in silico studies or in vitro experiments.


Asunto(s)
Simulación de Dinámica Molecular , Proteína Disulfuro Isomerasas , Humanos , Oxidación-Reducción , Proteína Disulfuro Isomerasas/metabolismo , Dominios Proteicos , Vitamina K/metabolismo , Vitamina K Epóxido Reductasas/química
19.
Nature ; 604(7907): 771-778, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35418677

RESUMEN

Adhesion G protein-coupled receptors (aGPCRs) constitute an evolutionarily ancient family of receptors that often undergo autoproteolysis to produce α and ß subunits1-3. A tethered agonism mediated by the 'Stachel sequence' of the ß subunit has been proposed to have central roles in aGPCR activation4-6. Here we present three cryo-electron microscopy structures of aGPCRs coupled to the Gs heterotrimer. Two of these aGPCRs are activated by tethered Stachel sequences-the ADGRG2-ß-Gs complex and the ADGRG4-ß-Gs complex (in which ß indicates the ß subunit of the aGPCR)-and the other is the full-length ADGRG2 in complex with the exogenous ADGRG2 Stachel-sequence-derived peptide agonist IP15 (ADGRG2(FL)-IP15-Gs). The Stachel sequences of both ADGRG2-ß and ADGRG4-ß assume a U shape and insert deeply into the seven-transmembrane bundles. Constituting the FXφφφXφ motif (in which φ represents a hydrophobic residue), five residues of ADGRG2-ß or ADGRG4-ß extend like fingers to mediate binding to the seven-transmembrane domain and activation of the receptor. The structure of the ADGRG2(FL)-IP15-Gs complex reveals the structural basis for the improved binding affinity of IP15 compared with VPM-p15 and indicates that rational design of peptidic agonists could be achieved by exploiting aGPCR-ß structures. By converting the 'finger residues' to acidic residues, we develop a method to generate peptidic antagonists towards several aGPCRs. Collectively, our study provides structural and biochemical insights into the tethered activation mechanism of aGPCRs.


Asunto(s)
Péptidos , Receptores Acoplados a Proteínas G , Microscopía por Crioelectrón , Humanos , Péptidos/metabolismo , Dominios Proteicos , Receptores Acoplados a Proteínas G/metabolismo
20.
Nature ; 604(7907): 763-770, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35418678

RESUMEN

Adhesion G-protein-coupled receptors (aGPCRs) are important for organogenesis, neurodevelopment, reproduction and other processes1-6. Many aGPCRs are activated by a conserved internal (tethered) agonist sequence known as the Stachel sequence7-12. Here, we report the cryogenic electron microscopy (cryo-EM) structures of two aGPCRs in complex with Gs: GPR133 and GPR114. The structures indicate that the Stachel sequences of both receptors assume an α-helical-bulge-ß-sheet structure and insert into a binding site formed by the transmembrane domain (TMD). A hydrophobic interaction motif (HIM) within the Stachel sequence mediates most of the intramolecular interactions with the TMD. Combined with the cryo-EM structures, biochemical characterization of the HIM motif provides insight into the cross-reactivity and selectivity of the Stachel sequences. Two interconnected mechanisms, the sensing of Stachel sequences by the conserved 'toggle switch' W6.53 and the constitution of a hydrogen-bond network formed by Q7.49/Y7.49 and the P6.47/V6.47φφG6.50 motif (φ indicates a hydrophobic residue), are important in Stachel sequence-mediated receptor activation and Gs coupling. Notably, this network stabilizes kink formation in TM helices 6 and 7 (TM6 and TM7, respectively). A common Gs-binding interface is observed between the two aGPCRs, and GPR114 has an extended TM7 that forms unique interactions with Gs. Our structures reveal the detailed mechanisms of aGPCR activation by Stachel sequences and their Gs coupling.


Asunto(s)
Péptidos , Receptores Acoplados a Proteínas G , Sitios de Unión , Microscopía por Crioelectrón , Dominios Proteicos , Estructura Secundaria de Proteína , Receptores Acoplados a Proteínas G/metabolismo , Relación Estructura-Actividad
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