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1.
Nat Commun ; 12(1): 6064, 2021 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-34663815

RESUMO

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. Here, to discover calcineurin-regulated processes we examined an understudied isoform, CNAß1. We show that unlike canonical cytosolic calcineurin, CNAß1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAß1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a calcineurin-regulated signaling pathway which highlights the PI4KA complex as a regulatory target and reveals that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAß1.

2.
Nat Commun ; 12(1): 5885, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620873

RESUMO

Pathogenic fungi exhibit a heavy burden on medical care and new therapies are needed. Here, we develop the fungal specific enzyme sterylglucosidase 1 (Sgl1) as a therapeutic target. Sgl1 converts the immunomodulatory glycolipid ergosterol 3ß-D-glucoside to ergosterol and glucose. Previously, we found that genetic deletion of Sgl1 in the pathogenic fungus Cryptococcus neoformans (Cn) results in ergosterol 3ß-D-glucoside accumulation, renders Cn non-pathogenic, and immunizes mice against secondary infections by wild-type Cn, even in condition of CD4+ T cell deficiency. Here, we disclose two distinct chemical classes that inhibit Sgl1 function in vitro and in Cn cells. Pharmacological inhibition of Sgl1 phenocopies a growth defect of the Cn Δsgl1 mutant and prevents dissemination of wild-type Cn to the brain in a mouse model of infection. Crystal structures of Sgl1 alone and with inhibitors explain Sgl1's substrate specificity and enable the rational design of antifungal agents targeting Sgl1.

3.
Sci Adv ; 7(35)2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34452907

RESUMO

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein-coupled receptors. Here, we report the cryo-electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gßγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gßγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ-adaptor binding domain interfaces enhanced Gßγ activation. A nanobody that specifically binds to the p101-Gßγ interface blocks activation, providing a novel tool to study and target p110γ-p101-specific signaling events in vivo.

4.
Structure ; 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34348129

RESUMO

There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G protein-coupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kγ binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development.

5.
Proc Natl Acad Sci U S A ; 118(33)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34385319

RESUMO

The protein kinase Akt is one of the primary effectors of growth factor signaling in the cell. Akt responds specifically to the lipid second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] via its PH domain, leading to phosphorylation of its activation loop and the hydrophobic motif of its kinase domain, which are critical for activity. We have now determined the crystal structure of Akt1, revealing an autoinhibitory interface between the PH and kinase domains that is often mutated in cancer and overgrowth disorders. This interface persists even after stoichiometric phosphorylation, thereby restricting maximum Akt activity to PI(3,4,5)P3- or PI(3,4)P2-containing membranes. Our work helps to resolve the roles of lipids and phosphorylation in the activation of Akt and has wide implications for the spatiotemporal control of Akt and potentially lipid-activated kinase signaling in general.

6.
Nat Commun ; 12(1): 4718, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354069

RESUMO

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.


Assuntos
Fosfatidato Fosfatase/química , Células 3T3-L1 , Adipogenia , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Sequência Conservada , Cristalografia por Raios X , Células HEK293 , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Genética
7.
J Mol Biol ; 433(18): 167145, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34229011

RESUMO

Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.

8.
Science ; 373(6557): 871-876, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34282049

RESUMO

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.


Assuntos
Aprendizado Profundo , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Proteínas ADAM/química , Sequência de Aminoácidos , Simulação por Computador , Microscopia Crioeletrônica , Cristalografia por Raios X , Bases de Dados de Proteínas , Proteínas de Membrana/química , Modelos Moleculares , Complexos Multiproteicos/química , Redes Neurais de Computação , Subunidades Proteicas/química , Proteínas/fisiologia , Receptores Acoplados a Proteínas G/química , Esfingosina N-Aciltransferase/química
9.
Nat Commun ; 12(1): 4028, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188059

RESUMO

CNNM/CorB proteins are a broadly conserved family of integral membrane proteins with close to 90,000 protein sequences known. They are associated with Mg2+ transport but it is not known if they mediate transport themselves or regulate other transporters. Here, we determine the crystal structure of an archaeal CorB protein in two conformations (apo and Mg2+-ATP bound). The transmembrane DUF21 domain exists in an inward-facing conformation with a Mg2+ ion coordinated by a conserved π-helix. In the absence of Mg2+-ATP, the CBS-pair domain adopts an elongated dimeric configuration with previously unobserved domain-domain contacts. Hydrogen-deuterium exchange mass spectrometry, analytical ultracentrifugation, and molecular dynamics experiments support a role of the structural rearrangements in mediating Mg2+-ATP sensing. Lastly, we use an in vitro, liposome-based assay to demonstrate direct Mg2+ transport by CorB proteins. These structural and functional insights provide a framework for understanding function of CNNMs in Mg2+ transport and associated diseases.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Hydrogenophilaceae/metabolismo , Magnésio/metabolismo , Methanomicrobiaceae/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte de Cátions/genética , Cristalografia por Raios X , Medição da Troca de Deutério , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios Proteicos
10.
J Biol Chem ; 297(2): 100919, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34181950

RESUMO

Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is a serine/threonine protein kinase activated by the phospholipid phosphatidylinositol 3-phosphate (PI3P) downstream of growth factor signaling via class I phosphatidylinositol 3-kinase (PI3K) signaling and by class III PI3K/Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers; however, the precise mechanism of activation of Sgk3 is unknown. Here, we use a wide range of cell biological, biochemical, and biophysical techniques, including hydrogen-deuterium exchange mass spectrometry, to investigate the mechanism of activation of Sgk3 by PI3P. We show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation. We demonstrate that binding of Sgk3 to PI3P via its regulatory phox homology (PX) domain induces large conformational changes in Sgk3 associated with its activation and that the PI3P-binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstitute Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to identifying the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.

11.
Methods Mol Biol ; 2263: 465-485, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33877613

RESUMO

Cellular membranes are a central hub for initiation and execution of many signaling processes. Integral to these processes being accomplished appropriately is the highly controlled recruitment and assembly of proteins at membrane surfaces. The study of the molecular mechanisms that mediate protein-membrane interactions can be facilitated by utilizing hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS is a robust analytical technique that allows for the measurement of the exchange rate of backbone amide hydrogens with solvent to make inferences about protein structure and conformation. This chapter discusses the use of HDX-MS as a tool to study the conformational changes that occur within peripheral membrane proteins upon association with membrane. Particular reference will be made to the analysis of the protein kinase Akt and its activation upon binding phosphatidylinositol (3,4,5) tris-phosphate (PIP3)-containing membranes to illustrate specific methodological principles.


Assuntos
Membrana Celular/metabolismo , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fenômenos Biofísicos , Membrana Celular/química , Espectrometria de Massa com Troca Hidrogênio-Deutério , Simulação de Dinâmica Molecular , Fosfatos de Fosfatidilinositol/química , Ligação Proteica , Conformação Proteica , Proteômica
12.
Foot Ankle Int ; 42(8): 969-975, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33926279

RESUMO

BACKGROUND: Previous studies have demonstrated success in using autogenous bone graft for arthrodesis in patients with failed surgeries of the hallux. These patients have several causes for pain and dysfunction preoperatively, including a shortened first ray, nonunion, and poor hallux alignment. METHODS: In this study, a consecutive series of 36 patients (38 procedures) were treated with a patellar wedge interposition structural allograft to salvage bone loss from great toe arthrodesis malunion, painful joint replacement, failed osteotomy, or infection of the great toe metatarsophalangeal (MP) joint with shortening of the first ray. The goals of the surgery were to restore length to the first ray and provide a stable MP joint fusion to relieve pain. The 38 treated toes were evaluated for preoperative and postoperative American Orthopaedic Foot & Ankle Society (AOFAS) MP scores, subjective patient outcome scores, and clinically successful fusion of the hallux. RESULTS: At a minimum 1-year follow-up (mean, 3.2 years), all but 2 feet healed with a solid fusion, and all healed patients reported good or excellent outcomes. AOFAS MP scores averaged 43.5 preoperatively and 77.2 postoperatively. Three patients with infection as cause for nonunion of the initial procedure were treated with staged procedures, including the use of a temporary antibiotic spacer and mini external fixator; all 3 healed without recurrent infection. One patient had a fracture of her allograft following her interposition arthrodesis, but it fused successfully after a second interposition arthrodesis surgery. Two patients developed a nonunion of the revision arthrodesis. CONCLUSION: The use of an interposition patellar wedge allograft can restore length to the first ray and provide successful salvage of arthrodesis nonunions and bone loss from failed hemiarthroplasty and total joint implants of the great toe MP joint. LEVEL OF EVIDENCE: Level IV, retrospective case series.

13.
Elife ; 102021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33661099

RESUMO

Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the class IB PI3K catalytic subunit (p110γ) playing key roles in immune signalling. p110γ is a key factor in inflammatory diseases and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the C-terminus can inactivate or activate enzyme activity. Screening of inhibitors using HDX-MS revealed that activation loop-binding inhibitors induce allosteric conformational changes that mimic those in the R1021C mutant. Structural analysis of advanced PI3K inhibitors in clinical development revealed novel binding pockets that can be exploited for further therapeutic development. Overall, this work provides unique insights into regulatory mechanisms that control PI3Kγ kinase activity and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

14.
Commun Biol ; 3(1): 735, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33277614

RESUMO

The TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.

15.
Adv Exp Med Biol ; 1274: 203-222, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894512

RESUMO

The lipid kinases that generate the lipid signalling phosphoinositides have been established as fundamental signalling enzymes that control numerous aspects of how cells respond to their extracellular environment. In addition, they play critical roles in regulating membrane trafficking and lipid transport within the cell. The class I phosphoinositide kinases which generate the critical lipid signal PIP3 are hyperactivated in numerous human pathologies including cancer, overgrowth syndromes, and primary immunodeficiencies. The type III phosphatidylinositol 4-kinase beta isoform (PI4KB), which are evolutionarily similar to the class I PI3Ks, have been found to be essential host factors mediating the replication of numerous devastating pathogenic viruses. Finally, targeting the parasite variant of PI4KB has been established as one of the most promising strategies for the development of anti-malarial and anti-cryptosporidium strategies. Therefore, the development of targeted isoform selective inhibitors for these enzymes are of paramount importance. The first generation of PI3K inhibitors have recently been clinically approved for a number of different cancers, highlighting their therapeutic value. This review will examine the history of the class I PI3Ks, and the type III PI4Ks, their relevance to human disease, and the structural basis for their regulation and inhibition by potent and selective inhibitors.


Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Doenças do Sistema Imunitário/tratamento farmacológico , Neoplasias/tratamento farmacológico , Doenças Parasitárias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Doenças da Imunodeficiência Primária/tratamento farmacológico , Viroses/tratamento farmacológico , 1-Fosfatidilinositol 4-Quinase/metabolismo , Animais , Humanos , Doenças do Sistema Imunitário/enzimologia , Neoplasias/enzimologia , Doenças Parasitárias/enzimologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Doenças da Imunodeficiência Primária/enzimologia , Viroses/enzimologia
16.
ACS Infect Dis ; 6(11): 3048-3063, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-32966036

RESUMO

Plasmodium falciparum phosphatidylinositol 4-kinase (PfPI4K) has emerged as a promising new drug target for novel antimalarial therapeutics. In the absence of a reliable high-resolution three-dimensional structure, a homology model of PfPI4K was built as a tool for structure-based drug design. This homology model has been validated against three distinct chemical series of potent inhibitors using docking and energy minimizations to elucidate the interactions crucial for PI4K inhibition and potent antiplasmodium activity. Despite its potential as an antimalarial target, the similarity between PfPI4K and structurally related human kinases poses a risk for human off-target kinase activity and associated toxicity. Comparative docking between PfPI4K and human phosphoinositide kinases (PIKs) presents compelling evidence for the origins of selectivity. This in-depth analysis of the PfPI4K homology model, the binding modes of the inhibitors, and the interactions responsible for selectivity over human kinases provides a powerful template for future optimization of Plasmodium PI4K inhibitors.


Assuntos
Antimaláricos , Plasmodium , 1-Fosfatidilinositol 4-Quinase , Antimaláricos/farmacologia , Desenho de Fármacos , Humanos , Plasmodium falciparum
17.
J Am Soc Mass Spectrom ; 31(10): 2202-2209, 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-32869988

RESUMO

Filtering of nonspecifically binding contaminant proteins from affinity purification mass spectrometry (AP-MS) data is a well-established strategy to improve statistical confidence in identified proteins. The CRAPome (contaminant repository for affinity purification) describes the contaminating background content present in many purification strategies. However, full contaminant lists for nickel-nitrilotriacetic acid (NiNTA) and glutathione S-transferase (GST) affinity matrices are lacking. Similarly, no Spodoptera frugiperda (Sf9) contaminants are available, and only the FLAG-purified contaminants are described for Escherichia coli. For MS experiments that use recombinant protein, such as structural mass spectrometry experiments (hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking, and radical foot-printing), failing to include these contaminants in the search database during the initial tandem MS (MS/MS) identification stage can result in complications in peptide identification. We have created contaminant FASTA databases for Sf9 and E. coli NiNTA or GST purification strategies and show that the use of these databases can effectively improve HDX-MS protein coverage, fragment count, and confidence in peptide identification. This approach provides a robust strategy toward the design of contaminant databases for any purification approach that will expand the complexity of systems able to be interrogated by HDX-MS.

18.
Structure ; 28(7): 830-846.e9, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32433991

RESUMO

Liver receptor homolog-1 (LRH-1; NR5A2) is a nuclear receptor that regulates a diverse array of biological processes. In contrast to dimeric nuclear receptors, LRH-1 is an obligate monomer and contains a subtype-specific helix at the C terminus of the DNA-binding domain (DBD), termed FTZ-F1. Although detailed structural information is available for individual domains of LRH-1, it is unknown how these domains exist in the intact nuclear receptor. Here, we developed an integrated structural model of human full-length LRH-1 using a combination of HDX-MS, XL-MS, Rosetta computational docking, and SAXS. The model predicts the DBD FTZ-F1 helix directly interacts with ligand binding domain helix 2. We confirmed several other predicted inter-domain interactions via structural and functional analyses. Comparison between the LRH-1/Dax-1 co-crystal structure and the integrated model predicted and confirmed Dax-1 co-repressor to modulate LRH-1 inter-domain dynamics. Together, these data support individual LRH-1 domains interacting to influence receptor structure and function.

19.
Structure ; 28(7): 810-819.e5, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32402248

RESUMO

Phospholipase C (PLC) enzymes hydrolyze phosphoinositide lipids to inositol phosphates and diacylglycerol. Direct activation of PLCß by Gαq and/or Gßγ subunits mediates signaling by Gq and some Gi coupled G-protein-coupled receptors (GPCRs), respectively. PLCß isoforms contain a unique C-terminal extension, consisting of proximal and distal C-terminal domains (CTDs) separated by a flexible linker. The structure of PLCß3 bound to Gαq is known, however, for both Gαq and Gßγ; the mechanism for PLCß activation on membranes is unknown. We examined PLCß2 dynamics on membranes using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Gßγ caused a robust increase in dynamics of the distal C-terminal domain (CTD). Gαq showed decreased deuterium incorporation at the Gαq binding site on PLCß. In vitro Gßγ-dependent activation of PLC is inhibited by the distal CTD. The results suggest that disruption of autoinhibitory interactions with the CTD leads to increased PLCß hydrolase activity.

20.
Nat Commun ; 11(1): 1734, 2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-32242008

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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