RESUMO
Mitochondrial calcium uptake is crucial to the regulation of eukaryotic Ca2+ homeostasis and is mediated by the mitochondrial calcium uniporter (MCU). While MCU alone can transport Ca2+ in primitive eukaryotes, metazoans require an essential single membrane-spanning auxiliary component called EMRE to form functional channels; however, the molecular mechanism of EMRE regulation remains elusive. Here, we present the cryo-EM structure of the human MCU-EMRE complex, which defines the interactions between MCU and EMRE as well as pinpoints the juxtamembrane loop of MCU and extended linker of EMRE as the crucial elements in the EMRE-dependent gating mechanism among metazoan MCUs. The structure also features the dimerization of two MCU-EMRE complexes along an interface at the N-terminal domain (NTD) of human MCU that is a hotspot for post-translational modifications. Thus, the human MCU-EMRE complex, which constitutes the minimal channel components among metazoans, provides a framework for future mechanistic studies on MCU.
Assuntos
Canais de Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Complexos Multiproteicos/metabolismo , Multimerização Proteica/fisiologia , Canais de Cálcio/genética , Células HEK293 , Humanos , Complexos Multiproteicos/genética , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
Maintaining a highly acidic lysosomal pH is central to cellular physiology. Here, we use functional proteomics, single-particle cryo-EM, electrophysiology, and in vivo imaging to unravel a key biological function of human lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in regulating lysosomal pH homeostasis. Despite being widely used as a lysosomal marker, the physiological functions of the LAMP proteins have long been overlooked. We show that LAMP-1 and LAMP-2 directly interact with and inhibit the activity of the lysosomal cation channel TMEM175, a key player in lysosomal pH homeostasis implicated in Parkinson's disease. This LAMP inhibition mitigates the proton conduction of TMEM175 and facilitates lysosomal acidification to a lower pH environment crucial for optimal hydrolase activity. Disrupting the LAMP-TMEM175 interaction alkalinizes the lysosomal pH and compromises the lysosomal hydrolytic function. In light of the ever-increasing importance of lysosomes to cellular physiology and diseases, our data have widespread implications for lysosomal biology.
Assuntos
Doença de Parkinson , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Canais de Potássio/metabolismoRESUMO
Transient receptor potential mucolipin 1 (TRPML1) is a Ca2+-permeable, nonselective cation channel ubiquitously expressed in the endolysosomes of mammalian cells and its loss-of-function mutations are the direct cause of type IV mucolipidosis (MLIV), an autosomal recessive lysosomal storage disease. TRPML1 is a ligand-gated channel that can be activated by phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] as well as some synthetic small-molecule agonists. Recently, rapamycin has also been shown to directly bind and activate TRPML1. Interestingly, both PI(3,5)P2 and rapamycin have low efficacy in channel activation individually but together they work cooperatively and activate the channel with high potency. To reveal the structural basis underlying the synergistic activation of TRPML1 by PI(3,5)P2 and rapamycin, we determined the high-resolution cryoelectron microscopy (cryo-EM) structures of the mouse TRPML1 channel in various states, including apo closed, PI(3,5)P2-bound closed, and PI(3,5)P2/temsirolimus (a rapamycin analog)-bound open states. These structures, combined with electrophysiology, elucidate the molecular details of ligand binding and provide structural insight into how the TRPML1 channel integrates two distantly bound ligand stimuli and facilitates channel opening.
Assuntos
Fosfatos de Fosfatidilinositol/farmacologia , Sirolimo/farmacologia , Canais de Potencial de Receptor Transitório/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Canais de Potencial de Receptor Transitório/genéticaRESUMO
The organellar two-pore channel (TPC) functions as a homodimer, in which each subunit contains two homologous Shaker-like six-transmembrane (6-TM)-domain repeats. TPCs belong to the voltage-gated ion channel superfamily and are ubiquitously expressed in animals and plants. Mammalian TPC1 and TPC2 are localized at the endolysosomal membrane, and have critical roles in regulating the physiological functions of these acidic organelles. Here we present electron cryo-microscopy structures of mouse TPC1 (MmTPC1)-a voltage-dependent, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2)-activated Na+-selective channel-in both the apo closed state and ligand-bound open state. Combined with functional analysis, these structures provide comprehensive structural insights into the selectivity and gating mechanisms of mammalian TPC channels. The channel has a coin-slot-shaped ion pathway in the filter that defines the selectivity of mammalian TPCs. Only the voltage-sensing domain from the second 6-TM domain confers voltage dependence on MmTPC1. Endolysosome-specific PtdIns(3,5)P2 binds to the first 6-TM domain and activates the channel under conditions of depolarizing membrane potential. Structural comparisons between the apo and PtdIns(3,5)P2-bound structures show the interplay between voltage and ligand in channel activation. These MmTPC1 structures reveal lipid binding and regulation in a 6-TM voltage-gated channel, which is of interest in light of the emerging recognition of the importance of phosphoinositide regulation of ion channels.
Assuntos
Canais de Cálcio/metabolismo , Canais de Cálcio/ultraestrutura , Microscopia Crioeletrônica , Ativação do Canal Iônico/efeitos dos fármacos , Fosfolipídeos/farmacologia , Sequência de Aminoácidos , Animais , Canais de Cálcio/química , Canais de Cálcio/genética , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Domínios Proteicos/efeitos dos fármacosRESUMO
The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel localized to the inner mitochondrial membrane. Here, we describe the structure of an MCU orthologue from the fungus Neosartorya fischeri (NfMCU) determined to 3.8 Å resolution by phase-plate cryo-electron microscopy. The channel is a homotetramer with two-fold symmetry in its amino-terminal domain (NTD) that adopts a similar structure to that of human MCU. The NTD assembles as a dimer of dimers to form a tetrameric ring that connects to the transmembrane domain through an elongated coiled-coil domain. The ion-conducting pore domain maintains four-fold symmetry, with the selectivity filter positioned at the start of the pore-forming TM2 helix. The aspartate and glutamate sidechains of the conserved DIME motif are oriented towards the central axis and separated by one helical turn. The structure of NfMCU offers insights into channel assembly, selective calcium permeation, and inhibitor binding.
Assuntos
Canais de Cálcio/química , Canais de Cálcio/ultraestrutura , Microscopia Crioeletrônica , Neosartorya/química , Sítios de Ligação , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Modelos Moleculares , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Compostos de Rutênio/farmacologia , SolubilidadeRESUMO
In this Article, ref. 15 has been replaced and references 32 to 50 have been renumbered online.
RESUMO
Immune checkpoint blockade therapy has been successful in treating some types of cancer but has not shown clinical benefits for treating leukaemia1. This result suggests that leukaemia uses unique mechanisms to evade this therapy. Certain immune inhibitory receptors that are expressed by normal immune cells are also present on leukaemia cells. Whether these receptors can initiate immune-related primary signalling in tumour cells remains unknown. Here we use mouse models and human cells to show that LILRB4, an immunoreceptor tyrosine-based inhibition motif-containing receptor and a marker of monocytic leukaemia, supports tumour cell infiltration into tissues and suppresses T cell activity via a signalling pathway that involves APOE, LILRB4, SHP-2, uPAR and ARG1 in acute myeloid leukaemia (AML) cells. Deletion of LILRB4 or the use of antibodies to block LILRB4 signalling impeded AML development. Thus, LILRB4 orchestrates tumour invasion pathways in monocytic leukaemia cells by creating an immunosuppressive microenvironment. LILRB4 represents a compelling target for the treatment of monocytic AML.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Evasão Tumoral/imunologia , Animais , Apolipoproteínas E/metabolismo , Arginase/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Movimento Celular , Proliferação de Células , Feminino , Humanos , Tolerância Imunológica/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores Imunológicos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Evasão Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Arabidopsis thaliana two-pore channel AtTPC1 is a voltage-gated, Ca2+-modulated, nonselective cation channel that is localized in the vacuolar membrane and responsible for generating slow vacuolar (SV) current. Under depolarizing membrane potential, cytosolic Ca2+ activates AtTPC1 by binding at the EF-hand domain, whereas luminal Ca2+ inhibits the channel by stabilizing the voltage-sensing domain II (VSDII) in the resting state. Here, we present 2.8 to 3.3 Å cryoelectron microscopy (cryo-EM) structures of AtTPC1 in two conformations, one in closed conformation with unbound EF-hand domain and resting VSDII and the other in a partially open conformation with Ca2+-bound EF-hand domain and activated VSDII. Structural comparison between the two different conformations allows us to elucidate the structural mechanisms of voltage gating, cytosolic Ca2+ activation, and their coupling in AtTPC1. This study also provides structural insight into the general voltage-gating mechanism among voltage-gated ion channels.
Assuntos
Proteínas de Arabidopsis/metabolismo , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Canais de Cálcio/genética , Cátions/metabolismo , Microscopia Crioeletrônica/métodos , Citosol/metabolismo , Ativação do Canal Iônico , Potenciais da Membrana/fisiologia , Vacúolos/metabolismoRESUMO
TRPM4 is a calcium-activated, phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) -modulated, non-selective cation channel that belongs to the family of melastatin-related transient receptor potential (TRPM) channels. Here we present the electron cryo-microscopy structures of the mouse TRPM4 channel with and without ATP. TRPM4 consists of multiple transmembrane and cytosolic domains, which assemble into a three-tiered architecture. The N-terminal nucleotide-binding domain and the C-terminal coiled-coil participate in the tetrameric assembly of the channel; ATP binds at the nucleotide-binding domain and inhibits channel activity. TRPM4 has an exceptionally wide filter but is only permeable to monovalent cations; filter residue Gln973 is essential in defining monovalent selectivity. The S1-S4 domain and the post-S6 TRP domain form the central gating apparatus that probably houses the Ca2+- and PtdIns(4,5)P2-binding sites. These structures provide an essential starting point for elucidating the complex gating mechanisms of TRPM4 and reveal the molecular architecture of the TRPM family.
Assuntos
Microscopia Crioeletrônica , Canais de Cátion TRPM/ultraestrutura , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Sítios de Ligação , Cálcio/metabolismo , Camundongos , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína , Especificidade por Substrato , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/química , Canais de Cátion TRPM/metabolismoRESUMO
TMEM175 is a lysosomal K+ channel that is important for maintaining the membrane potential and pH stability in lysosomes. It contains two homologous copies of a six-transmembrane-helix (6-TM) domain, which has no sequence homology to the canonical tetrameric K+ channels and lacks the TVGYG selectivity filter motif found in these channels. The prokaryotic TMEM175 channel, which is present in a subset of bacteria and archaea, contains only a single 6-TM domain and functions as a tetramer. Here, we present the crystal structure of a prokaryotic TMEM175 channel from Chamaesiphon minutus, CmTMEM175, the architecture of which represents a completely different fold from that of canonical K+ channels. All six transmembrane helices of CmTMEM175 are tightly packed within each subunit without undergoing domain swapping. The highly conserved TM1 helix acts as the pore-lining inner helix, creating an hourglass-shaped ion permeation pathway in the channel tetramer. Three layers of hydrophobic residues on the carboxy-terminal half of the TM1 helices form a bottleneck along the ion conduction pathway and serve as the selectivity filter of the channel. Mutagenesis analysis suggests that the first layer of the highly conserved isoleucine residues in the filter is primarily responsible for channel selectivity. Thus, the structure of CmTMEM175 represents a novel architecture of a tetrameric cation channel whose ion selectivity mechanism appears to be distinct from that of the classical K+ channel family.
Assuntos
Lisossomos/química , Canais de Potássio/química , Canais de Potássio/metabolismo , Estrutura Quaternária de Proteína , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Isoleucina/metabolismo , Modelos MolecularesRESUMO
Transient receptor potential mucolipin 1 (TRPML1) is a cation channel located within endosomal and lysosomal membranes. Ubiquitously expressed in mammalian cells, its loss-of-function mutations are the direct cause of type IV mucolipidosis, an autosomal recessive lysosomal storage disease. Here we present the single-particle electron cryo-microscopy structure of the mouse TRPML1 channel embedded in nanodiscs. Combined with mutagenesis analysis, the TRPML1 structure reveals that phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2) binds to the N terminus of the channel-distal from the pore-and the helix-turn-helix extension between segments S2 and S3 probably couples ligand binding to pore opening. The tightly packed selectivity filter contains multiple ion-binding sites, and the conserved acidic residues form the luminal Ca2+-blocking site that confers luminal pH and Ca2+ modulation on channel conductance. A luminal linker domain forms a fenestrated canopy atop the channel, providing several luminal ion passages to the pore and creating a negative electrostatic trap, with a preference for divalent cations, at the luminal entrance. The structure also reveals two equally distributed S4-S5 linker conformations in the closed channel, suggesting an S4-S5 linker-mediated PtdInsP2 gating mechanism among TRPML channels.
Assuntos
Microscopia Crioeletrônica , Endossomos/química , Lisossomos/química , Nanoestruturas/química , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/ultraestrutura , Animais , Sítios de Ligação , Cálcio , Concentração de Íons de Hidrogênio , Transporte de Íons , Ligantes , Camundongos , Modelos Moleculares , Mutação , Fosfatos de Fosfatidilinositol/metabolismo , Conformação Proteica , Eletricidade Estática , Canais de Potencial de Receptor Transitório/genéticaRESUMO
Protein kinase C has been implicated in the phosphorylation of the erythrocyte/brain glucose transporter, GLUT1, without a clear understanding of the site(s) of phosphorylation and the possible effects on glucose transport. Through in vitro kinase assays, mass spectrometry, and phosphospecific antibodies, we identify serine 226 in GLUT1 as a PKC phosphorylation site. Phosphorylation of S226 is required for the rapid increase in glucose uptake and enhanced cell surface localization of GLUT1 induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Endogenous GLUT1 is phosphorylated on S226 in primary endothelial cells in response to TPA or VEGF. Several naturally occurring, pathogenic mutations that cause GLUT1 deficiency syndrome disrupt this PKC phosphomotif, impair the phosphorylation of S226 in vitro, and block TPA-mediated increases in glucose uptake. We demonstrate that the phosphorylation of GLUT1 on S226 regulates glucose transport and propose that this modification is important in the physiological regulation of glucose transport.
Assuntos
Erros Inatos do Metabolismo dos Carboidratos/genética , Transportador de Glucose Tipo 1/metabolismo , Proteínas de Transporte de Monossacarídeos/deficiência , Proteína Quinase C-alfa/fisiologia , Sequência de Aminoácidos , Animais , Transporte Biológico , Erros Inatos do Metabolismo dos Carboidratos/enzimologia , Linhagem Celular , Células Endoteliais/metabolismo , Eritrócitos/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 1/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/genética , Mutação de Sentido Incorreto , Fosforilação , Processamento de Proteína Pós-Traducional , Ratos , Xenopus laevisRESUMO
Two-pore channels (TPCs) belong to the family of voltage-gated tetrameric cation channels and are ubiquitously expressed in organelles of animals and plants. These channels are believed to be evolutionary intermediates between homotetrameric voltage-gated potassium/sodium channels and the four-domain, single subunit, voltage-gated sodium/calcium channels. Each TPC subunit contains 12 transmembrane segments that can be divided into two homologous copies of an S1-S6 Shaker-like 6-TM domain. A functional TPC channel assembles as a dimer - the equivalent of a voltage-gated tetrameric cation channel. The plant TPC channel is localized in the vacuolar membrane and is also called the SV channel for generating the slow vacuolar (SV) current observed long before its molecular identification. Three subfamilies of mammalian TPC channels have been defined - TPC1, 2, and 3 - with the first two being ubiquitously expressed in animals and TPC3 being expressed in some animals but not in humans. Mammalian TPC1 and TPC2 are localized to the endolysosomal membrane and their functions are associated with various physiological processes. TPC3 is localized in the plasma membrane and its physiological function is not well defined.
Assuntos
Canais de Cálcio , Canais de Sódio Disparados por Voltagem , Humanos , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Ativação do Canal Iônico , Endossomos/metabolismo , Cátions/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Mamíferos/metabolismoRESUMO
Two-pore channels (TPCs) contain two copies of a Shaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in both animals and plants as organellar cation channels. Here we present the crystal structure of a vacuolar two-pore channel from Arabidopsis thaliana, AtTPC1, which functions as a homodimer. AtTPC1 activation requires both voltage and cytosolic Ca(2+). Ca(2+) binding to the cytosolic EF-hand domain triggers conformational changes coupled to the pair of pore-lining inner helices from the first 6-TM domains, whereas membrane potential only activates the second voltage-sensing domain, the conformational changes of which are coupled to the pair of inner helices from the second 6-TM domains. Luminal Ca(2+) or Ba(2+) can modulate voltage activation by stabilizing the second voltage-sensing domain in the resting state and shift voltage activation towards more positive potentials. Our Ba(2+)-bound AtTPC1 structure reveals a voltage sensor in the resting state, providing hitherto unseen structural insight into the general voltage-gating mechanism among voltage-gated channels.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Canais de Cálcio/química , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Bário/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Cálcio/farmacologia , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Cristalografia por Raios X , Citosol/metabolismo , Motivos EF Hand , Condutividade Elétrica , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Modelos Moleculares , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismoRESUMO
Organellar two-pore channels (TPCs) contain two copies of a Shaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in plants and animals. Interestingly, plant and animal TPCs share high sequence similarity in the filter region, yet exhibit drastically different ion selectivity. Plant TPC1 functions as a nonselective cation channel on the vacuole membrane, whereas mammalian TPC channels have been shown to be endo/lysosomal Na+-selective or Ca2+-release channels. In this study, we performed systematic characterization of the ion selectivity of TPC1 from Arabidopsis thaliana (AtTPC1) and compared its selectivity with the selectivity of human TPC2 (HsTPC2). We demonstrate that AtTPC1 is selective for Ca2+ over Na+, but nonselective among monovalent cations (Li+, Na+, and K+). Our results also confirm that HsTPC2 is a Na+-selective channel activated by phosphatidylinositol 3,5-bisphosphate. Guided by our recent structure of AtTPC1, we converted AtTPC1 to a Na+-selective channel by mimicking the selectivity filter of HsTPC2 and identified key residues in the TPC filters that differentiate the selectivity between AtTPC1 and HsTPC2. Furthermore, the structure of the Na+-selective AtTPC1 mutant elucidates the structural basis for Na+ selectivity in mammalian TPCs.
Assuntos
Proteínas de Arabidopsis/metabolismo , Canais de Cálcio/metabolismo , Cátions Monovalentes/metabolismo , Ativação do Canal Iônico , Sódio/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Canais de Cálcio/química , Canais de Cálcio/genética , Sequência Consenso , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Conformação Proteica , Proteínas Recombinantes/química , Alinhamento de Sequência , Especificidade da Espécie , Especificidade por SubstratoRESUMO
The TrkH/TrkG/KtrB proteins mediate K(+) uptake in bacteria and probably evolved from simple K(+) channels by multiple gene duplications or fusions. Here we present the crystal structure of a TrkH from Vibrio parahaemolyticus. TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to those of K(+) channels but significantly shorter, is lined by backbone and side-chain oxygen atoms. Functional studies showed that TrkH is selective for permeation of K(+) and Rb(+) over smaller ions such as Na(+) or Li(+). Immediately intracellular to the selectivity filter are an intramembrane loop and an arginine residue, both highly conserved, which constrict the permeation pathway. Substituting the arginine with an alanine significantly increases the rate of K(+) flux. These results reveal the molecular basis of K(+) selectivity and suggest a novel gating mechanism for this large and important family of membrane transport proteins.
Assuntos
Canais de Potássio/química , Canais de Potássio/metabolismo , Vibrio parahaemolyticus/química , Transportadores de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Ativação do Canal Iônico , Transporte de Íons , Modelos Moleculares , Dados de Sequência Molecular , Potássio/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
A better understanding of the interaction between extrinsic factors and surface receptors on stem cells will greatly benefit stem cell research and applications. Recently, we showed that several angiopoietin-like proteins (Angptls) bind and activate the immune inhibitory receptor human leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) to support ex vivo expansion of hematopoietic stem cells (HSCs) and leukemia development. However, the molecular basis for the interaction between Angptls and LILRB2 was unclear. Here, we demonstrate that Angptl2 expressed in mammalian cells forms high-molecular-weight species and that ligand multimerization is required for activation of LILRB2 for downstream signaling. A novel motif in the first and fourth Ig domains of LILRB2 was identified that is necessary for the receptor to be bound and activated by Angptl2. The binding of Angptl2 to LILRB2 is more potent than and not completely overlapped with the binding of another ligand, HLA-G. Immobilized anti-LILRB2 antibodies induce a more potent activation of LILRB2 than Angptl2, and we developed a serum-free culture containing defined cytokines and immobilized anti-LILRB2 that supports a net expansion of repopulating human cord blood HSCs. Our elucidation of the mode of Angptl binding to LILRB2 enabled the development of a new approach for ex vivo expansion of human HSCs.
Assuntos
Angiopoietinas/química , Angiopoietinas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/genética , Animais , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Xenoenxertos , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Imunológicos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
Large-conductance Ca(2+)-gated K(+) (BK) channels are essential for many biological processes such as smooth muscle contraction and neurotransmitter release. This group of channels can be activated synergistically by both voltage and intracellular Ca(2+), with the large carboxy-terminal intracellular portion being responsible for Ca(2+) sensing. Here we present the crystal structure of the entire cytoplasmic region of the human BK channel in a Ca(2+)-free state. The structure reveals four intracellular subunits, each comprising two tandem RCK domains, assembled into a gating ring similar to that seen in the MthK channel and probably representing its physiological assembly. Three Ca(2+) binding sites including the Ca(2+) bowl are mapped onto the structure based on mutagenesis data. The Ca(2+) bowl, located within the second RCK domain, forms an EF-hand-like motif and is strategically positioned close to the assembly interface between two subunits. The other two Ca(2+) (or Mg(2+)) binding sites, Asp 367 and Glu 374/Glu 399, are located on the first RCK domain. The Asp 367 site has high Ca(2+) sensitivity and is positioned in the groove between the amino- and carboxy-terminal subdomains of RCK1, whereas the low-affinity Mg(2+)-binding Glu 374/Glu 399 site is positioned on the upper plateau of the gating ring and close to the membrane. Our structure also contains the linker connecting the transmembrane and intracellular domains, allowing us to dock a voltage-gated K(+) channel pore of known structure onto the gating ring with reasonable accuracy and generate a structural model for the full BK channel.
Assuntos
Ativação do Canal Iônico , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Sítios de Ligação , Cálcio/análise , Cálcio/metabolismo , Cristalografia por Raios X , Citoplasma/metabolismo , Humanos , Ativação do Canal Iônico/fisiologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Magnésio/metabolismo , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismoRESUMO
The Na(+)-Ca(2+) exchanger (NCX) is a ubiquitously expressed plasma membrane protein. It plays a fundamental role in Ca(2+) homeostasis by moving Ca(2+) out of the cell using the electrochemical gradient of Na(+) as the driving force. Recent structural studies of a homologous archaebacterial exchanger, NCX_Mj, revealed its outward configuration with two potential ion permeation pathways exposed to the extracellular environment. Based on the symmetry of NCX_Mj structure, an atomic model of an inward-facing conformation was generated showing similar pathways but directed to the cytoplasm. The presence of these water-filled cavities has yet to be confirmed experimentally, and it is unknown if the mammalian exchanger adopts the same structure. In this study, we mutated multiple residues within transmembrane segments 2 and 7 of NCX1.1 (cardiac isoform) to cysteines, allowing us to investigate their sensitivity to membrane-impermeable sulfhydryl reagents as exchanger current block. By trapping NCX1.1 in the inward-facing configuration, we have mapped two differently sized cytoplasmic aqueous cavities, the access of which is modified during exchange. This data reveals movements of the protein associated with ion transport. Electrophysiological characterization shows that the conserved residues within transmembrane segments 2 and 7, coordinating Na(+) and Ca(2+) ions in NCX_Mj, play a fundamental role in NCX1.1. Our results suggest a similar architecture between the mammalian and archaebacterial exchangers.
Assuntos
Citoplasma/metabolismo , Miocárdio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sequência de Aminoácidos , Animais , Cisteína/genética , Citoplasma/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Íons , Mesilatos/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Trocador de Sódio e Cálcio/química , XenopusRESUMO
The structural and functional conversion of the nonselective NaK channel to a K(+) selective channel (NaK2K) allows us to identify two key residues, Tyr and Asp in the filter sequence of TVGYGD, that participate in interactions central to stabilizing the K(+) channel selectivity filter. By using protein crystallography and channel electrophysiology, we demonstrate that the K(+) channel filter exists as an energetically strained structure and requires these key protein interactions working in concert to hold the filter in the precisely defined four-sited configuration that is essential for selective K(+) permeation. Disruption of either interaction, as tested on both the NaK2K and eukaryotic K(v)1.6 channels, can reduce or completely abolish K(+) selectivity and in some cases may also lead to channel inactivation due to conformational changes at the filter. Additionally, on the scaffold of NaK we recapitulate the protein interactions found in the filter of the Kir channel family, which uses a distinct interaction network to achieve similar stabilization of the filter.