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2.
PLoS Comput Biol ; 17(7): e1009232, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34280187

RESUMO

The T cell receptor (TCR-CD3) initiates T cell activation by binding to peptides of Major Histocompatibility Complexes (pMHC). The TCR-CD3 topology is well understood but the arrangement and dynamics of its cytoplasmic tails remains unknown, limiting our grasp of the signalling mechanism. Here, we use molecular dynamics simulations and modelling to investigate the entire TCR-CD3 embedded in a model membrane. Our study demonstrates conformational changes in the extracellular and transmembrane domains, and the arrangement of the TCR-CD3 cytoplasmic tails. The cytoplasmic tails formed highly interlaced structures while some tyrosines within the immunoreceptor tyrosine-based activation motifs (ITAMs) penetrated the hydrophobic core of the membrane. Interactions between the cytoplasmic tails and phosphatidylinositol phosphate lipids in the inner membrane leaflet led to the formation of a distinct anionic lipid fingerprint around the TCR-CD3. These results increase our understanding of the TCR-CD3 dynamics and the importance of membrane lipids in regulating T cell activation.


Assuntos
Modelos Moleculares , Complexo Receptor-CD3 de Antígeno de Linfócitos T/química , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Biologia Computacional , Simulação por Computador , Microscopia Crioeletrônica , Citoplasma/química , Citoplasma/metabolismo , Humanos , Ativação Linfocitária , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Complexo Receptor-CD3 de Antígeno de Linfócitos T/ultraestrutura , Eletricidade Estática , Linfócitos T/imunologia , Linfócitos T/metabolismo
3.
Cell Rep ; 36(2): 109375, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34260912

RESUMO

The mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRß or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαß and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαß cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαß cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαß to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation.

4.
Biophys J ; 120(8): 1510-1521, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33582135

RESUMO

Piezo1 is a mechanosensitive channel involved in many cellular functions and responsible for sensing shear stress and pressure forces in cells. Piezo1 has a unique trilobed topology with a curved membrane region in the closed state. It has been suggested that upon activation Piezo1 adopts a flattened conformation, but the molecular and structural changes underpinning the Piezo1 gating and opening mechanisms and how the channel senses forces in the membrane remain elusive. Here, we used molecular dynamics simulations to reveal the structural rearrangements that occur when Piezo1 moves from a closed to an open state in response to increased mechanical tension applied to a model membrane. We find that membrane stretching causes Piezo1 to flatten and expand its blade region, resulting in tilting and lateral movement of the pore-lining transmembrane helices 37 and 38. This is associated with the opening of the channel and movement of lipids out of the pore region. Our results reveal that because of the rather loose packing of Piezo1 pore region, movement of the lipids outside the pore region is critical for the opening of the pore. Our simulations also suggest synchronous flattening of the Piezo1 blades during Piezo1 activation. The flattened structure lifts the C-terminal extracellular domain up, exposing it more to the extracellular space. Our studies support the idea that it is the blade region of Piezo1 that senses tension in the membrane because pore opening failed in the absence of the blades. Additionally, our simulations reveal that upon opening, water molecules occupy lateral fenestrations in the cytosolic region of Piezo1, which might be likely paths for ion permeation. Our results provide a model for how mechanical force opens the Piezo1 channel and thus how it might couple mechanical force to biological response.


Assuntos
Ativação do Canal Iônico , Canais Iônicos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Mecanotransdução Celular , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína
5.
Biophys J ; 120(8): 1343-1356, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33582137

RESUMO

Piezo1 forms a mechanically activated calcium-permeable nonselective cation channel that is functionally important in many cell types. Structural data exist for C-terminal regions, but we lack information about N-terminal regions and how the entire channel interacts with the lipid bilayer. Here, we use computational approaches to predict the three-dimensional structure of the full-length Piezo1 and simulate it in an asymmetric membrane. A number of novel insights are suggested by the model: 1) Piezo1 creates a trilobed dome in the membrane that extends beyond the radius of the protein, 2) Piezo1 changes the lipid environment in its vicinity via preferential interactions with cholesterol and phosphatidylinositol 4,5-bisphosphate (PIP2) molecules, and 3) cholesterol changes the depth of the dome and PIP2 binding preference. In vitro alteration of cholesterol concentration inhibits Piezo1 activity in a manner complementing some of our computational findings. The data suggest the importance of N-terminal regions of Piezo1 for dome structure and membrane cholesterol and PIP2 interactions.


Assuntos
Canais Iônicos , Bicamadas Lipídicas , Colesterol , Canais Iônicos/genética , Fosfatidilinositóis
6.
Commun Biol ; 3(1): 766, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318620

RESUMO

The ß-barrel assembly machinery (BAM) catalyses the folding and insertion of ß-barrel outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria by mechanisms that remain unclear. Here, we present an ensemble of cryoEM structures of the E. coli BamABCDE (BAM) complex in lipid nanodiscs, determined using multi-body refinement techniques. These structures, supported by single-molecule FRET measurements, describe a range of motions in the BAM complex, mostly localised within the periplasmic region of the major subunit BamA. The ß-barrel domain of BamA is in a 'lateral open' conformation in all of the determined structures, suggesting that this is the most energetically favourable species in this bilayer. Strikingly, the BAM-containing lipid nanodisc is deformed, especially around BAM's lateral gate. This distortion is also captured in molecular dynamics simulations, and provides direct structural evidence for the lipid 'disruptase' activity of BAM, suggested to be an important part of its functional mechanism.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Bicamadas Lipídicas , Lipídeos , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Nanoestruturas , Multimerização Proteica , Proteínas da Membrana Bacteriana Externa/metabolismo , Catálise , Complexos Multiproteicos/metabolismo , Conformação Proteica , Dobramento de Proteína , Proteolipídeos/metabolismo
7.
Cell Rep ; 33(1): 108225, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33027663

RESUMO

Endogenous PIEZO1 channels of native endothelium lack the hallmark inactivation often seen when these channels are overexpressed in cell lines. Because prior work showed that the force of shear stress activates sphingomyelinase in endothelium, we considered if sphingomyelinase is relevant to endogenous PIEZO1. Patch clamping was used to quantify PIEZO1-mediated signals in freshly isolated murine endothelium exposed to the mechanical forces caused by shear stress and membrane stretch. Neutral sphingomyelinase inhibitors and genetic disruption of sphingomyelin phosphodiesterase 3 (SMPD3) cause PIEZO1 to switch to profoundly inactivating behavior. Ceramide (a key product of SMPD3) rescues non-inactivating channel behavior. Its co-product, phosphoryl choline, has no effect. In contrast to ceramide, sphingomyelin (the SMPD3 substrate) does not affect inactivation but alters channel force sensitivity. The data suggest that sphingomyelinase activity, ceramide, and sphingomyelin are determinants of native PIEZO gating that enable sustained activity.


Assuntos
Canais Iônicos/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Humanos , Camundongos
8.
Nat Commun ; 11(1): 2155, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358557

RESUMO

The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but its mechanisms of client binding and chaperone function have remained unclear. Here, we use chemical cross-linking, hydrogen-deuterium exchange mass spectrometry, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and interrogate the role of conformational dynamics in OMP recognition. We demonstrate that SurA samples an array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested in the SurA crystal structure. OMP binding sites are located primarily in the core domain, and OMP binding results in conformational changes between the core/P1 domains. Together, the results suggest that unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between domains assisting OMP recognition, binding and release.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Sítios de Ligação , Proteínas de Transporte/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Espectrometria de Massas , Chaperonas Moleculares/genética , Peptidilprolil Isomerase/genética , Ligação Proteica
10.
Sci Adv ; 6(8): eaay5736, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32128410

RESUMO

Association of peripheral proteins with lipid bilayers regulates membrane signaling and dynamics. Pleckstrin homology (PH) domains bind to phosphatidylinositol phosphate (PIP) molecules in membranes. The effects of local PIP enrichment on the interaction of PH domains with membranes is unclear. Molecular dynamics simulations allow estimation of the binding energy of GRP1 PH domain to PIP3-containing membranes. The free energy of interaction of the PH domain with more than two PIP3 molecules is comparable to experimental values, suggesting that PH domain binding involves local clustering of PIP molecules within membranes. We describe a mechanism of PH binding proceeding via an encounter state to two bound states which differ in the orientation of the protein relative to the membrane, these orientations depending on the local PIP concentration. These results suggest that nanoscale clustering of PIP molecules can control the strength and orientation of PH domain interaction in a concentration-dependent manner.


Assuntos
Sítios de Ligação , Membrana Celular/química , Lipídeos/química , Fosfatidilinositóis/química , Domínios de Homologia à Plecstrina , Algoritmos , Membrana Celular/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Modelos Teóricos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo
11.
Front Mol Biosci ; 6: 132, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824962

RESUMO

Membrane integral pyrophosphatases (mPPases) are responsible for the hydrolysis of pyrophosphate. This enzymatic mechanism is coupled to the pumping of H+ or Na+ across membranes in a process that can be K+ dependent or independent. Understanding the movements and dynamics throughout the mPPase catalytic cycle is important, as this knowledge is essential for improving or impeding protein function. mPPases have been shown to play a crucial role in plant maturation and abiotic stress tolerance, and so have the potential to be engineered to improve plant survival, with implications for global food security. mPPases are also selectively toxic drug targets, which could be pharmacologically modulated to reduce the virulence of common human pathogens. The last few years have seen the publication of many new insights into the function and structure of mPPases. In particular, there is a new body of evidence that the catalytic cycle is more complex than originally proposed. There are structural and functional data supporting a mechanism involving half-of-the-sites reactivity, inter-subunit communication, and exit channel motions. A more advanced and in-depth understanding of mPPases has begun to be uncovered, leaving the field of research with multiple interesting avenues for further exploration and investigation.

12.
Biophys J ; 117(7): 1364-1379, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31540709

RESUMO

Anion exchanger 1 (AE1) is responsible for the exchange of bicarbonate and chloride across the erythrocyte plasma membrane. Human AE1 consists of a cytoplasmic and a membrane domain joined by a 33-residue flexible linker. Crystal structures of the individual domains have been determined, but the intact AE1 structure remains elusive. In this study, we use molecular dynamics simulations and modeling to build intact AE1 structures in a complex lipid bilayer that resembles the native erythrocyte plasma membrane. AE1 models were evaluated using available experimental data to provide an atomistic view of the interaction and dynamics of the cytoplasmic domain, the membrane domain, and the connecting linker in a complete model of AE1 in a lipid bilayer. Anionic lipids were found to interact strongly with AE1 at specific amino acid residues that are linked to diseases and blood group antigens. Cholesterol was found in the dimeric interface of AE1, suggesting that it may regulate subunit interactions and anion transport.


Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/química , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Lipídeos/química , Simulação de Dinâmica Molecular , Ânions , Humanos , Ligação Proteica , Domínios Proteicos , Multimerização Proteica
13.
Arterioscler Thromb Vasc Biol ; 39(11): 2228-2239, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31533470

RESUMO

Mechanical forces are fundamental in cardiovascular biology, and deciphering the mechanisms by which they act remains a testing frontier in cardiovascular research. Here, we raise awareness of 2 recently discovered proteins, Piezo1 and Piezo2, which assemble as transmembrane triskelions to combine exquisite force sensing with regulated calcium influx. There is emerging evidence for their importance in endothelial shear stress sensing and secretion, NO generation, vascular tone, angiogenesis, atherosclerosis, vascular permeability and remodeling, blood pressure regulation, insulin sensitivity, exercise performance, and baroreceptor reflex, and there are early suggestions of relevance to cardiac fibroblasts and myocytes. Human genetic analysis points to significance in lymphatic disease, anemia, varicose veins, and potentially heart failure, hypertension, aneurysms, and stroke. These channels appear to be versatile force sensors, used creatively to inform various force-sensing situations. We discuss emergent concepts and controversies and suggest that the potential for new important understanding is substantial.


Assuntos
Canais de Cálcio/metabolismo , Doenças Cardiovasculares/fisiopatologia , Endotélio Vascular/fisiologia , Canais Iônicos/fisiologia , Mecanotransdução Celular , Animais , Fenômenos Fisiológicos Cardiovasculares , Humanos , Canais Iônicos/genética , Mutação
14.
Structure ; 27(8): 1336-1346.e2, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31204251

RESUMO

Phosphatidylinositol phosphates (PIPs) are lipid signaling molecules that play key roles in many cellular processes. PIP5K1A kinase catalyzes phosphorylation of PI4P to form PIP2, which in turn interacts with membrane and membrane-associated proteins. We explore the mechanism of membrane binding by the PIP5K1A kinase using a multiscale molecular dynamics approach. Coarse-grained simulations show binding of monomeric PIP5K1A to a model cell membrane containing PI4P. PIP5K1A did not bind to zwitterionic or anionic membranes lacking PIP molecules. Initial encounter of kinase and bilayer was followed by reorientation to enable productive binding to the PI4P-containing membrane. The simulations suggest that unstructured regions may be important for the preferred orientation for membrane binding. Atomistic simulations indicated that the dimeric kinase could not bind to the membrane via both active sites at the same time, suggesting a conformational change in the protein and/or bilayer distortion may be needed for dual-site binding to occur.


Assuntos
Membrana Celular/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Humanos , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína
15.
J Cell Sci ; 132(11)2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31076511

RESUMO

Endothelial cell (EC) sensing of fluid shear stress direction is a critical determinant of vascular health and disease. Unidirectional flow induces EC alignment and vascular homeostasis, whereas bidirectional flow has pathophysiological effects. ECs express several mechanoreceptors that respond to flow, but the mechanism for sensing shear stress direction is poorly understood. We determined, by using in vitro flow systems and magnetic tweezers, that ß1 integrin is a key sensor of force direction because it is activated by unidirectional, but not bidirectional, shearing forces. ß1 integrin activation by unidirectional force was amplified in ECs that were pre-sheared in the same direction, indicating that alignment and ß1 integrin activity has a feedforward interaction, which is a hallmark of system stability. En face staining and EC-specific genetic deletion studies in the murine aorta revealed that ß1 integrin is activated and is essential for EC alignment at sites of unidirectional flow but is not activated at sites of bidirectional flow. In summary, ß1 integrin sensing of unidirectional force is a key mechanism for decoding blood flow mechanics to promote vascular homeostasis.This article has an associated First Person interview with the first author of the paper.


Assuntos
Aorta/fisiologia , Integrina beta1/metabolismo , Fluxo Sanguíneo Regional/fisiologia , Animais , Linhagem Celular , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrina beta1/genética , Mecanorreceptores/fisiologia , Camundongos , Camundongos Knockout , Estresse Fisiológico/fisiologia
16.
PLoS Comput Biol ; 14(7): e1006284, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30011272

RESUMO

The Band 3 (AE1, SLC4A1) membrane protein is found in red blood cells and in kidney where it functions as an electro-neutral chloride/bicarbonate exchanger. In this study, we have used molecular dynamics simulations to provide the first realistic model of the dimeric membrane domain of human Band 3 in an asymmetric lipid bilayer containing a full complement of phospholipids, including phosphatidylinositol 4,5-bisphosphate (PIP2) and cholesterol, and its partner membrane protein Glycophorin A (GPA). The simulations show that the annular layer in the inner leaflet surrounding Band 3 was enriched in phosphatidylserine and PIP2 molecules. Cholesterol was also enriched around Band 3 but also at the dimer interface. The interaction of these lipids with specific sites on Band 3 may play a role in the folding and function of this anion transport membrane protein. GPA associates with Band 3 to form the Wright (Wr) blood group antigen, an interaction that involves an ionic bond between Glu658 in Band 3 and Arg61 in GPA. We were able to recreate this complex by performing simulations to allow the dimeric transmembrane portion of GPA to interact with Band 3 in a model membrane. Large-scale simulations showed that the GPA dimer can bridge Band 3 dimers resulting in the dynamic formation of long strands of alternating Band 3 and GPA dimers.


Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Antígenos de Grupos Sanguíneos/metabolismo , Glicoforinas/metabolismo , Fosfolipídeos/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/química , Ânions , Arginina/metabolismo , Antígenos de Grupos Sanguíneos/química , Colesterol/metabolismo , Dimerização , Ácido Glutâmico/metabolismo , Glicoforinas/química , Humanos , Bicamadas Lipídicas , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Dobramento de Proteína
17.
Structure ; 26(7): 1025-1034.e2, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29887500

RESUMO

EphA2 is a member of the receptor tyrosine kinase family. Interactions of the cytoplasmic region of EphA2 with the cell membrane are functionally important and yet remain incompletely characterized. Molecular dynamics simulations combined with biochemical studies reveal the interactions of the transmembrane, juxtamembrane (JM), and kinase domains with the membrane. We describe how the kinase domain is oriented relative to the membrane and how the JM region can modulate this interaction. We highlight the role of phosphatidylinositol phosphates (PIPs) in mediating the interaction of the kinase domain with the membrane and, conversely, how positively charged patches at the kinase surface and in the JM region induce the formation of nanoclusters of PIP molecules in the membrane. Integration of these results with those from previous studies enable computational reconstitution of a near complete EphA2 receptor within a membrane, suggesting a role for receptor-lipid interactions in modulation of EphA2.


Assuntos
Fosfatos de Fosfatidilinositol/metabolismo , Receptor EphA2/química , Receptor EphA2/metabolismo , Sítios de Ligação , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica
18.
J Biol Chem ; 293(24): 9335-9344, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29724824

RESUMO

Kidney- and brain-expressed protein (KIBRA), a multifunctional scaffold protein with around 20 known binding partners, is involved in memory and cognition, organ size control via the Hippo pathway, cell polarity, and membrane trafficking. KIBRA includes tandem N-terminal WW domains, a C2 domain, and motifs for binding atypical PKC and PDZ domains. A naturally occurring human KIBRA variant involving residue changes at positions 734 (Met-to-Ile) and 735 (Ser-to-Ala) within the C2 domain affects cognitive performance. We have elucidated 3D structures and calcium- and phosphoinositide-binding properties of human KIBRA C2 domain. Both WT and variant C2 adopt a canonical type I topology C2 domain fold. Neither Ca2+ nor any other metal ion was bound to WT or variant KIBRA C2 in crystal structures, and Ca2+ titration produced no significant reproducible changes in NMR spectra. NMR and X-ray diffraction data indicate that KIBRA C2 binds phosphoinositides via an atypical site involving ß-strands 5, 2, 1, and 8. Molecular dynamics simulations indicate that KIBRA C2 interacts with membranes via primary and secondary sites on the same domain face as the experimentally identified phosphoinositide-binding site. Our results indicate that KIBRA C2 domain association with membranes is calcium-independent and involves distinctive C2 domain-membrane relative orientations.


Assuntos
Cálcio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfatidilinositóis/metabolismo , Fosfoproteínas/metabolismo , Domínios C2 , Membrana Celular/metabolismo , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/genética , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Conformação Proteica
19.
Cell Chem Biol ; 25(7): 840-848.e4, 2018 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-29681524

RESUMO

The role of membrane lipids in modulating eukaryotic transporter assembly and function remains unclear. We investigated the effect of membrane lipids in the structure and transport activity of the purine transporter UapA from Aspergillus nidulans. We found that UapA exists mainly as a dimer and that two lipid molecules bind per UapA dimer. We identified three phospholipid classes that co-purified with UapA: phosphatidylcholine, phosphatidylethanolamine (PE), and phosphatidylinositol (PI). UapA delipidation caused dissociation of the dimer into monomers. Subsequent addition of PI or PE rescued the UapA dimer and allowed recovery of bound lipids, suggesting a central role of these lipids in stabilizing the dimer. Molecular dynamics simulations predicted a lipid binding site near the UapA dimer interface. Mutational analyses established that lipid binding at this site is essential for formation of functional UapA dimers. We propose that structural lipids have a central role in the formation of functional, dimeric UapA.


Assuntos
Eucariotos/química , Proteínas Fúngicas/química , Proteínas de Membrana Transportadoras/química , Fosfolipídeos/química , Sítios de Ligação , Eucariotos/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Simulação de Dinâmica Molecular , Estrutura Molecular , Fosfolipídeos/metabolismo
20.
J Mol Biol ; 430(3): 372-388, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29273202

RESUMO

Pleckstrin homology (PH) domains mediate protein-membrane interactions by binding to phosphatidylinositol phosphate (PIP) molecules. The structural and energetic basis of selective PH-PIP interactions is central to understanding many cellular processes, yet the molecular complexities of the PH-PIP interactions are largely unknown. Molecular dynamics simulations using a coarse-grained model enables estimation of free-energy landscapes for the interactions of 12 different PH domains with membranes containing PIP2 or PIP3, allowing us to obtain a detailed molecular energetic understanding of the complexities of the interactions of the PH domains with PIP molecules in membranes. Distinct binding modes, corresponding to different distributions of cationic residues on the PH domain, were observed, involving PIP interactions at either the "canonical" (C) and/or "alternate" (A) sites. PH domains can be grouped by the relative strength of their C- and A-site interactions, revealing that a higher affinity correlates with increased C-site interactions. These simulations demonstrate that simultaneous binding of multiple PIP molecules by PH domains contributes to high-affinity membrane interactions, informing our understanding of membrane recognition by PH domains in vivo.


Assuntos
Bicamadas Lipídicas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Domínios de Homologia à Plecstrina , Termodinâmica , Animais , Sítios de Ligação , Bases de Dados de Proteínas , Humanos , Cinética , Bicamadas Lipídicas/química , Membranas Artificiais , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fosfatos de Fosfatidilinositol/química , Ligação Proteica
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