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1.
Biophys J ; 123(17): 2934-2955, 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-38932456

RESUMO

Biomolecules often exhibit complex free energy landscapes in which long-lived metastable states are separated by large energy barriers. Overcoming these barriers to robustly sample transitions between the metastable states with classical molecular dynamics (MD) simulations presents a challenge. To circumvent this issue, collective variable (CV)-based enhanced sampling MD approaches are often employed. Traditional CV selection relies on intuition and prior knowledge of the system. This approach introduces bias, which can lead to incomplete mechanistic insights. Thus, automated CV detection is desired to gain a deeper understanding of the system/process. Analysis of MD data with various machine-learning algorithms, such as principal component analysis (PCA), support vector machine, and linear discriminant analysis (LDA) based approaches have been implemented for automated CV detection. However, their performance has not been systematically evaluated on structurally and mechanistically complex biological systems. Here, we applied these methods to MD simulations of the MFSD2A (Major Facilitator Superfamily Domain 2A) lysolipid transporter in multiple functionally relevant metastable states with the goal of identifying optimal CVs that would structurally discriminate these states. Specific emphasis was on the automated detection and interpretive power of LDA-based CVs. We found that LDA methods, which included a novel gradient descent-based multiclass harmonic variant, termed GDHLDA, we developed here, outperform PCA in class separation, exhibiting remarkable consistency in extracting CVs critical for distinguishing metastable states. Furthermore, the identified CVs included features previously associated with conformational transitions in MFSD2A. Specifically, conformational shifts in transmembrane helix 7 and in residue Y294 on this helix emerged as critical features discriminating the metastable states in MFSD2A. This highlights the effectiveness of LDA-based approaches in automatically extracting from MD trajectories CVs of functional relevance that can be used to drive biased MD simulations to efficiently sample conformational transitions in the molecular system.


Assuntos
Simportadores , Automação , Análise Discriminante , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Simulação de Dinâmica Molecular , Análise de Componente Principal , Simportadores/química
2.
J Biol Chem ; 298(7): 102058, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35605664

RESUMO

There is substantial evidence for extensive nonvesicular sterol transport in cells. For example, lipid transfer by the steroidogenic acute regulator-related proteins (StarD) containing a StarT domain has been shown to involve several pathways of nonvesicular trafficking. Among the soluble StarT domain-containing proteins, StarD4 is expressed in most tissues and has been shown to be an effective sterol transfer protein. However, it was unclear whether the lipid composition of donor or acceptor membranes played a role in modulating StarD4-mediated transport. Here, we used fluorescence-based assays to demonstrate a phosphatidylinositol phosphate (PIP)-selective mechanism by which StarD4 can preferentially extract sterol from liposome membranes containing certain PIPs (especially, PI(4,5)P2 and to a lesser degree PI(3,5)P2). Monophosphorylated PIPs and other anionic lipids had a smaller effect on sterol transport. This enhancement of transport was less effective when the same PIPs were present in the acceptor membranes. Furthermore, using molecular dynamics (MD) simulations, we mapped the key interaction sites of StarD4 with PIP-containing membranes and identified residues that are important for this interaction and for accelerated sterol transport activity. We show that StarD4 recognizes membrane-specific PIPs through specific interaction with the geometry of the PIP headgroup as well as the surrounding membrane environment. Finally, we also observed that StarD4 can deform membranes upon longer incubations. Taken together, these results suggest a mechanism by which PIPs modulate cholesterol transfer activity via StarD4.


Assuntos
Proteínas de Membrana Transportadoras , Esteróis , Transporte Biológico , Lipossomos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fosfatos de Fosfatidilinositol , Esteróis/metabolismo
3.
J Colloid Interface Sci ; 674: 813-822, 2024 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-38955012

RESUMO

A heterojunction of trace Co3O4 bonded on oxygen vacancies (OVs)-rich ZnO (OVs-ZnO/Co3O4) was synthesized via defect-assisted method to promote peroxymonosulfate (PMS) activation and pollutants degradation. Experiments and theoretical calculations demonstrated that electrons could efficiently transfer from OVs-ZnO to Co3O4. OVs-ZnO and Co3O4 played different roles in activating PMS. PMS was easily adsorbed on the OVs-ZnO to form PMS* complex and mediated electron transfer to oxide ciprofloxacin (CIP), whereas, Co3O4 facilitated breakup of peroxide bond to produce radicals. The optimal OVs-ZnO/Co3O4 with Co content of 1.34% exhibited good PMS decomposition ability (94.2% in 30 min) compared to unmodified ZnO (24.2%), stability and anti-interference feature in removing CIP, 96.9% CIP (10 ppm) and 79.6% of total organic carbon were removed in 30 min. Moreover, the OVs-ZnO/Co3O4 achieved 91.2% CIP removal ratio with 1.0 mM PMS via a flow-through device in 180 min. This study proposes a new strategy to enhance PMS activation of ZnO and provides new viewpoint in PMS activation way.

4.
Front Mol Biosci ; 10: 1197154, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37275961

RESUMO

Complex mechanisms regulate the cellular distribution of cholesterol, a critical component of eukaryote membranes involved in regulation of membrane protein functions directly and through the physiochemical properties of membranes. StarD4, a member of the steroidogenic acute regulator-related lipid-transfer (StART) domain (StARD)-containing protein family, is a highly efficient sterol-specific transfer protein involved in cholesterol homeostasis. Its mechanism of cargo loading and release remains unknown despite recent insights into the key role of phosphatidylinositol phosphates in modulating its interactions with target membranes. We have used large-scale atomistic Molecular dynamics (MD) simulations to study how the dynamics of cholesterol bound to the StarD4 protein can affect interaction with target membranes, and cargo delivery. We identify the two major cholesterol (CHL) binding modes in the hydrophobic pocket of StarD4, one near S136&S147 (the Ser-mode), and another closer to the putative release gate located near W171, R92&Y117 (the Trp-mode). We show that conformational changes of StarD4 associated directly with the transition between these binding modes facilitate the opening of the gate. To understand the dynamics of this connection we apply a machine-learning algorithm for the detection of rare events in MD trajectories (RED), which reveals the structural motifs involved in the opening of a front gate and a back corridor in the StarD4 structure occurring together with the spontaneous transition of CHL from the Ser-mode of binding to the Trp-mode. Further analysis of MD trajectory data with the information-theory based NbIT method reveals the allosteric network connecting the CHL binding site to the functionally important structural components of the gate and corridor. Mutations of residues in the allosteric network are shown to affect the performance of the allosteric connection. These findings outline an allosteric mechanism which prepares the CHL-bound StarD4 to release and deliver the cargo when it is bound to the target membrane.

5.
ACS Chem Neurosci ; 13(7): 987-1001, 2022 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-35258946

RESUMO

Abnormal aggregation of amyloid ß (Aß) peptides into fibrils plays a critical role in the development of Alzheimer's disease. A two-stage "dock-lock" model has been proposed for the Aß fibril elongation process. However, the mechanisms of the Aß monomer-fibril binding process have not been elucidated with the necessary molecular-level precision, so it remains unclear how the lock phase dynamics leads to the overall in-register binding of the Aß monomer onto the fibril. To gain mechanistic insights into this critical step during the fibril elongation process, we used molecular dynamics (MD) simulations with a physics-based coarse-grained UNited-RESidue (UNRES) force field and sampled extensively the dynamics of the lock phase process, in which a fibril-bound Aß(9-40) peptide rearranged to establish the native docking conformation. Analysis of the MD trajectories with Markov state models was used to quantify the kinetics of the rearrangement process and the most probable pathways leading to the overall native docking conformation of the incoming peptide. These revealed a key intermediate state in which an intra-monomer hairpin is formed between the central core amyloidogenic patch 18VFFA21 and the C-terminal hydrophobic patch 34LMVG37. This hairpin structure is highly favored as a transition state during the lock phase of the fibril elongation. We propose a molecular mechanism for facilitation of the Aß fibril elongation by amyloidogenic hydrophobic patches.


Assuntos
Peptídeos beta-Amiloides , Fragmentos de Peptídeos , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Cinética , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/metabolismo
6.
Elife ; 82019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31343409

RESUMO

FcγRIIB binding to its ligand suppresses immune cell activation. A single-nucleotide polymorphic (SNP) change, I232T, in the transmembrane (TM) domain of FcγRIIB loses its suppressive function, which is clinically associated with systemic lupus erythematosus (SLE). Previously, we reported that I232T tilts FcγRIIB's TM domain. In this study, combining with molecular dynamics simulations and single-cell FRET assay, we further reveal that such tilting by I232T unexpectedly bends the FcγRIIB's ectodomain toward plasma membrane to allosterically impede FcγRIIB's ligand association. I232T substitution reduces in situ two-dimensional binding affinities and association rates of FcγRIIB to interact with its ligands, IgG1, IgG2 and IgG3 by three to four folds. This allosteric regulation by an SNP provides an intrinsic molecular mechanism for the functional loss of FcγRIIB-I232T in SLE patients.


Assuntos
Imunoglobulina G/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Receptores de IgG/genética , Receptores de IgG/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Receptores de IgG/química
7.
Sci Adv ; 5(7): eaaw0315, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31328160

RESUMO

B cell activation is regulated by the stimulatory or inhibitory co-receptors of B cell receptors (BCRs). Here, we investigated the signaling mechanism of Fc receptor-like 1 (FcRL1), a newly identified BCR co-receptor. FcRL1 was passively recruited into B cell immunological synapses upon BCR engagement in the absence of FcRL1 cross-linking, suggesting that FcRL1 may intrinsically regulate B cell activation and function. BCR cross-linking alone led to the phosphorylation of the intracellular Y281ENV motif of FcRL1 to provide a docking site for c-Abl, an SH2 domain-containing kinase. The FcRL1 and c-Abl signaling module, in turn, potently augmented B cell activation and proliferation. FcRL1-deficient mice exhibited markedly impaired formation of extrafollicular plasmablasts and germinal centers, along with decreased antibody production upon antigen stimulation. These findings reveal a critical BCR signal-enhancing function of FcRL1 through its intrinsic recruitment to B cell immunological synapses and subsequent recruitment of c-Abl upon BCR cross-linking.


Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Deleção de Genes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Domínios de Homologia de src
8.
Sci Immunol ; 3(29)2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30504208

RESUMO

Protective antibody responses to vaccination or infection depend on affinity maturation, a process by which high-affinity germinal center (GC) B cells are selected on the basis of their ability to bind, gather, and present antigen to T follicular helper (Tfh) cells. Here, we show that human GC B cells have intrinsically higher-affinity thresholds for both B cell antigen receptor (BCR) signaling and antigen gathering as compared with naïve B cells and that these functions are mediated by distinct cellular structures and pathways that ultimately lead to antigen affinity- and Tfh cell-dependent differentiation to plasma cells. GC B cells bound antigen through highly dynamic, actin- and ezrin-rich pod-like structures that concentrated BCRs. The behavior of these structures was dictated by the intrinsic antigen affinity thresholds of GC B cells. Low-affinity antigens triggered continuous engagement and disengagement of membrane-associated antigens, whereas high-affinity antigens induced stable synapse formation. The pod-like structures also mediated affinity-dependent antigen internalization by unconventional pathways distinct from those of naïve B cells. Thus, intrinsic properties of human GC B cells set thresholds for affinity selection.


Assuntos
Afinidade de Anticorpos/imunologia , Apresentação de Antígeno/imunologia , Linfócitos B/imunologia , Centro Germinativo/citologia , Centro Germinativo/imunologia , Reações Antígeno-Anticorpo , Humanos
9.
J Cell Biol ; 217(7): 2565-2582, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29685902

RESUMO

B lymphocytes use B cell receptors (BCRs) to sense the chemical and physical features of antigens. The activation of isotype-switched IgG-BCR by mechanical force exhibits a distinct sensitivity and threshold in comparison with IgM-BCR. However, molecular mechanisms governing these differences remain to be identified. In this study, we report that the low threshold of IgG-BCR activation by mechanical force is highly dependent on tethering of the cytoplasmic tail of the IgG-BCR heavy chain (IgG-tail) to the plasma membrane. Mechanistically, we show that the positively charged residues in the IgG-tail play a crucial role by highly enriching phosphatidylinositol (4,5)-biphosphate (PI(4,5)P2) into the membrane microdomains of IgG-BCRs. Indeed, manipulating the amounts of PI(4,5)P2 within IgG-BCR membrane microdomains significantly altered the threshold and sensitivity of IgG-BCR activation. Our results reveal a lipid-dependent mechanism for determining the threshold of IgG-BCR activation by mechanical force.


Assuntos
Linfócitos B/imunologia , Ativação Linfocitária/imunologia , Fenômenos Mecânicos , Receptores de IgG/imunologia , Animais , Linfócitos B/química , Membrana Celular/química , Membrana Celular/imunologia , Humanos , Camundongos , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de IgG/química , Transdução de Sinais/imunologia
10.
Sci Immunol ; 2(17)2017 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-29150438

RESUMO

Lymphocytes have evolved sophisticated signaling amplification mechanisms to efficiently activate downstream signaling after detection of rare ligands in their microenvironment. B cell receptor microscopic clusters (BCR microclusters) are assembled on the plasma membrane and recruit signaling molecules for the initiation of lymphocyte signaling after antigen binding. We identified a signaling amplification loop derived from phosphatidylinositol 4,5-biphosphate (PIP2) for the sustained B cell activation. Upon antigen recognition, PIP2 was depleted by phospholipase C-γ2 (PLC-γ2) within the BCR microclusters and was regenerated by phosphatidic acid-dependent type I phosphatidylinositol 4-phosphate 5-kinase outside the BCR microclusters. The hydrolysis of PIP2 inside the BCR microclusters induced a positive feedback mechanism for its synthesis outside the BCR microclusters. The falling gradient of PIP2 across the boundary of BCR microclusters was important for the efficient formation of BCR microclusters. Our results identified a PIP2-derived amplification loop that fuels the sustained initiation of B cell activation.


Assuntos
Linfócitos B/imunologia , Ativação Linfocitária/imunologia , Fosfatidilinositol 4,5-Difosfato/imunologia , Transdução de Sinais/imunologia , Animais , Linfócitos B/metabolismo , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Microscopia de Fluorescência , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase C gama/imunologia , Fosfolipase C gama/metabolismo , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo
11.
J Exp Med ; 213(12): 2707-2727, 2016 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-27799621

RESUMO

FcγRIIB functions to suppress the activation of immune cells. A single-nucleotide polymorphism in the transmembrane (TM) domain of FcγRIIB, FcγRIIB-T232, is associated with lupus. In this study, we investigated the pathogenic mechanism of FcγRIIB-T232 at both functional and structural levels. Our results showed that FcγRIIB-T232 exhibited significantly reduced lateral mobility compared with FcγRIIB-I232 and was significantly less enriched into the microclusters of immune complexes (ICs) after stimulation. However, if sufficient responding time is given for FcγRIIB-T232 to diffuse and interact with the ICs, FcγRIIB-T232 can restore its inhibitory function. Moreover, substituting the FcγRIIB-T232 TM domain with that of a fast floating CD86 molecule restored both the rapid mobility and the inhibitory function, which further corroborated the importance of fast mobility for FcγRIIB to function. Mechanistically, the crippled lateral mobility of FcγRIIB-T232 can be explained by the structural changes of the TM domain. Both atomistic simulations and nuclear magnetic resonance measurement indicated that the TM helix of FcγRIIB-T232 exhibited a more inclined orientation than that of FcγRIIB-I232, thus resulting in a longer region embedded in the membrane. Therefore, we conclude that the single-residue polymorphism T232 enforces the inclination of the TM domain and thereby reduces the lateral mobility and inhibitory functions of FcγRIIB.


Assuntos
Lúpus Eritematoso Sistêmico/genética , Polimorfismo de Nucleotídeo Único/genética , Receptores de IgG/química , Receptores de IgG/genética , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo/metabolismo , Linfócitos B/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Difusão , Recuperação de Fluorescência Após Fotodegradação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Lipídeos de Membrana/metabolismo , Modelos Biológicos , Simulação de Dinâmica Molecular , Monócitos/metabolismo , Estrutura Secundária de Proteína , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de IgG/metabolismo , Imagem Individual de Molécula
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