RESUMO
Theoretical work suggests that collective spatiotemporal behavior of integral membrane proteins should be modulated by boundary lipids sheathing their membrane anchors. Here, we show evidence for this prediction while investigating the mechanism for maintaining a steady amount of the active form of integral membrane protein Lck kinase (LckA) by Lck trans-autophosphorylation regulated by the phosphatase CD45. We used super-resolution microscopy, flow cytometry, and pharmacological and genetic perturbation to gain insight into the spatiotemporal context of this process. We found that LckA is generated exclusively at the plasma membrane, where CD45 maintains it in a ceaseless dynamic equilibrium with its unphosphorylated precursor. Steady LckA shows linear dependence, after an initial threshold, over a considerable range of Lck expression levels. This behavior fits a phenomenological model of trans-autophosphorylation that becomes more efficient with increasing LckA. We then challenged steady LckA formation by genetically swapping the Lck membrane anchor with structurally divergent ones, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45 that were expected to drastically modify Lck boundary lipids. We observed small but significant changes in LckA generation, except for the CD45 transmembrane domain that drastically reduced LckA due to its excessive lateral proximity to CD45. Comprehensively, LckA formation and maintenance can be best explained by lipid bilayer critical density fluctuations rather than liquid-ordered phase-separated nanodomains, as previously thought, with "like/unlike" boundary lipids driving dynamical proximity and remoteness of Lck with itself and with CD45.
Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Processamento de Proteína Pós-Traducional , Antígenos Comuns de Leucócito/metabolismo , Bicamadas Lipídicas/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Fosforilação , Domínios ProteicosRESUMO
Several essential ionotropic neurotransmitter receptors, including the nicotinic acetylcholine receptor (nAChR) and gamma-aminobutyric acid (GABA) type A receptor (GABAAr), belong to the family of pentameric ligand-gated ion channels (pLGICs). Function of these receptors is particularly sensitive to their lipid environment, including cholesterol and cholesterol-derived neurosteroids. Direct structural data investigating interactions between sterols and pLGICs, as well as their role in modulatory mechanisms, are largely unavailable. Physics-based computational approaches can serve a vital role in interpretation of more indirect data as well as hypothesis generation and experimental design. In this chapter, I report several examples in which computational approaches were used to predict direct binding interactions of steroids and pLGICs, evaluate the relative likelihood of possible interpretations of experimental data, and present rationally designed simple experiments. I conclude by offering several predictions that could be tested by future experiments.
Assuntos
Colesterol/metabolismo , Biologia Computacional/métodos , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Multimerização Proteica , Animais , Humanos , Ligação Proteica , Estrutura Quaternária de ProteínaRESUMO
Amphiphilic molecules with one or more perfluoroalkyl groups (Rf, CnF2n+1), which show peculiar interfacial properties, are attracting much attention in membrane protein science. We recently have developed a partially fluorinated dimyristoylphosphatidylcholine (DMPC) with a perfluorobutyl group in the hydrophobic chain terminal (F4-DMPC) and demonstrated that F4-DMPC is a promising material for incorporating membrane proteins. Moreover, we have found out that membrane properties of a series of partially fluorinated DMPCs with different Rf chain lengths (Fn-DMPCs) vary in a significant Rf chain length-dependent manner. In the present study, structural and functional properties of a membrane protein bacteriorhodopsin (bR) in the Fn-DMPC (n = 4, 6, and 8) membranes (bR/Fn-DMPC) are investigated using several physicochemical techniques. Regardless of the Rf chain lengths, bR/Fn-DMPCs retain native-like structural and functional properties at 30 °C, unlike bR molecules in DMPC vesicles. In particular, bR/F6-DMPC, which is in the fluid phase at 30 °C, shows flash-induced transient absorption changes very similar to the native purple membrane (PM) and very high thermal stability of bR trimers comparable to the PM. Structural and functional properties of bR/Fn-DMPCs are discussed compared to the PM and bR/DMPC.
Assuntos
Bacteriorodopsinas/química , Dimiristoilfosfatidilcolina/química , Fluorocarbonos/química , Lipossomos , Biopolímeros/química , Dicroísmo Circular , Halogenação , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Luz , Conformação Proteica , Estabilidade Proteica , Espectrofotometria UltravioletaRESUMO
We have investigated the perturbation of influenza A M2TM in DMPC bilayers. We have shown that (a) DSC and SAXS detect changes in membrane organization caused by small changes (micromolar) in M2TM or aminoadamantane concentration and aminoadamantane structure, by comparison of amantadine and spiro[pyrrolidine-2,2'-adamantane] (AK13), (b) that WAXS and MD can suggest details of ligand topology. DSC and SAXS show that at a low M2TM micromolar concentration in DPMC bilayers, two lipid domains are observed, which likely correspond to M2TM boundary lipids and bulk-like lipids. At higher M2TM concentrations, one domain only is identified, which constitutes essentially all of the lipid molecules behaving as boundary lipids. According to SAXS, WAXS, and DSC in the absence of M2TM, both aminoadamantane drugs exert a similar perturbing effect on the bilayer at low concentrations. At the same concentrations of the drug when M2TM is present, amantadine and, to a lesser extent, AK13 cause, according to WAXS, a significant disordering of chain-stacking, which also leads to the formation of two lipid domains. This effect is likely due, according to MD simulations, to the preference of the more lipophilic AK13 to locate closer to the lateral surfaces of M2TM when compared to amantadine, which forms stronger ionic interactions with phosphate groups. The preference of AK13 to concentrate inside the lipid bilayer close to the exterior of the hydrophobic M2TM helices may contribute to its higher binding affinity compared to amantadine.