Modeling fatty acid delivery from intestinal fatty acid binding protein to a membrane.
ABSTRACT: Intestinal fatty acid binding protein (IFABP) interacts with biological membranes and delivers fatty acid (FA) into them via a collisional mechanism. However, the membrane-bound structure of the protein and the pathway of FA transfer are not precisely known. We used molecular dynamics (MD) simulations with an implicit membrane model to determine the optimal orientation of apo- and holo-IFABP (bound with palmitate) on an anionic membrane. In this orientation, the helical portal region, delimited by the alphaII helix and the betaC-betaD and betaE-betaF turns, is oriented toward the membrane whereas the putative beta-strand portal, delimited by the betaB-betaC, betaF-betaG, betaH-betaI turns and the N terminus, is exposed to solvent. Starting from the MD structure of holo-IFABP in the optimal orientation relative to the membrane, we examined the release of palmitate via both pathways. Although the domains can widen enough to allow the passage of palmitate, fatty acid release through the helical portal region incurs smaller conformational changes and a lower energetic cost.
Project description:Intestinal FABP (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms of fatty acid transfer to acceptor model membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP transfer occurs by diffusion through the aqueous phase. In addition, transfer from IFABP is markedly faster than from LFABP. The overall goal of this study was to further explore the structural differences between IFABP and LFABP which underlie their large functional differences in ligand transport. In particular, we addressed the role of the alphaI-helix domain in the unique transport properties of intestinal FABP. A chimeric protein was engineered with the 'body' (ligand binding domain) of IFABP and the alphaI-helix of LFABP (alpha(I)LbetaIFABP), and the fatty acid transfer properties of the chimeric FABP were examined using a fluorescence resonance energy transfer assay. The results showed a significant decrease in the absolute rate of FA transfer from alpha(I)LbetaIFABP compared to IFABP. The results indicate that the alphaI-helix is crucial for IFABP collisional FA transfer, and further indicate the participation of the alphaII-helix in the formation of a protein-membrane "collisional complex". Photo-crosslinking experiments with a photoactivable reagent demonstrated the direct interaction of IFABP with membranes and further support the importance of the alphaI helix of IFABP in its physical interaction with membranes.
Project description:We crystallized human liver fatty acid-binding protein (LFABP) in apo, holo, and intermediate states of palmitic acid engagement. Structural snapshots of fatty acid recognition, entry, and docking within LFABP support a heads-in mechanism for ligand entry. Apo-LFABP undergoes structural remodeling, where the first palmitate ingress creates the atomic environment for placement of the second palmitate. These new mechanistic insights will facilitate development of pharmacological agents against LFABP.
Project description:Interleukin (IL)-5 exerts hematopoietic functions through binding to the IL-5 receptor subunits, alpha and betac. Specific assembly steps of full-length subunits as they occur in cell membranes, ultimately leading to receptor activation, are not well understood. We tracked the oligomerization of IL-5 receptor subunits using fluorescence resonance energy transfer (FRET) imaging. Full-length IL-5Ralpha and betac were expressed in Phoenix cells as chimeric proteins fused to enhanced cyan or yellow fluorescent protein (CFP or YFP, respectively). A time- and dose-dependent increase in FRET signal between IL-5Ralpha-CFP and betac-YFP was observed in response to IL-5, indicative of heteromeric receptor alpha-betac subunit interaction. This response was inhibited by AF17121, a peptide antagonist of IL-5Ralpha. Substantial FRET signals with betac-CFP and betac-YFP co-expressed in the absence of IL-5Ralpha demonstrated that betac subunits exist as preformed homo-oligomers. IL-5 had no effect on this betac-alone FRET signal. Interestingly, the addition of IL-5 to cells co-expressing betac-CFP, betac-YFP, and nontagged IL-5Ralpha led to further increase in FRET efficiency. Observation of preformed betac oligomers fits with the view that this form can lead to rapid cellular responses upon IL-5 stimulation. The IL-5-induced effects on betac assembly in the presence of nontagged IL-5Ralpha provide direct evidence that IL-5 can cause higher order rearrangements of betac homo-oligomers. These results suggest that IL-5 and perhaps other betac cytokines (IL-3 and granulocyte/macrophage colony-stimulating factor) trigger cellular responses by the sequential binding of cytokine ligand to the specificity receptor (subunit alpha), followed by binding of the ligand-subunit alpha complex to, and consequent rearrangement of, a ground state form of betac oligomers.
Project description:Receptor activation by IL5 and GM-CSF is a sequential process that depends on their interaction with a cytokine-specific subunit alpha and recruitment of a common signaling subunit beta (betac). In order to elucidate the assembly dynamics of these receptor subunits, we performed kinetic interaction analysis of the cytokine-receptor complex formation by a surface plasmon resonance biosensor. Using the extracellular domains of receptor fused with C-terminal V5-tag, we developed an assay method to co-anchor alpha and betac subunits on the biosensor surface. We demonstrated that dissociation of the cytokine-receptor complexes was slower when both subunits were co-anchored on the biosensor surface than when alpha subunit alone was anchored. The slow-dissociation effect of betac had a similar impact on GM-CSF receptor stabilization to that of IL5. The effects were abolished by alanine replacement of either Tyr18 or Tyr344 residue in betac, which together constitute key parts of a cytokine binding epitope. The data argue that betac plays an important role in preventing the ligand-receptor complexes from rapidly dissociating. This slow-dissociation effect of betac explains how, when multiple betac cytokine receptor alpha subunits are present on the same cell surface, selective betac usage can be controlled by sequestration in stabilized cytokine-alpha-betac complexes.
Project description:1. Simultaneous measurements of the entry rates of palmitate and glucose have been made in Merino sheep (wethers), starved for 24hr., by using constant infusions of [9,10-(3)H(2)]palmitate and [U-(14)C]glucose. 2. The infusion of glucose into the peripheral circulation of the sheep lowered the endogenous entry of both glucose and palmitate. Since palmitate is roughly metabolically representative of the free fatty acid fraction, there was no marked change in the calories available to the sheep. 3. The infusion of insulin into either the peripheral or portal circulation increased the uptake of glucose and decreased the uptake of palmitate by the tissues of the sheep. 4. The infusion of insulin into the peripheral circulation produced a depression in glucose entry after about 80min., whereas the infusion of insulin into the portal circulation produced an almost immediate depression in glucose entry. 5. The hypoglycaemia produced gave rise to an increase in free fatty acid production followed by an increase in glucose production. 6. No direct effect of insulin on the metabolism of free fatty acids has been demonstrated by the techniques used. The effect of insulin on the metabolism of free fatty acids is apparently mediated through its effect on glucose metabolism.
Project description:Hydrogen sulfide, which causes oral malodour, is generally produced from L-cysteine by the action of betaC-S lyase from oral bacteria. The betaC-S lyases from two oral bacteria, Streptococcus anginosus and S. gordonii, have been cloned, overproduced, purified and crystallized. X-ray diffraction data were collected from the two types of crystals using synchrotron radiation. The crystal of S. anginosus betaC-S lyase belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.0, b = 111.1, c = 216.4 A, and the crystal of S. gordonii betaC-S lyase belonged to the same space group, with unit-cell parameters a = 58.0, b = 73.9. c = 187.6 A. The structures of the betaC-S lyases were solved by molecular-replacement techniques.
Project description:Intestinal and liver fatty acid binding proteins (IFABP and LFABP, respectively) are cytosolic soluble proteins with the capacity to bind and transport hydrophobic ligands between different sub-cellular compartments. Their functions are still not clear but they are supposed to be involved in lipid trafficking and metabolism, cell growth, and regulation of several other processes, like cell differentiation. Here we investigated the interaction of these proteins with different models of phospholipid membrane vesicles in order to achieve further insight into their specificity within the enterocyte. A combination of biophysical and biochemical techniques allowed us to determine affinities of these proteins to membranes, the way phospholipid composition and vesicle size and curvature modulate such interaction, as well as the effect of protein binding on the integrity of the membrane structure. We demonstrate here that, besides their apparently opposite ligand transfer mechanisms, both LFABP and IFABP are able to interact with phospholipid membranes, but the factors that modulate such interactions are different for each protein, further implying different roles for IFABP and LFABP in the intracellular context. These results contribute to the proposed central role of intestinal FABPs in the lipid traffic within enterocytes as well as in the regulation of more complex cellular processes.
Project description:The intestinal fatty acid binding protein (IFABP) is composed of two beta-sheets with a large hydrophobic cavity into which ligands bind. After eight 4-(19)F-phenylalanines were incorporated into the protein, the acid state of both apo- and holo-IFABP (at pH 2.8 and 2.3) was characterized by means of (1)H NMR diffusion measurements, circular dichroism, and (19)F NMR. Diffusion measurements show a moderately increased hydrodynamic radius while near- and far-UV CD measurements suggest that the acid state has substantial secondary structure as well as persistent tertiary interactions. At pH 2.8, these tertiary interactions have been further characterized by (19)F NMR and show an NOE cross-peak between residues that are located on different beta-strands. Side chain conformational heterogeneity on the millisecond time scale was captured by phase-sensitive (19)F-(19)F NOESY. At pH 2.3, native NMR peaks are mostly gone, but the protein can still bind fatty acid to form the holoprotein. An exchange cross-peak of one phenylalanine in the holoprotein is attributed to increased motional freedom of the fatty acid backbone caused by the slight opening of the binding pocket at pH 2.8. In the acid environment Phe128 and Phe17 show dramatic line broadening and chemical shift changes, reflecting greater degrees of motion around these residues. We propose that there is a separation of specific regions of the protein that gives rise to the larger radius of hydration. Temperature and urea unfolding studies indicate that persistent hydrophobic clusters are nativelike and may account for the ability of ligand to bind and induce nativelike structure, even at pH 2.3.
Project description:Fatty acid binding proteins (FABPs) carry fatty acids (FAs) and other lipids in the cellular environment, and are thus involved in processes such as FA uptake, transport, and oxidation. These proteins bind either one or two ligands in a binding site, which appears to be inaccessible from the bulk. Thus, the entry of the substrate necessitates a conformational change, whose nature is still unknown. A possible description of the ligand binding process is given by the portal hypothesis, which suggests that the FA enters the protein through a dynamic area known as the portal region. On the other hand, recent simulations of the adipocyte lipid binding protein (ALBP) suggested a different entry site (the alternative portal). In this article, we discuss molecular dynamics simulations of the apo-intestinal-FABP (I-FABP) in the presence of palmitate molecule(s) in the simulation box. The simulations were carried out to study whether the FA can enter the protein during the simulations (as in the ALBP) and where the ligand entry site is (the portal region, the alternative portal or a different domain). The analysis of the simulations revealed a clear difference between the ALBP and the I-FABP. In the latter case, the palmitate preferentially adsorbed to the portal region, which was more mobile than the rest of the protein. However, no ligand entry was observed in the multi-nanosecond-long simulations, in contrast to ALBP. These findings suggest that, although the main structural motif of the FABPs is common, the fine details of each individual protein structure grossly modulate its reactivity.
Project description:P2 is a fatty acid-binding protein expressed in vertebrate peripheral nerve myelin, where it may function in bilayer stacking and lipid transport. P2 binds to phospholipid membranes through its positively charged surface and a hydrophobic tip, and accommodates fatty acids inside its barrel structure. The structure of human P2 refined at the ultrahigh resolution of 0.93?Å allows detailed structural analyses, including the full organization of an internal hydrogen-bonding network. The orientation of the bound fatty-acid carboxyl group is linked to the protonation states of two coordinating arginine residues. An anion-binding site in the portal region is suggested to be relevant for membrane interactions and conformational changes. When bound to membrane multilayers, P2 has a preferred orientation and is stabilized, and the repeat distance indicates a single layer of P2 between membranes. Simulations show the formation of a double bilayer in the presence of P2, and in cultured cells wild-type P2 induces membrane-domain formation. Here, the most accurate structural and functional view to date on P2, a major component of peripheral nerve myelin, is presented, showing how it can interact with two membranes simultaneously while going through conformational changes at its portal region enabling ligand transfer.