Juan C. Gómez-Fernández

A New Phosphatidylinositol 4,5-Bisphosphate-binding Site Located in the C2 Domain of Protein Kinase Cα

In view of the interest shown in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) as a second messenger, we studied the activation of protein kinase Cα by this phosphoinositide. By using two double mutants from two different sites located in the C2 domain of protein kinase Cα, we have determined and characterized the PtdIns(4,5)P2-binding site in the protein, which was found to be important for its activation. Thus, there are two distinct sites in the C2 domain: the first, the lysine-rich cluster located in the β3- and β4-sheets and which activates the enzyme through direct binding of PtdIns(4,5)P2; and the second, the already well described site formed by the Ca2+-binding region, which also binds phosphatidylserine and a result of which the enzyme is activated. The results obtained in this work point to a sequential activation model, in which protein kinase Cα needs Ca2+ before the PtdIns(4,5)P2-dependent activation of the enzyme can occur.

Structural and mechanistic insights into the association of PKCα-C2 domain to PtdIns(4,5)P2

C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca2+-binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKCα-C2 domain in complex with Ca2+, 1,2-dihexanoyl-sn-glycero-3-[phospho-l-serine] (PtdSer), and 1,2-diayl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns(4,5)P2] shows that PtdSer binds specifically to the calcium-binding region, whereas PtdIns(4,5)P2 occupies the concave surface of strands β3 and β4. Strikingly, the structure reveals a PtdIns(4,5)P2-C2 domain-binding mode in which the aromatic residues Tyr-195 and Trp-245 establish direct interactions with the phosphate moieties of the inositol ring. Mutations that abrogate Tyr-195 and Trp-245 recognition of PtdIns(4,5)P2 severely impaired the ability of PKCα to localize to the plasma membrane. Notably, these residues are highly conserved among C2 domains of topology I, and a general mechanism of C2 domain-membrane docking mediated by PtdIns(4,5)P2 is presented.

Influence of liposome charge and composition on their interaction with human blood serum proteins

Lipid composition and specially their electrostatic properties, were found to greatly influence the stability of liposomes in human blood serum. The amount and type of serum proteins bound to the liposomes were also clearly influenced by lipid composition and charge of liposomes. a good correlation was found between the amount of serum proteins adsorbed to a given type of liposome and its instability as measured by the release of an encapsulated fluorescent probe. Liposomes that bind the highest amount of protein were the least stable, except for the case of liposomes containing gangliosides, which were fairly stable even at a high amount of bound protein. Liposomes with neutral charge containing phosphatidylcholine were the most stable and bound the lowest amount of protein. Liposomes with positive charge behaved similarly to those with neutral charge. However, the stability of negatively charged liposomes was very dependent on their composition. Those liposomes containing only one class of negatively charged phospholipids bound a great amount of protein and were very unstable. However, those liposomes containing also phosphatidylcholine bound less protein and were more stable. The examination of the electrophoresis patterns of serum proteins bound to the different types of liposomes indicated the presence of specific proteins which correlated with liposome instability. (Mol Cell Biochem120: 119–126, 1993)

Signaling through C2 domains: More than one lipid target

C2 domains are membrane-binding modules that share a common overall fold: a single compact Greek-key motif organized as an eight-stranded anti-parallel β-sandwich consisting of a pair of four-stranded β-sheets. A myriad of studies have demonstrated that in spite of sharing the common structural β-sandwich core, slight variations in the residues located in the interconnecting loops confer C2 domains with functional abilities to respond to different Ca(2+) concentrations and lipids, and to signal through protein-protein interactions as well. This review summarizes the main structural and functional findings on Ca(2+) and lipid interactions by C2 domains, including the discovery of the phosphoinositide-binding site located in the β3-β4 strands. The wide variety of functions, together with the different Ca(2+) and lipid affinities of these domains, converts this superfamily into a crucial player in many functions in the cell and more to be discovered. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cells Physiology, Pathology and Therapy.

A differential scanning calorimetry study of the interaction of α-tocopherol with mixtures of phospholipids

When alpha-tocopherol was included in multibilayer vesicles of dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine it induced a broadening of the main transition and a displacement of this transition to lower temperatures, as seen by differential scanning calorimetry. This effect was quantitatively more important in the samples of distearoylphosphatidylcholine than in those of the other phosphatidylcholines. Alpha-Tocopherol when present in equimolar mixtures of dimyristoylphosphatidylcholine and diastearoylphosphatidylcholine, which show monotectic behaviour, preferentially partitions in the most fluid phase. The effect of alpha-tocopherol on the phase transition of dilauroylphosphatidylethanolamine and dipalmitoylphosphatidylethanolamine is qualitatively different of that observed on phosphatidylcholines, and several peaks are observed in the calorimetric profile, probably indicating the formation of separated phases with different contents in alpha-tocopherol. The effect was more apparent in dipalmitoylphosphatidylethanolamine than in dilauroylphosphatidylethanolamine. The inclusion of alpha-tocopherol in equimolar mixtures of dilauroylphosphatidylethanolamine and dipalmitoylphosphatidylcholine, which show cocrystallization, only produced a broadening of the phase transition and a shift to lower temperatures. However, in the case of equimolar mixtures of dipalmitoylphosphatidylcholine which also show cocrystallization, the effect was to cause lateral phase separation with the formation of different mixtures of phospholipids and alpha-tocopherol. Alpha-Tocopherol was also included in equimolar mixtures of phosphatidylethanolamine and phosphatidylcholine showing monotectic behaviour, and in this case alpha-tocopherol preferentially partitioned in the most fluid phase, independently of whether this was composed mainly of phosphatidylcholine or of phosphatidylethanolamine.

Additional binding sites for anionic phospholipids and calcium ions in the crystal structures of complexes of the C2 domain of protein kinase Cα

The C2 domain of protein kinase Calpha (PKCalpha) corresponds to the regulatory sequence motif, found in a large variety of membrane trafficking and signal transduction proteins, that mediates the recruitment of proteins by phospholipid membranes. In the PKCalpha isoenzyme, the Ca2+-dependent binding to membranes is highly specific to 1,2-sn-phosphatidyl-l-serine. Intrinsic Ca2+ binding tends to be of low affinity and non-cooperative, while phospholipid membranes enhance the overall affinity of Ca2+ and convert it into cooperative binding. The crystal structure of a ternary complex of the PKCalpha-C2 domain showed the binding of two calcium ions and of one 1,2-dicaproyl-sn-phosphatidyl-l-serine (DCPS) molecule that was coordinated directly to one of the calcium ions. The structures of the C2 domain of PKCalpha crystallised in the presence of Ca2+ with either 1,2-diacetyl-sn-phosphatidyl-l-serine (DAPS) or 1,2-dicaproyl-sn-phosphatidic acid (DCPA) have now been determined and refined at 1.9 A and at 2.0 A, respectively. DAPS, a phospholipid with short hydrocarbon chains, was expected to facilitate the accommodation of the phospholipid ligand inside the Ca2+-binding pocket. DCPA, with a phosphatidic acid (PA) head group, was used to investigate the preference for phospholipids with phosphatidyl-l-serine (PS) head groups. The two structures determined show the presence of an additional binding site for anionic phospholipids in the vicinity of the conserved lysine-rich cluster. Site-directed mutagenesis, on the lysine residues from this cluster that interact directly with the phospholipid, revealed a substantial decrease in C2 domain binding to vesicles when concentrations of either PS or PA were increased in the absence of Ca2+. In the complex of the C2 domain with DAPS a third Ca2+, which binds an extra phosphate group, was identified in the calcium-binding regions (CBRs). The interplay between calcium ions and phosphate groups or phospholipid molecules in the C2 domain of PKCalpha is supported by the specificity and spatial organisation of the binding sites in the domain and by the variable occupancies of ligands found in the different crystal structures. Implications for PKCalpha activity of these structural results, in particular at the level of the binding affinity of the C2 domain to membranes, are discussed.

A differential scanning calorimetry study of the interaction of free fatty acids with phospholipid membranes

Mixtures of stearic, arachic, oleic and linoleic acids with dimyristoylphosphatidylcholine (DMPC) and distearylphosphatidylcholine (DSPC) have been studied by means of differential scanning calorimetry (DSC). The mixtures of stearic (SA) and arachic acids (AA) with DMPC and DSPC show phase diagrams of the peritectic type, with a region of solid phase immiscibility from 0 to 28.5 mol% of fatty acid. A pure component, with a stoichiometry fatty acid/phospholipid (2:1) seems to be formed except for the system AA/DSPC. The mixtures of oleic (OA) and linoleic acids (LA) show complex phase diagrams. In the case of OA, different regions where a phase separation exists can be observed and for the mixture of OA with DMPC, a pure component seems to be formed with a stoichiometry OA/DMPC (1:2). LA shows different behaviour in the mixtures with DMPC and with DSPC. For the mixture, LA/DMPC, a fluid phase immiscibility region is observed over the same range of concentration as for the mixture with OA, however, the mixture with DSPC shows a solid phase immiscibility for the samples containing 45 mol% or more of LA. The interaction of the different free fatty acids with equimolar mixtures of DMPC and DSPC, showing monotectic behaviour, has also been analyzed. From our results it can be clearly concluded that saturated fatty acids partition preferentially into solid-like domains, while cis-unsaturated fatty acids partition preferentially into fluid-like domains.

Edelfosine Is Incorporated into Rafts and Alters Their Organization

The effect of edelfosine (1- O-octadecyl-2- O-methyl-rac-glycero-3-phosphocholine or ET-18-OCH3) on model membranes containing 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine/sphingomyelin/cholesterol (POPC/SM/cholesterol) was studied by several physical techniques. The sample POPC/SM (1:1 molar ratio) showed a broad phase transition as seen by DSC, X-ray diffraction, and 2H NMR. The addition of edelfosine to this sample produced isotropic structures at temperatures above the phase transition, as seen by 2H NMR and by 31P NMR. When cholesterol was added to give a POPC/SM/cholesterol (at a molar ratio 1:1:1), no transition was observed by DSC nor X-ray diffraction, and 2H NMR indicated the presence of a liquid ordered phase. The addition of 10 mol % edelfosine increased the thickness of the membrane as seen by X-ray diffraction and led to bigger differences in the values of the molecular order of the membrane detected at high and low temperatures, as detected through the M 1 first spectral moment from 2H NMR. These differences were even greater when 20 mol % edelfosine was added, and a transition was now clearly visible by DSC. In addition, a gel phase was clearly indicated by X-ray diffraction at low temperatures. The same technique pointed to greater membrane thickness in this mixture and to the appearance of a second membrane structure, indicating the formation of two separated phases in the presence of edelfosine. All of these data strongly suggest that edelfosine associating with cholesterol alter the phase status present in a POPC/SM/cholesterol (1:1:1 molar ratio) mixture, which is reputed to be a model of a raft structure. However, cell experiments showed that edelfosine colocalizes in vivo with rafts and that it may reach concentrations higher than 20 mol % of total lipid, indicating that the concentrations used in the biophysical experiments were within what can be expected in a cell membrane. The conclusion is that molecular ways of action of edelfosine in cells may involve the modification of the structure of rafts.

Fluorescence study of the location and dynamics of α-tocopherol in phospholipid vesicles

The intrinsic fluorescence of α-tocopherol has been used as a tool to study the location and dynamics of the molecule in phospholipid vesicles made of egg yolk phosphatidylcholine using steady-state and time-resolved techniques. From absorption spectra it was concluded that most α-tocopherol molecules are hydrogen bonded, although the aggregates formed are fluorescent. By calculating several fluorescence parameters in different solvents it was concluded that α-tocopherol should be situated in a polar region of the membrane. From the results obtained in measurements of fluorescence quenching and resonance energy transfer it was deduced that th chromanol moiety of the molecule is located in a position close to that occupied by the probes 7-(9-anthroxyloxy)stearic acid (7-AS) and 5-(N-oxy-4,4-dimethyloxazolidin-2-yl)stearic acid (5-NS) in the membrane. The lateral diffusion coefficient of α-tocopherol in phospholipid vesicles was calculated through quenching of its fluorescence by the spin probe 5-NS, and a value of 4.8 · 10−6 cm2 · s−1 was found, indicating a very high lateral diffusion of α-tocopherol.

Infrared spectroscopic study of the interaction of diacylglycerol with phosphatidylserine in the presence of calcium

The interaction of 1,2-dipalmitoylglycerol (DPG) with dipalmitoylphosphatidylserine (DPPS) has been studied in aqueous dispersion in the presence and in the absence of Ca2+ by using Fourier transform infrared spectroscopy (FT-IR) and 45Ca(2+)-binding. FT-IR showed that DPG increased the phase transition of DPPS and induced a rigidification of the DPPS/DPG-Ca2+ complex. In the absence of Ca2+, the incorporation of DPG produced an increase in the proportion of dehydrated carbonyl groups in the mixture of DPPS plus DPG whereas, in the presence of Ca2+, DPG suppressed the solid-solid phase transition of phosphatidylserine-Ca2+ complexes. The phosphate band of DPPS was analyzed using a multivariate statistical analysis, indicating that DPG induced a higher dehydration of the PO2- group in the presence of subsaturating Ca2+ concentrations. Even very low concentrations of DPG, such as 2 mol%, already produced a significant effect. In the presence of both DPG and Ca2+, dehydration of DPPS increased, so that full dehydration was reached at a DPPS/Ca2+ molar ratio of 2.94 instead of 2.04 as observed for pure DPPS. However, the stoichiometry of the binding of Ca2+ to DPPS was not significantly altered by the inclusion of DPG as revealed by 45Ca(2+)-binding experiments, indicating that, in this situation, full dehydration of the PO2- groups of DPPS was reached when approx. 2 out of every 3 molecules of DPPS were binding Ca2+. The effects reported here for the interaction of DPG with DPPS may be significant for a number of biological situations where Ca2+, phosphatidylserine and diacylglycerols are involved, such as fusion of membranes or the activation of protein kinase C, where the dehydration effect produced by diacylglycerols may explain, at least in part, their effects.