The cPLA2 C2alpha domain in solution: structure and dynamics of its Ca2+-activated and cation-free states.

Cytosolic phospholipase A2 is involved in several signal transduction pathways where it catalyses release of arachidonic acid from intracellular lipid membranes. Its membrane insertion is facilitated by its independently folding C2alpha domain, which is activated by the binding of two intracellular Ca2+ ions. However, the details of its membrane-insertion mechanism, including its Ca2+-activation mechanism, are not understood. There are several unresolved issues, including the following. There are two experimentally resolved structures of the Ca2+-activated state of its isolated C2alpha domain, one determined using x-ray crystallography and the other determined using NMR spectroscopy, which differ from each other significantly in the spatial region that inserts into the membrane. This by itself adds to ambiguities associated with investigations targeting its mechanism of membrane insertion. Furthermore, there is no experimentally determined structure of its cation-free state, which hinders investigations associated with its cation-activation mechanism. In this work, we generate several unrestrained molecular dynamics trajectories of its isolated C2alpha domain in solution (equivalent to approximately 60 ns) and investigate these issues. Our main results are as follows: a), the Ca2+ coordination scheme of the domain is consistent with the x-ray structure and with previous mutagenesis studies; b), the helical segment of the Ca2+-binding loop, CBL-I, undergoes nanosecond timescale flexing (but not an unwinding), as can be inferred from physiological temperature NMR data and in contrast to low temperature x-ray data; and c), removal of the two activating Ca2+ ions from their binding pockets does not alter the backbone structure of the domain, a result consistent with electron paramagnetic resonance data.

Self-induced docking site of a deeply embedded peripheral membrane protein.

As a first step toward understanding the principles of the targeting of C2 domains to membranes, we have carried out a molecular dynamics simulation of the C2 domain of cytosolic phospholipase A2 (cPLA2-C2) in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer at constant pressure and temperature (NPT, 300 K and 1 atm). Using the high-resolution crystal structure of cPLA2-C2 as a starting point, we embedded two copies of the C2 domain into a pre-equilibrated membrane at the depth and orientation previously defined by electron paramagnetic resonance (EPR). Noting that in the membrane-bound state the three calcium binding loops are complexed to two calcium ions, we initially restrained the calcium ions at the membrane depth determined by EPR. But the depth and orientation of the domains remained within EPR experimental errors when the restraints were later removed. We find that the thermally disordered, chemically heterogeneous interfacial zones of phosphatidylcholine bilayers allow local lipid remodeling to produce a nearly perfect match to the shape and polarity of the C2 domain, thereby enabling the C2 domain to assemble and optimize its own lipid docking site. The result is a cuplike docking site with a hydrophobic bottom and hydrophilic rim. Contrary to expectations, we did not find direct interactions between the protein-bound calcium ions and lipid headgroups, which were sterically excluded from the calcium binding cleft. Rather, the lipid phosphate groups provided outer-sphere calcium coordination through intervening water molecules. These results show that the combined use of high-resolution protein structures, EPR measurements, and molecular dynamics simulations provides a general approach for analyzing the molecular interactions between membrane-docked proteins and lipid bilayers.

Regulation of cytosolic phospholipase A(2) in a new perspective: recruitment of active monomers from an inactive clustered pool.

cPLA(2) plays a key role in many signal transduction cascades by hydrolyzing arachidonic acid from membrane phospholipids. Tight control of cPLA(2) activity by a number of regulatory mechanisms is essential to its cellular function. We recently described the localization of cPLA(2) in clusters in fibroblasts and now propose that these clusters reflect a localized inactive pool from which active monomers can be recruited to keep cPLA(2) activity under control on the subcellular level. Using an electron microscopic in vitro approach, we show that cPLA(2) monomers, but not the clusters, bind to membranes in a Ca(2+)-dependent manner. This binding is accompanied by hydrolytic activity. The present data combined with our previous observation of a relative abundance of clusters over monomers in fixed fibroblasts [Bunt, G., de Wit, J., van den Bosch, H., Verkleij, A., and Boonstra, J. (1997) J. Cell Sci. 110, 2449-2459] gives rise to a concept of cPLA(2) regulation in which small amounts of active monomers are recruited to fulfill their function upon stimulation. This is in contrast to processes described for inflammatory cells, where a substantial part of the cytoplasmically localized cPLA(2) translocates to the perinuclear region upon stimulation to become active. Small-scale regulation of cPLA(2) by the proposed cluster-monomer cycle allows local and strictly confined control of cPLA(2) activity, apparently necessary for its cellular role in fibroblasts.

A 7.4-A projection structure of outer membrane phospholipase A from Escherichia coli by electron crystallography.

Outer membrane phospholipase A (OMPLA) is one of the few enzymes present in the outer membrane of Escherichia coli. Two-dimensional crystals of OMPLA were grown by reconstitution of purified protein into lipid bilayers via detergent dialysis and were studied by electron crystallography. A 7.4-A projection map reveals OMPLA molecules exhibiting an oval-shaped domain of 30 x 20 A resembling the beta-barrel structure characteristic of porins, which is associated with a 25-A elongated domain of lower density.

Direct imaging by cryo-TEM shows membrane break-up by phospholipase A2 enzymatic activity.

Phospholipid hydrolysis to free fatty acid and 1-lyso-phospholipid by water-soluble phospholipase A2 (PLA2) at the surface of lipid membranes exhibits a poorly understood transition from a low-activity lag phase to a burst regime of rapid hydrolysis. Understanding this kinetic phenomenon may increase our insight into the function of PLA2 under physiological conditions as well as into general interfacial catalysis. In the present study we apply for the first time cryo-transmission electron microscopy (cryo-TEM) and high-performance liquid chromatography (HPLC) to characterize the PLA2 hydrolysis of phospholipid vesicles with respect to changes in lipid composition and morphology. Our direct experimental results show that the initial reaction conditions are strongly perturbed during the course of hydrolysis. Most strikingly, cryo-TEM reveals that starting in the lag phase, vesicles become perforated and degrade into open vesicles, bilayer fragments, and micelles. This structural instability extends throughout the system in the activity burst regime. In agreement with earlier reported correlations between initial phospholipase activity and substrate morphology, our results suggest that the lag-burst phenomenon reflects a cascade process. The PLA2-induced changes in lipid composition transform the morphology which in turn results in an acceleration of the rate of hydrolysis because of a strong coupling between the PLA2 activity and the morphology of the lipid suspension.

Differential regulation of human T cell cytokine patterns and IgE and IgG4 responses by conformational antigen variants.

Bee venom phospholipase A2 (PLA) represents the major allergen and antigen in allergic and non-allergic individuals sensitized to bee sting. We have studied specific activation of peripheral T cells by different structural and conformational variants of PLA and secretion of cytokines regulating IgE and IgG4 antibody (Ab) formation. PLA molecules expressing the correctly folded tertiary structure, which show high affinity to membrane phospholipids and were recognized by Ab from bee sting allergic patients, induced high IL-4, IL-5 and IL-13 production in peripheral blood mononuclear cell cultures. In contrast, non-refolded recombinant PLA (rPLA) and reduced and alkylated native PLA (nPLA) induced more IFN-gamma and IL-2 and higher proliferative responses. Differences in proliferation and cytokine patterns among correctly folded and non-refolded PLA resulted from conformation-dependent involvement of different antigen-presenting cell (APC) types. Antigen (Ag)-presenting B cells recognized PLA only in its natural conformation, stimulated Th2 type cytokines and induced IgE Ab. Non-refolded PLA was recognized, processed and presented exclusively by monocytes and induced a Th1 dominant cytokine profile leading to IgG4 production by B cells. The possibility that production of particular cytokine patterns and Ig isotype was influenced by the enzymatic activity of PLA was excluded by using enzymatically inactive H34Q point-mutated, refolded rPLA. These findings demonstrate the decisive role of specific Ag recognition by different APC, depending on structural features, membrane phospholipid binding property and the existence of conformational B cell epitopes, in the differential regulation of memory IgE and IgG4 Ab. Furthermore, they show that a change from IgE-mediated allergy to normal immunity against a major allergen can be induced by rPLA variants that are not recognized by specific Ab and B cells but still carry the T cell epitopes. These features may enable new applications for safer immunotherapy.

Ultrastructural localization of cPLA2 in unstimulated and EGF/A23187-stimulated fibroblasts.

The 85 kDa cytosolic phospholipase A2 is the key enzyme in the release of arachidonic acid. To gain insight into cytosolic phospholipase A2 action in mitogen-activated cells, the localization of the phospholipase was investigated in fibroblasts upon stimulation with epidermal growth factor and the calcium ionophore A23187. By the use of indirect immunofluorescence microscopy, staining of endogenous cytosolic phospholipase A2 resulted in a punctate labeling pattern randomly distributed throughout the cytoplasm of the cell. Immunogold electron microscopy revealed that this punctate labeling pattern exhibited the presence of the 85 kDa phospholipase A2 in small clusters. These clusters were found in the cytosol in the vicinity of all organellar membranes, except for the Golgi system. The enzyme showed no preference for the nuclear envelope, the endoplasmic reticulum or the plasma membrane. Stimulation of cells with epidermal growth factor or A23187 or both did not change the punctate immunofluorescence labeling pattern. Furthermore, a similar labeling pattern was observed by the artificial introduction of extremely low or high intracellular calcium concentrations. Even by electron microscopy, translocation of cytosolic phospholipase A2 to membranes was not observed after stimulation of cells with epidermal growth factor and A23187. From these results it is concluded that cytosolic phospholipase A2 is localized in clusters close to membranes in stimulated as well as unstimulated fibroblasts, without preference for a specific organellar membrane.

Crystallographic and biochemical studies of the (inactive) Lys-49 phospholipase A2 from the venom of Agkistridon piscivorus piscivorus.

Chemical, genetic, and structural studies have defined a critical role for Asp-49 in the calcium-mediated activation of extracellular phospholipases A2 (PLA2). In 1984, a new class of PLA2 was isolated in which this invariant aspartate was replaced with a lysine (Maragnore, J.M., Merutka, G., Cho, W., Welches, W., Kezdy, F.J., and Heinrikson, R.L. (1984) J. Biol. Chem. 259, 13839-13843; Maragnore, J.M., and Heinrikson, R.L. (1986) J. Biol. Chem. 261, 4797-4804). The enzymatic activity of Lys-49 PLA2s has been questioned based on biochemical, mutational, and structural studies (van den Bergh, C.J., Slotboom, A.J., Verheij, H.M., and de Haas, G.H. (1988) Eur. J. Biochem. 176, 353-357). In this paper, we describe the structures of two crystal forms of the Lys-49 PLA2 isolated from the venom of Agkistridon piscivorus piscivorus. The refined models, along with complementary biochemical analysis, clarify the structural basis for the enzymatic inactivity of Lys-49 proteins.

Phospholipase A2 engineering. Structural and functional roles of highly conserved active site residues tyrosine-52 and tyrosine-73.

Site-directed mutagenesis was used to probe the structural and functional roles of two highly conserved residues, Tyr-52 and Tyr-73, in interfacial catalysis by bovine pancreatic phospholipase A2 (PLA2, overproduced in Escherichia coli). According to crystal structures, the side chains of these two active site residues form H-bonds with the carboxylate of the catalytic residue Asp-99. Replacement of either or both Tyr residues by Phe resulted in only very small changes in catalytic rates, which suggests that the hydrogen bonds are not essential for catalysis by PLA2. Substitution of either Tyr residue by nonaromatic amino acids resulted in substantial decreases in the apparent kcat toward 1,2-dioctanoyl-sn-glycero-3-phosphocholine (DC8PC) micelles and the v(o) (turnover number at maximal substrate concentration, i.e., mole fraction = 1) toward 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DC14PM) vesicles in scooting mode kinetics [Berg, O. G., Yu, B.-Z., Rogers, J., & Jain, M. K. (1991) Biochemistry 30, 7283-7297]. The Y52V mutant was further analyzed in detail by scooting mode kinetics: the E to E* equilibrium was examined by fluorescence; the dissociation constants of E*S, E*P, and E*I (KS*, KP*, and KI*, respectively) in the presence of Ca2+ were measured by protection of histidine-48 modification and by difference UV spectroscopy; the Michaelis constant KM* was calculated from initial rates of hydrolysis in the absence and presence of competitive inhibitors; and the turnover number under saturating conditions (kcat, which is a theoretical value since the enzyme may not be saturated at the interface) was calculated from the vo and KM* values. The results indicated little perturbation in the interfacial binding step (E to E*) but ca. 10-fold increases in KS*, KP*, KI*, and KM* and a less than 10-fold decrease in kcat. Such changes in the function of Y52V are not due to global conformational changes since the proton NMR properties of Y52V closely resemble those of wild-type PLA2; instead, it is likely to be caused by perturbed enzyme-substrate interactions at the active site. Tyr-73 appears to play an important structural role. The conformational stability of all Tyr-73 mutants decreased by 4-5 kcal/mol relative to that of the wild-type PLA2. The proton NMR properties of Y73A suggested significant conformational changes and substantially increased conformational flexibility. These detailed structural and functional analyses represent a major advancement in the structure-function study of an enzyme involved in interfacial catalysis.

Structures of free and inhibited human secretory phospholipase A2 from inflammatory exudate.

Phospholipase A2 (PLA2) participates in a wide range of cellular processes including inflammation and transmembrane signaling. A human nonpancreatic secretory PLA2 (hnps-PLA2) has been identified that is found in high concentrations in the synovial fluid of patients with rheumatoid arthritis and in the plasma of patients with septic shock. This enzyme is secreted from certain cell types in response to the proinflammatory cytokines, tumor necrosis factor or interleukin-1. The crystal structures of the calcium-bound form of this enzyme have been determined at physiological pH both in the presence [2.1 angstrom (A) resolution] and absence (2.2 A resolution) of a transition-state analogue. Although the critical features that suggest the chemistry of catalysis are identical to those inferred from the crystal structures of other extracellular PLA2s, the shape of the hydrophobic channel of hnps-PLA2 is uniquely modulated by substrate binding.