The mechanism of binding of two promising anticancer agents (the cytotoxic alkylphospholipids perifosine and miltefosine) to the Akt PH domain is investigated by high-resolution field-cycling 31P nuclear magnetic resonance (NMR) spectroscopy using a spin-labeled recombinant PH domain. These results strongly indicate that there are two discrete amphiphile binding sites on the domain: (i) the cationic site that binds phosphoinositides and some alkylphospholipids and (ii) a second site that is occupied by only the alkylphospholipids. The identification of this second site for amphiphiles on the Akt1 PH domain provides a new target for drug development as well as insights into the regulation of the activity of the intact Akt1 protein. The field-cycling NMR methodology could be used to define discrete phospholipid or amphiphile binding sites on a wide variety of peripheral membrane proteins.

Staphylococcus aureus secretes a phosphatidylinositol-specific phospholipase C (PI-PLC) as a virulence factor that is unusual in exhibiting higher activity at acidic pH values than other enzymes in this class. We have determined the crystal structure of this enzyme at pH 4.6 and pH 7.5. Under slightly basic conditions, the S. aureus PI-PLC structure closely follows the conformation of other bacterial PI-PLCs. However, when crystallized under acidic conditions, a large section of mobile loop at the αβ-barrel rim in the vicinity of the active site shows ∼10 Å shift. This loop displacement at acidic pH is the result of a titratable intramolecular π-cation interaction between His258 and Phe249. This was verified by a structure of the mutant protein H258Y crystallized at pH 4.6, which does not exhibit the large loop shift. The intramolecular π-cation interaction for S. aureus PI-PLC provides an explanation for the activity of the enzyme at acid pH and also suggests how phosphatidylcholine, as a competitor for Phe249, may kinetically activate this enzyme.

Itaconic acid (ITA), or methylenesuccinic acid, is not generally classified as a mammalian metabolite. Using NMR-based metabolomics and 13C-labeling, we have detected ITA in both macrophage-like VM-M3 and RAW 264.7 tumor cell lines as well as stimulated and unstimulated primary murine macrophages. Macrophage activation by addition of lipopolysaccharide and IFN-γ markedly increased ITA production and secretion. Crude cell extracts synthesize ITA via decarboxylation of cis-aconitate, indicative of a novel mammalian cis-aconitic decarboxylase activity. Our results highlight a previously unidentified biosynthetic pathway related to TCA cycle metabolism in mammalian cells and a novel metabolite that likely plays a role in macrophage-based immune response.

The phosphatidylinositol-specific phospholipase C (PI-PLC) from Listeria monocytogenes forms aggregates with anionic lipids leading to low activity. The specific activity of the enzyme can be enhanced by dilution of the protein or by addition of both zwitterionic and neutral amphiphiles (e.g., diheptanoylphosphatidylcholine or Triton X-100) or 0.1−0.2 M inorganic salts. Activation by amphiphiles occurs with both micellar (phosphatidylinositol dispersed in detergents) and monomeric [dibutroylphosphatidylinositol (diC4PI)] phosphotransferase substrates and inositol 1,2-(cyclic)-phosphate (cIP), the phosphodiesterase substrate. The presence of zwitterionic and neutral amphiphiles (to which the protein binds weakly) dilutes the surface concentration of the interfacial anionic substrate and thereby reduces the level of enzyme−phospholipid particle aggregation. Zwitterionic amphiphiles also can bind directly to the protein and enhance catalysis since they enhance both diC4PI and cIP hydrolysis. In contrast to activation by amphiphiles, the rate enhancement by salt occurs for only the phosphotransferase step of the reaction. Added salt has a synergistic effect with zwitterionic phospholipids, leading to high specific activities for PI cleavage with only moderate dilution of the anionic substrate in the interface. This kinetic activation correlates with weakening of strong PI-PLC hydrophobic interactions with the interface as monitored by a decrease in the maximum monolayer surface pressure for insertion of the protein. Several point mutations of surface hydrophobic residues (W49A, L51A, L235A, and F237W) can dramatically alter the unusual kinetics of this secreted enzyme. The high affinity of PI-PLC for anionic phospholipids along with a strong hydrophobic interaction, which gives rise to the unusual kinetic behavior, is considered in terms of how it might contribute to the role of this phospholipase in L. monocytogenes infectivity.

31P NMR relaxation studies from 0.005 to 11.7 T are used to monitor water-soluble inositol 1,2-(cyclic) phosphate (cIP) binding to phosphatidylinositol-specific phospholipase C spin-labeled at H82C, a position near the active site of the enzyme, and to determine how activating phosphatidylcholine (PC) molecules affect this interaction. We show that, in the absence of an interface, cIP binding to the protein is not rate-limiting, and that lower activation by PC vesicles as opposed to micelles is likely due to hindered product release. The methodology is general and could be used for determining distances in other weakly binding small molecule ligand−protein interactions.

The Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), an interfacial enzyme associated with prokaryotic infectivity, is activated by binding to zwitterionic surfaces, particularly phosphatidycholine (PC). Two tryptophan residues (Trp47 in the two-turn helix B and Trp242 in a disordered loop) at the rim of the barrel structure are critical for this interaction. The helix B region (Ile43 to Gly48) in wild-type PI-PLC orients the side chains of Ile43 and Trp47 so that they pack together and form a hydrophobic protrusion from the protein surface that likely facilitates initial membrane binding. In previous studies we reported that in the crystal structure of the dimeric W47A/W242A mutant, which is unable to bind to PC, the helix B region has been reorganized by the mutation into an extended loop. Here we report the construction and characterization (catalytic activity, fluorescence, and NMR studies) of a series of PI-PLC mutants targeting helix B residues and surrounding regions to explore what is needed to stabilize the “membrane-active” conformation of the helix B region. Results strongly suggest that, while hydrophobic groups and presumably an intact helix B are critical for the initial binding of PI-PLC to membranes, disruption of helix B to allow enzyme dimerization is what leads to the activated PI-PLC conformation.

Methanococcus jannaschii accumulates α- and β-glutamate as osmolytes. The effect of these and other solutes on the thermostability of two multisubunit metabolic enzymes from M. jannaschii, aspartate transcarbamoylase catalytic trimer (ATCase C3) and glutamine synthetase (GS), has been measured and compared to solute effects on bacterial mesophilic counterparts in order to explore if osmolytes accumulated by each organism can preferentially stabilize the proteins to thermal unfolding. For both ATCase enzymes and for the B. subtilis GS, the solutes normally accumulated by the organism were very effective in protecting the enzyme from losing activity at high temperatures, although solute effects on loss of secondary structure did not necessarily correlate with this thermoprotection of activity. The recombinant M. jannaschii GS exhibited quite different behavior. The pure enzyme had a thermal unfolding transition with a midpoint temperature (Tm) less than 60 °C, well under the growth temperature of the organism (85 °C). None of the small molecule solutes tested (including the K+-glutamate isomers accumulated by M. jannaschii) significantly stabilized the protein to incubation at 85 °C. Instead, protein–protein interactions, as illustrated by E. coli GroEL or ribosomal protein L2 stabilization of GS, appeared to be the dominant factor in stabilizing this archaeal enzyme at the growth temperature.

•NMR shows cytotoxic and non-toxic PIAs bind to the same Akt1 PH domain site.•MCF-7 cells exhibit reduced levels of constitutively active Akt.•d-3-deoxy-diC8PI induces apoptosis in MCF-7 cells.•d-3-deoxy-diC8PI downregulates the D-type cyclin-retinoblastoma protein pathway.•The effect of this PIA is independent of its interaction with Akt.Phosphatidylinositol analogs (PIAs) were originally designed to bind competitively to the Akt PH domain and prevent membrane translocation and activation. d-3-Deoxy-dioctanoylphosphatidylinositol (d-3-deoxy-diC8PI), but not compounds with altered inositol stereochemistry (e.g., l-3-deoxy-diC8PI and l-3,5-dideoxy-diC8PI), is cytotoxic. However, high resolution NMR field cycling relaxometry shows that both cytotoxic and non-toxic PIAs bind to the Akt1 PH domain at the site occupied by the cytotoxic alkylphospholipid perifosine. This suggests that another mechanism for cytotoxicity must account for the difference in efficacy of the synthetic short-chain PIAs. In MCF-7 breast cancer cells, with little constitutively active Akt, d-3-deoxy-diC8PI (but not l-compounds) decreases viability concomitant with increased cleavage of PARP and caspase 9, indicative of apoptosis. d-3-Deoxy-diC8PI also induces a decrease in endogenous levels of cyclins D1 and D3 and blocks downstream retinoblastoma protein phosphorylation. siRNA-mediated depletion of cyclin D1, but not cyclin D3, reduces MCF-7 cell proliferation. Thus, growth arrest and cytotoxicity induced by the soluble d-3-deoxy-diC8PI occur by a mechanism that involves downregulation of the D-type cyclin-pRb pathway independent of its interaction with Akt. This ability to downregulate D-type cyclins contributes, at least in part, to the anti-proliferative activity of d-3-deoxy-diC8PI and may be a common feature of other cytotoxic phospholipids.Download high-res image (92KB)Download full-size image

The magnetic field dependence of the 31P spin-lattice relaxation rate, R1, of phospholipids can be used to differentiate motions for these molecules in a variety of unilamellar vesicles. In particular, internal motion with a 5- to 10-ns correlation time has been attributed to diffusion-in-a-cone of the phosphodiester region, analogous to motion of a cylinder in a liquid hydrocarbon. We use the temperature dependence of 31P R1 at low field (0.03–0.08 T), which reflects this correlation time, to explore the energy barriers associated with this motion. Most phospholipids exhibit a similar energy barrier of 13.2 ± 1.9 kJ/mol at temperatures above that associated with their gel-to-liquid-crystalline transition (Tm); at temperatures below Tm, this barrier increases dramatically to 68.5 ± 7.3 kJ/mol. This temperature dependence is broadly interpreted as arising from diffusive motion of the lipid axis in a spatially rough potential energy landscape. The inclusion of cholesterol in these vesicles has only moderate effects for phospholipids at temperatures above their Tm, but significantly reduces the energy barrier (to 17 ± 4 kJ/mol) at temperatures below the Tm of the pure lipid. Very-low-field R1 data indicate that cholesterol inclusion alters the averaged disposition of the phosphorus-to-glycerol-proton vector (both its average length and its average angle with respect to the membrane normal) that determines the 31P relaxation.

The Aichi RNA virus remodels host membranes by conscripting two host proteins, PI4KIIIβ (to generate PI4P in the remodeled vesicle) and ACBD3 (that tightly binds PI4KIIIβ), and localizing them on target membranes via Aichi protein 3A. In this issue of Structure, McPhail et al. (2017) reveal structural glimpses of the interfaces involved in this protein threesome using HDX-MS.