Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: role of a conserved aromatic cage

Spider venom GDPD-like phospholipases D ( SicTox ) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the α clade and the β clade. Most α clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in β clade toxins vary and include preference for substrates containing ethanolamine headgroups ( Sicarius terrosus, St_βIB1). A structural comparison of available PLDs structures reveals a conserved aromatic cage in the α clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular-dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the α clade, Loxosceles intermedia αIA1 (Li_αIA) and Loxosceles laeta αIII1 (Ll_αIII1), and one from the β clade, St_βIB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_βIB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the α clade PLDs. Here, we confirmed the membrane binding site of α and β clade PLDs on choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the α clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.

Nanodiscs-based proteomics identify Caj1 as an Hsp40 with affinity for phosphatidic acid lipids

Many soluble proteins interact with membranes to perform important biological functions, including signal transduction, regulation, transport, trafficking and biogenesis. Despite their importance, these protein-membrane interactions are difficult to characterize due to their often-transient nature as well as phospholipids' poor solubility in aqueous solution. Here, we employ nanodiscs - small, water-soluble patches of lipid bilayer encircled with amphipathic scaffold proteins - along with quantitative proteomics to identify lipid-binding proteins in S. cerevisiae. Using nanodiscs reconstituted with yeast total lipid extracts or only phosphatidylethanolamine (PE-nanodiscs), we capture several known membrane-interacting proteins, including the Rab GTPases Sec4 and Ypt1, which play key roles in vesicle trafficking. Utilizing PE-nanodiscs enriched with phosphatidic acid (PEPA-nanodiscs), we specifically capture a member of the Hsp40/J-protein family, Caj1, whose function has recently been linked to membrane protein quality control. We show that Caj1 interaction with liposomes containing PA is modulated by pH and PE lipids, and depends on two patches of positively charged residues near the C-terminus of the protein. The protein Caj1 is the first example of an Hsp40/J-domain protein with affinity for membranes and phosphatidic acid lipid specificity. These findings highlight the utility of the nanodisc system to identify and characterize protein-lipid interactions that may not be evident using other methods.

The Profound Influence of Lipid Composition on the Catalysis of the Drug Target NADH Type II Oxidoreductase

Lipids play a pivotal role in cellular respiration, providing the natural environment in which an oxidoreductase interacts with the quinone pool. To date, it is generally accepted that negatively charged lipids play a major role in the activity of quinone oxidoreductases. By changing lipid compositions when assaying a type II NADH:quinone oxidoreductase, we demonstrate that phosphatidylethanolamine has an essential role in substrate binding and catalysis. We also reveal the importance of acyl chain composition, specifically c14:0, on membrane-bound quinone-mediated catalysis. This demonstrates that oxidoreductase lipid specificity is more diverse than originally thought and that the lipid environment plays an important role in the physiological catalysis of membrane-bound oxidoreductases.

The ESCRT-III isoforms CHMP2A and CHMP2B display different effects on membranes upon polymerization

Background ESCRT-III proteins are involved in many membrane remodeling processes including multivesicular body biogenesis as first discovered in yeast. In humans, ESCRT-III CHMP2 exists as two isoforms, CHMP2A and CHMP2B, but their physical characteristics have not been compared yet. Results Here, we use a combination of techniques on biomimetic systems and purified proteins to study their affinity and effects on membranes. We establish that CHMP2B binding is enhanced in the presence of PI(4,5)P2 lipids. In contrast, CHMP2A does not display lipid specificity and requires CHMP3 for binding significantly to membranes. On the micrometer scale and at moderate bulk concentrations, CHMP2B forms a reticular structure on membranes whereas CHMP2A (+CHMP3) binds homogeneously. Thus, CHMP2A and CHMP2B unexpectedly induce different mechanical effects to membranes: CHMP2B strongly rigidifies them while CHMP2A (+CHMP3) has no significant effect. Conclusions We therefore conclude that CHMP2B and CHMP2A exhibit different mechanical properties and might thus contribute differently to the diverse ESCRT-III-catalyzed membrane remodeling processes.

Lipid specificity of the immune effector perforin

Perforin is a pore forming protein used by cytotoxic T lymphocytes to remove cancerous or virus-infected cells during immune response. During the response, the lymphocyte membrane becomes refractory to perforin function by accumulating densely ordered lipid rafts and externalizing negatively charged lipid species. The dense membrane packing lowers the capacity of perforin to bind, and negatively charged lipids scavenge any residual protein before pore formation. Using atomic force microscopy on model membrane systems, we here provide insight into the molecular basis of perforin lipid specificity.

Lipid specificity of the immune effector perforin

Perforin is a pore forming protein used by cytotoxic T lymphocytes to remove cancerous or virus-infected cells during immune response. During the response, the lymphocyte membrane becomes refractory to perforin...

The ESCRT-III Isoforms CHMP2A And CHMP2B Display Different Effects On Membranes Upon Polymerization

ABSTRACTESCRT-III proteins are involved in many membrane remodeling processes including multivesicular body biogenesis as first discovered in yeast. In humans, CHMP2 exists as two potential isoforms, CHMP2A and CHMP2B, but their physical characteristics have not been compared yet. Here, we use a combination of technics on biomimetic systems and purified proteins to study their affinity and effects on membranes. We establish that CHMP2B binding is enhanced in the presence of PI(4,5)P2 lipids. In contrast, CHMP2A does not display lipid specificity and requires CHMP3 for binding significantly to membranes. On the micrometer scale and at moderate bulk concentrations, CHMP2B forms a reticular structure on membranes whereas CHMP2A (+CHMP3) binds homogeneously. Eventually, CHMP2A and CHMP2B unexpectedly induce different mechanical effects to membranes: CHMP2B strongly rigidifies them while CHMP2A (+CHMP3) has no significant effect. Altogether, we conclude that CHMP2B and CHMP2A cannot be considered as isoforms and might thus contribute differently to membrane remodeling processes.