(A) Sequence alignment of C2-domain calcium-binding loop (CBL) regions in cPLA2α from different eukaryotes compared to human PKCs and Syt1. Residues that bind Ca2+ are green. Residues interacting …
In the C2-Domains, Ca2+ binding residues are cyan, DHPC binding residues are yellow; C1P binding residues are beige, and membrane interaction residues are burgundy. In the Catalytic Domains, active …
(Upper left panel) Cross-sectional view of three cPLA2α C2-PC complexes in the asymmetric unit (labeled A, B, C) and three complexes in the neighboring asymmetric unit (A*, B* and C*) that together …
(A) Coordination of three bound Ca2+ ions observed in the C2-domain–DHPC complex. Residues that interact with Ca2+ ions are labeled in black with their side-chains (cyan) depicted in a stick …
C2-domain and DHPC are shown in lime green and beige, respectively. Nitrogen, oxygen, and phosphorus atoms are blue, red, and orange, respectively. Numeric labels for dashed lines equal distances in …
(A) SPR binding isotherms showing point mutant and control protein equilibrium adsorption to immobilized 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) vesicles saturating a L1 sensor chip at 5 …
Membrane partitioning data for cPLA2α C2-domains mutated in PC binding region.
Activity data for cPLA2α C2-domains mutated in PC binding region.
(A) Phosphoglyceride structural formulas. (B) Relative affinities of the C2-domain (1 μM) for different phosphoglycerides obtained by SPR. Molar ratios for PS/PE, PC/PS and PC/PE mixed composition …
cPLA2α C2-domain binding affinity for phosphoglyceride and sphingomyelin (SM) vesicles.
Head group chemical formulas of various non-PC membrane phosphoglycerides are (A) phosphatidylethanolamine, PE; (B) phosphatidylserine, PS; (C) phosphatidylglycerol, PG; (D) phosphatidylinositol, PI;…
(Upper panel) SPR data showing cPLA2α C2-domain adsorption/desorption to/from immobilized POPC vesicles at varying protein concentrations (0.1, 0.2, 0.4, 0.6, 1, 2, and 4 μM; bottom to top) and 5 …
(A) Interaction of the C2-domain–DHPC structural complex with a PC membrane interface produced by ad hoc modeling. The dashed horizontal lines represent planar boundaries for the lipid headgroup and …
(A) cPLA2α C2-domain bound to DHPC determined in this study. (B) PKCα C2-domain bound to phosphatidylserine (PDB 1DSY). (C) Synaptotagmin-1 C2B-domain bound to phosphoserine (PDB 2YOA). For …
cPLA2α C2-domain complexed with DHPC. cPLA2α C2-domain (green) is shown with bound Ca2+ (green) and bound DHPC (yellow). Both Ca4 and CaPC are directly involved in DHPC binding. Synaptotagin-1 C2A …
β-strand sequences (arrows) as well as CBL2 and CBL3 sequences (bracketed) are shown above the alignment. Green highlights represent identical residues. Cyan highlights represent similar residues. …
Interaction distances (Å) associated with bound calcium in the lipid-free cPLA2α C2-domain structure (2.4 Å resolution; PDB 1RLW) of Perisic et al. (1998) and with bound calcium and DHPC in the …
1RLW | Ca1 | Ca4 | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
Asp40 | 2.3/3.4 | 2.3 | ||||||||
Asp43 | 2.1 | 2.6/2.2 | ||||||||
Asp93 | 2.7/2.5 | |||||||||
Asn65 | 2.1 | |||||||||
Asn95 | 2.2 | |||||||||
C2/DHPC | C2 | DHPC | ||||||||
Ca1 | Ca4 | CaPC | N+(CH3)3 | PO4 | sn-2 C=O | sn-1 C=O | sn-1 chain | |||
Asp40 | 2.4/3.4 | 2.4 | ||||||||
Asp43 | 2.3 | 2.6/2.7 | ||||||||
Asp93 | 2.7/2.8 | |||||||||
Asn65 | 2.4 | 3.1 | ||||||||
Asn95 | 2.3 | |||||||||
Tyr96 | ~4.0 | |||||||||
Ala94 | 3.6 | |||||||||
His62 | ~5.0 | ~8.5 | ||||||||
Asn64 | 3.6 | |||||||||
Leu39 | 5.4 | |||||||||
Ca1 | 5.5 | 5.7 | ||||||||
Ca4 | 6.3 | 2.1 | ||||||||
CaPC | 8.5 | 3.6 | 3.2 | 3.1 |
Protein | Kd (M) | Fold increase* |
---|---|---|
WT-C2-domain | (4.2 ± 0.8)×10−7 | ---- |
Y96F-C2-domain | (4.3 ± 0.5)×10−7 | 1 |
Y96A-C2-domain | (2.4 ± 0.4)×10−6 | 5.7 |
N65D-C2-domain | (2.2 ± 0.5)×10−6 | 5.2 |
*Fold increase in Kd relative to the C2-domain binding to POPC vesicles. Kd values were determined from the normalized saturation binding responses (Req) at the protein concentrations shown in Figure 4—figure supplement 1 after fitting by nonlinear least squares analysis using Req = Rmax/(1 + Kd/C).
Protein | KsA (μM) | Vmax (nmol/min/mg) |
---|---|---|
WT-cPLA2α | 182.8 ± 12.5 | 4.053 ± 0.092 |
Y96F-cPLA2α | 205.8 ± 12.7 | 4.930 ± 0.111 |
Y96A-cPLA2α | 467.5 ± 31.6 | 3.438 ± 0.177 |
N65D-cPLA2α | 394.9 ± 29.8 | 3.203 ± 0.166 |
N64A-cPLA2α | 207.7 ± 9.63 | 4.328 ± 0.069 |
*Analyses for data shown in Figure 3E.
Protein | K0.5 (mole fraction) | Vmax (nmol/min/mg) |
---|---|---|
WT-cPLA2α | 0.130 ± 0.007 | 4.352 ± 0.183 |
Y96F-cPLA2α | 0.132 ± 0.007 | 4.362 ± 0.172 |
Y96A-cPLA2α | 0.139 ± 0.006 | 2.510 ± 0.098 |
N65D-cPLA2α | 0.143 ± 0.005 | 1.791 ± 0.050 |
N64A-cPLA2α | 0.142 ± 0.008 | 3.436 ± 0.168 |
*Analyses for data shown in Figure 3F
Reagent (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Escherichia coli) | BL21 (DE) Star competent cells | ThermoFisher Scientific | SKU# C6010-03 | Cells for protein expression |
Transfected construct (E. coli) | pET SUMO | Snapgene | https://www.snapgene.com/resources/plasmid-files/?set=ta_and_gc_cloning_vectors&plasmid=pET_SUMO _(linearized) | Protein expression vector |
Commercial assay or kit | JCSG Core Suites | Qiagen | https://www.qiagen.com/us/shop/sample-technologies/protein/crystallization/the-jcsg-core-suites/#orderinginformation | Protein crystallization; crystallization screening kit |
Chemical compound, drug | 1,2-dihexanoyl -sn-glycero-3- phosphocholine | Avanti Polar Lipids | https://avantilipids.com/product/850305/ | DHPC |
Chemical compound, drug | 1-palmitoyl-2- oleoyl-glycero-3- phosphocholine | Avanti Polar Lipids | https://avantilipids.com/product/850457/ | POPC |
Chemical compound, drug | 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine | Avanti Polar Lipids | https://avantilipids.com/product/840034/ | POPS |
Chemical compound, drug | 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine | Avanti Polar Lipids | https://avantilipids.com/product/850757 | POPE |
Chemical compound, drug | 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphate | Avanti Polar Lipids | https://avantilipids.com/product/840857 | POPA |
Chemical compound, drug | 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) | Avanti Polar Lipids | https://avantilipids.com/product/840457 | POPG |
Chemical compound, drug | N-oleoyl-D-erythro-sphingosylphosphorylcholine | Avanti Polar Lipids | https://avantilipids.com/product/860587 | 18:1 SM |
Chemical compound, drug | 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl) | Avanti Polar Lipids | https://avantilipids.com/product/810145 | NBD-PE |
Software, algorithm | GeneCards | http://genecards.org | RRID:SCR_002773 | Orthologs; retrieval of protein sequences for human, mouse, and chicken proteins |
Software, algorithm | UniProtKB | http://www.uniprot.org/help/uniprotkb | RRID:SCR_004426 | C2-domain sequences for various proteins and organisms |
Software, algorithm | NCBI Protein | http://www.ncbi.nlm.nih.gov/protein | RRID:SCR_003257 | Protein sequences for human, mouse, and chicken proteins |
Software, algorithm | Clustal Omega | http://www.ebi.ac.uk/Tools/msa/clustalo/ | RRID:SCR_001591 | Software package for multiple sequence alignment |
Software, algorithm | Clustal W2 | http://www.ebi.ac.uk/Tools/ msa/clustalw2/ | RRID:SCR_002909 | Multiple sequence alignment program for DNA or proteins. |
Software, algorithm | UCSF Chimera | http://plato.cgl.ucsf.edu/chimera/ | RRID:SCR_004097 | Program for interactive visualization and analysis of molecular structures |
Software, algorithm | Protein Data Bank (PDB) | http://www.wwpdb.org/ | RRID:SCR_006555 | Macromolecular structure archive that oversees and reviews deposition and processing data |
Software, algorithm | Coot | http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ | RRID:SCR_014222 | Software for macromolecular model building, completion and validation, and protein modeling using X-ray data |
Software, algorithm | PHENIX | https://www.phenix-online.org/ | RRID:SCR_014224 | Python-based software suite for determination of X-ray crystallographic molecular structures |
Software, algorithm | PyMol | http://www.pymol.org/ | RRID:SCR_000305 | Data processing, 3D visualization and rendering software |
Software, algorithm | PDBeFold | http://pdbe.org/fold/ | RRID:SCR_004312 | Co-alignment of compared structures |
Peptide, recombinant protein | Cytosolic phospholipase A2 | https://www.uniprot.org/uniprot/P47712 | Human cPLA2 sequence | |
Peptide, recombinant protein | Cytosolic phospholipase A2 | https://www.uniprot.org/uniprot/P47713 | Mouse cPLA2 sequence | |
Peptide, recombinant protein | Cytosolic phospholipase A2 | https://www.uniprot.org/uniprot/P49147 | Chicken cPLA2 sequence |
Native | |
---|---|
Data collection | |
Space group | C222 |
Cell dimensions | |
a, b, c (Å) | 108.3, 187.4, 68.8 |
Wavelength (Å) | 1.00000 |
Resolution (Å) * | 50–2.20 (2.24–2.20) |
Rsym* | 5.9 (36.3) |
I/σI* | 30.9 (1.9) |
Completeness (%)* | 99.5 (97.7) |
Redundancy* | 7.7 (6.6) |
Refinement | |
Resolution (Å) | 47–2.2 |
No. reflections | 35,185 |
Rwork/Rfree (%) | 22.4/24.9 |
No. atoms | |
Protein | 2998 |
Water | 82 |
Ion | 11 |
Ligand | 75 |
B-factor (Å2) | |
Protein | 59.9 |
Water | 56.6 |
Ion | 54.2 |
Ligand | 81.3 |
R.m.s. deviations | |
Bond lengths (Å) | 0.008 |
Bond angles (°) | 1.117 |
One crystal was used for each data set.
*Highest resolution shell is shown in parenthesis.