Structural basis of phosphatidylcholine recognition by the C2–domain of cytosolic phospholipase A2α

  1. Yoshinori Hirano
  2. Yong-Guang Gao
  3. Daniel J Stephenson
  4. Ngoc T Vu
  5. Lucy Malinina
  6. Dhirendra K Simanshu
  7. Charles E Chalfant  Is a corresponding author
  8. Dinshaw J Patel  Is a corresponding author
  9. Rhoderick E Brown  Is a corresponding author
  1. Memorial Sloan-Kettering Cancer Center, United States
  2. Nara Institute of Science and Technology (NAIST), Japan
  3. University of Minnesota, United States
  4. Virginia Commonwealth University Medical Center, United States
  5. University of South Florida, United States
  6. James A. Haley Veterans Hospital, United States
  7. The Moffitt Cancer Center, United States
7 figures, 6 tables and 1 additional file

Figures

Figure 1 with 2 supplements
Structure of cPLA2α C2‒domain containing bound DHPC and calcium.

(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 …

https://doi.org/10.7554/eLife.44760.002
Figure 1—figure supplement 1
Sequence alignment of cPLA2α for human, mouse, and chicken proteins.

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 …

https://doi.org/10.7554/eLife.44760.003
Figure 1—figure supplement 2
Tubular topology formed in the crystal lattice of the cPLA2α C2-domain–DHPC structural complex.

(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 …

https://doi.org/10.7554/eLife.44760.004
Figure 2 with 1 supplement
Structural interactions of the cPLA2α C2-domain complexed with Ca2+ and DHPC.

(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 …

https://doi.org/10.7554/eLife.44760.005
Figure 2—figure supplement 1
Zoomed views of the cPLA2α C2-domain complexed with Ca2+ and DHPC.

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 …

https://doi.org/10.7554/eLife.44760.006
Membrane partitioning of cPLA2α C2-domains and cPLA2α catalytic activities of point-mutated C2-domains in the PC-binding region.

(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 …

https://doi.org/10.7554/eLife.44760.008
Figure 3—source data 1

Membrane partitioning data for cPLA2α C2-domains mutated in PC binding region.

https://doi.org/10.7554/eLife.44760.009
Figure 3—source data 2

Activity data for cPLA2α C2-domains mutated in PC binding region.

https://doi.org/10.7554/eLife.44760.010
Figure 4 with 2 supplements
cPLA2α C2-domain binding affinity for phosphoglyceride and sphingomyelin (SM) vesicles.

(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 …

https://doi.org/10.7554/eLife.44760.014
Figure 4—source data 1

cPLA2α C2-domain binding affinity for phosphoglyceride and sphingomyelin (SM) vesicles.

https://doi.org/10.7554/eLife.44760.017
Figure 4—figure supplement 1
Assessment of C2-domain binding to different phosphoglycerides.

Head group chemical formulas of various non-PC membrane phosphoglycerides are (A) phosphatidylethanolamine, PE; (B) phosphatidylserine, PS; (C) phosphatidylglycerol, PG; (D) phosphatidylinositol, PI;…

https://doi.org/10.7554/eLife.44760.015
Figure 4—figure supplement 2
The concentration- and time-dependence of C2-domain adsorption/desorption to/from immobilized POPC and 18:1 SM vesicles measured by SPR.

(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 …

https://doi.org/10.7554/eLife.44760.016
Model of the cPLA2α C2-domain selectively interacting with the PC membrane interface.

(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 …

https://doi.org/10.7554/eLife.44760.018
Figure 6 with 1 supplement
Structures of various C2-domains bound to lipids.

(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 …

https://doi.org/10.7554/eLife.44760.019
Figure 6—figure supplement 1
Ca2+binding site differences in the synaptotagmin-1 C2A domain versus the cPLA2α C2-domain complexed with DHPC.

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 …

https://doi.org/10.7554/eLife.44760.020
C2-domain sequence alignment for five human cPLA2 isoforms showing the uniqueness of the Tyr96 residue in the cPLA2α C2-domain.

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. …

https://doi.org/10.7554/eLife.44760.021

Tables

Table 1
Interaction distances in cPLA2α C2-domain. 

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 …

https://doi.org/10.7554/eLife.44760.007
1RLWCa1Ca4
 Asp402.3/3.42.3
 Asp432.12.6/2.2
 Asp932.7/2.5
 Asn652.1
 Asn952.2
C2/DHPCC2 DHPC
Ca1Ca4CaPCN+(CH3)3PO4sn-2 C=Osn-1 C=Osn-1 chain
 Asp402.4/3.42.4
 Asp432.32.6/2.7
 Asp932.7/2.8
 Asn652.43.1
 Asn952.3
 Tyr96~4.0
 Ala943.6
 His62~5.0~8.5
 Asn643.6
 Leu395.4
 Ca15.55.7
 Ca46.32.1
 CaPC8.53.63.23.1
Table 2
Kd values determined by SPR.
https://doi.org/10.7554/eLife.44760.011
ProteinKd (M)Fold increase*
WT-C2-domain(4.2 ± 0.8)×10−7----
Y96F-C2-domain(4.3 ± 0.5)×10−71
Y96A-C2-domain(2.4 ± 0.4)×10−65.7
N65D-C2-domain(2.2 ± 0.5)×10−65.2
  1. *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).

Table 3
Kinetic activity parameters for point-mutated PC-binding-site residues in the C2-domain of cPLA2α*.
https://doi.org/10.7554/eLife.44760.012
ProteinKs(μM)Vmax (nmol/min/mg)
WT-cPLA2α182.8 ± 12.54.053 ± 0.092
Y96F-cPLA2α205.8 ± 12.74.930 ± 0.111
Y96A-cPLA2α467.5 ± 31.63.438 ± 0.177
N65D-cPLA2α394.9 ± 29.83.203 ± 0.166
N64A-cPLA2α207.7 ± 9.634.328 ± 0.069
  1. *Analyses for data shown in Figure 3E.

Table 4
Kinetic activity parameters for point-mutated PC-binding-site residues in the C2-domain of cPLA2α *
https://doi.org/10.7554/eLife.44760.013
Protein

K0.5
(mole fraction)
Vmax
(nmol/min/mg)
WT-cPLA2α0.130 ± 0.0074.352 ± 0.183
Y96F-cPLA2α0.132 ± 0.0074.362 ± 0.172
Y96A-cPLA2α0.139 ± 0.0062.510 ± 0.098
N65D-cPLA2α0.143 ± 0.0051.791 ± 0.050
N64A-cPLA2α0.142 ± 0.0083.436 ± 0.168
  1. *Analyses for data shown in Figure 3F

Key resources table
Reagent (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (Escherichia coli)BL21 (DE) Star competent cellsThermoFisher ScientificSKU# C6010-03Cells for protein
expression
Transfected construct (E. coli)pET SUMOSnapgenehttps://www.snapgene.com/resources/plasmid-files/?set=ta_and_gc_cloning_vectors&plasmid=pET_SUMO
_(linearized)
Protein expression vector
Commercial assay or kitJCSG Core SuitesQiagenhttps://www.qiagen.com/us/shop/sample-technologies/protein/crystallization/the-jcsg-core-suites/#orderinginformationProtein crystallization; crystallization
screening kit
Chemical compound, drug1,2-dihexanoyl
-sn-glycero-3-
phosphocholine
Avanti Polar Lipidshttps://avantilipids.com/product/850305/DHPC
Chemical compound, drug1-palmitoyl-2-
oleoyl-glycero-3-
phosphocholine
Avanti Polar Lipidshttps://avantilipids.com/product/850457/POPC
Chemical compound, drug1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serineAvanti Polar Lipidshttps://avantilipids.com/product/840034/POPS
Chemical compound, drug1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamineAvanti Polar Lipidshttps://avantilipids.com/product/850757POPE
Chemical compound, drug1-palmitoyl-2-
oleoyl-sn-glycero-3-phosphate
Avanti Polar Lipidshttps://avantilipids.com/product/840857POPA
Chemical compound, drug1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)Avanti Polar Lipidshttps://avantilipids.com/product/840457POPG
Chemical compound, drugN-oleoyl-D-erythro-sphingosylphosphorylcholineAvanti Polar Lipidshttps://avantilipids.com/product/86058718:1 SM
Chemical compound, drug1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl)Avanti Polar Lipidshttps://avantilipids.com/product/810145NBD-PE
Software, algorithmGeneCardshttp://genecards.orgRRID:SCR_002773Orthologs; retrieval of protein sequences for human, mouse, and chicken proteins
Software, algorithmUniProtKBhttp://www.uniprot.org/help/uniprotkbRRID:SCR_004426C2-domain sequences for various proteins and organisms
Software, algorithmNCBI Proteinhttp://www.ncbi.nlm.nih.gov/proteinRRID:SCR_003257Protein sequences for human, mouse, and chicken proteins
Software, algorithmClustal Omegahttp://www.ebi.ac.uk/Tools/msa/clustalo/RRID:SCR_001591Software package for multiple sequence alignment
Software, algorithmClustal W2http://www.ebi.ac.uk/Tools/
msa/clustalw2/
RRID:SCR_002909Multiple sequence alignment program for DNA or proteins.
Software, algorithmUCSF Chimerahttp://plato.cgl.ucsf.edu/chimera/RRID:SCR_004097Program for
interactive visualization and analysis of molecular structures
Software, algorithmProtein Data Bank (PDB)http://www.wwpdb.org/RRID:SCR_006555Macromolecular structure archive that oversees and reviews deposition and processing data
Software, algorithmCoothttp://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/RRID:SCR_014222Software for macromolecular model building, completion and
validation, and protein modeling using
X-ray data
Software, algorithmPHENIXhttps://www.phenix-online.org/RRID:SCR_014224Python-based
software suite for determination of X-ray
crystallographic molecular structures
Software, algorithmPyMolhttp://www.pymol.org/RRID:SCR_000305Data processing,
3D visualization and rendering
software
Software, algorithmPDBeFoldhttp://pdbe.org/fold/RRID:SCR_004312Co-alignment of
compared structures
Peptide, recombinant proteinCytosolic phospholipase A2https://www.uniprot.org/uniprot/P47712Human cPLA2 sequence
Peptide, recombinant proteinCytosolic
phospholipase A2
https://www.uniprot.org/uniprot/P47713Mouse cPLA2 sequence
Peptide, recombinant proteinCytosolic phospholipase A2https://www.uniprot.org/uniprot/P49147Chicken cPLA2 sequence
Table 5
X-ray data collection statistics.
https://doi.org/10.7554/eLife.44760.022
Native
Data collection
Space groupC222
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)
II*30.9 (1.9)
Completeness (%)*99.5 (97.7)
Redundancy*7.7 (6.6)
Refinement
Resolution (Å)47–2.2
No. reflections35,185
Rwork/Rfree (%)22.4/24.9
No. atoms
Protein2998
Water82
Ion11
Ligand75
B-factor (Å2)
Protein59.9
Water56.6
Ion54.2
Ligand81.3
R.m.s. deviations
Bond lengths (Å)0.008
Bond angles (°)1.117
  1. One crystal was used for each data set.

    *Highest resolution shell is shown in parenthesis.

Additional files

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