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Lys39-Lysophosphatidate Carbonyl Oxygen Interaction Locks LPA1 N-terminal Cap to the Orthosteric Site and partners Arg124 During Receptor Activation.

Omotuyi OI, Nagai J, Ueda H - Sci Rep (2015)

Bottom Line: In activated state, LPA-type agonists interact with Arg124 (R3.28), Gln125 (Q3.29), Lys294 (K7.36) and a novel N-terminal Lys39.Structurally, LPA-type agonist via Carbonyl-oxygen/Lys39 interaction facilitated the formation of a hypothetical N-terminal cap tightly packed over LPA1 heptahelical bundle.This packing may represent a key mechanism to distinguish an apo-receptor from bound LPA1.

View Article: PubMed Central - PubMed

Affiliation: 1] From the Department of Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, Japan [2] From the Center for Drug Discovery and Therapeutic Innovation, Nagasaki University, Japan.

ABSTRACT
Lysophosphatidic acid (LPA) receptor 1 (LPA1) is a member of the G protein-coupled receptors mediating the biological response to LPA species. Lack of detailed mechanism underlying LPA/LPA1 interaction has hampered the development of specific antagonists. Here, novel N-terminal Lys39 has been identified as a key residue during LPA-type agonist binding and LPA1 activation. Analysis of the molecular dynamics (MD) trajectories showed that LPA-type agonist but not VPC-32183 (antagonist) evolved structures with classical GPCR activation signatures such as reduced cytoplasmic transmembrane (TM) 3/TM6 dynamic network, ruptured ionic lock, and formation of a continuous and highly ordered internal water pathway was also observed. In activated state, LPA-type agonists interact with Arg124 (R3.28), Gln125 (Q3.29), Lys294 (K7.36) and a novel N-terminal Lys39. Site-directed mutagenesis showed complete loss of intracellular calcium mobilization in B103 cells expressing R3.28A and Lys39Ala when treated with LPA-type agonists. Structurally, LPA-type agonist via Carbonyl-oxygen/Lys39 interaction facilitated the formation of a hypothetical N-terminal cap tightly packed over LPA1 heptahelical bundle. This packing may represent a key mechanism to distinguish an apo-receptor from bound LPA1.

No MeSH data available.


Related in: MedlinePlus

Classical GPCR activation signature in LPA-type agonist bound LPA1.(a) Free-energy surface of apo-LPA1. Inset depicts intermediately activated LPA1 (green cartoon) superimposed on the starting LPA1 structure (purple cartoon). R3.50 and L6.33 are shown as stick representation. (b) Distribution of Cα-NPxxY (N7.49-Y7.53) rmsd from inactive and TM3(Cα-R3.50)-TM6 (Cα-L6.33) distance in structures generated within the last 100. (c, i–iv) Dynamic network between cytoplasmic ends of transmembrane helices in VPC32183, AGP18:1, LPA18:1 and LPA20:4 bound LPA1. (d, i–v) Average internal water density flow along LPA1 bound to strong agonists (LPA20:4, LPA18:1 and AGP18:1), weak agonists (LPA 16:0) and an antagonist (VPC32183). (e, i–viii) Free-energy surface representation of LPA1 P7.50 dihedral angle bound to strong and weak agonists and antagonist. Each dot in “b” represents the mean value of three independent simulations, the green arrow depecits the movement of TM6 away from the ionic lock zone.
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f1: Classical GPCR activation signature in LPA-type agonist bound LPA1.(a) Free-energy surface of apo-LPA1. Inset depicts intermediately activated LPA1 (green cartoon) superimposed on the starting LPA1 structure (purple cartoon). R3.50 and L6.33 are shown as stick representation. (b) Distribution of Cα-NPxxY (N7.49-Y7.53) rmsd from inactive and TM3(Cα-R3.50)-TM6 (Cα-L6.33) distance in structures generated within the last 100. (c, i–iv) Dynamic network between cytoplasmic ends of transmembrane helices in VPC32183, AGP18:1, LPA18:1 and LPA20:4 bound LPA1. (d, i–v) Average internal water density flow along LPA1 bound to strong agonists (LPA20:4, LPA18:1 and AGP18:1), weak agonists (LPA 16:0) and an antagonist (VPC32183). (e, i–viii) Free-energy surface representation of LPA1 P7.50 dihedral angle bound to strong and weak agonists and antagonist. Each dot in “b” represents the mean value of three independent simulations, the green arrow depecits the movement of TM6 away from the ionic lock zone.

Mentions: The initial LPA1 model shared similar seven transmembrane helices conformation with sphingosine 1 phosphate receptor 1 (S1PR1) in complex with an antagonist (PDB ID: 3V2Y)22. Since LPA species are LPA1 agonist, the initial model was simulated in an apo-state (150 ns) to generate intermediate or active-state features, such as breaking transmembrane (TM) 3-TM6 ionic lock (TM3~TM6 (intracellular) center of mass distance > 1.2 nm) and root mean square deviation (rmsd) of TM7 NPxxY motif from the inactive state (N(7.49)PxxY(7.53) rmsd to 3V2Y > 0.05 nm) as previously observed during β2-adrenergic receptor activation23. Apo-structures were preferentially trapped in the intermediate state (TM3-TM6 distance ≈ 1.0–1.5 nm, Cα−NPxxY rmsd to 3V2Y ≈ 0.05 nm) (Fig. 1a). Three substructures were harvested from the energy basin (ΔG ≈ 0 Kj/mol, colour bar represents energy) to investigate LPA-dependent LPA1 activation. Upon superimposition of one of the three starting structures (green cartoon) on the starting coordinate (purple cartoon), movement of TM6 (R3.50) away (green arrow) from the TM3 (L6.33) relative to the starting model was observed (inset).


Lys39-Lysophosphatidate Carbonyl Oxygen Interaction Locks LPA1 N-terminal Cap to the Orthosteric Site and partners Arg124 During Receptor Activation.

Omotuyi OI, Nagai J, Ueda H - Sci Rep (2015)

Classical GPCR activation signature in LPA-type agonist bound LPA1.(a) Free-energy surface of apo-LPA1. Inset depicts intermediately activated LPA1 (green cartoon) superimposed on the starting LPA1 structure (purple cartoon). R3.50 and L6.33 are shown as stick representation. (b) Distribution of Cα-NPxxY (N7.49-Y7.53) rmsd from inactive and TM3(Cα-R3.50)-TM6 (Cα-L6.33) distance in structures generated within the last 100. (c, i–iv) Dynamic network between cytoplasmic ends of transmembrane helices in VPC32183, AGP18:1, LPA18:1 and LPA20:4 bound LPA1. (d, i–v) Average internal water density flow along LPA1 bound to strong agonists (LPA20:4, LPA18:1 and AGP18:1), weak agonists (LPA 16:0) and an antagonist (VPC32183). (e, i–viii) Free-energy surface representation of LPA1 P7.50 dihedral angle bound to strong and weak agonists and antagonist. Each dot in “b” represents the mean value of three independent simulations, the green arrow depecits the movement of TM6 away from the ionic lock zone.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4542628&req=5

f1: Classical GPCR activation signature in LPA-type agonist bound LPA1.(a) Free-energy surface of apo-LPA1. Inset depicts intermediately activated LPA1 (green cartoon) superimposed on the starting LPA1 structure (purple cartoon). R3.50 and L6.33 are shown as stick representation. (b) Distribution of Cα-NPxxY (N7.49-Y7.53) rmsd from inactive and TM3(Cα-R3.50)-TM6 (Cα-L6.33) distance in structures generated within the last 100. (c, i–iv) Dynamic network between cytoplasmic ends of transmembrane helices in VPC32183, AGP18:1, LPA18:1 and LPA20:4 bound LPA1. (d, i–v) Average internal water density flow along LPA1 bound to strong agonists (LPA20:4, LPA18:1 and AGP18:1), weak agonists (LPA 16:0) and an antagonist (VPC32183). (e, i–viii) Free-energy surface representation of LPA1 P7.50 dihedral angle bound to strong and weak agonists and antagonist. Each dot in “b” represents the mean value of three independent simulations, the green arrow depecits the movement of TM6 away from the ionic lock zone.
Mentions: The initial LPA1 model shared similar seven transmembrane helices conformation with sphingosine 1 phosphate receptor 1 (S1PR1) in complex with an antagonist (PDB ID: 3V2Y)22. Since LPA species are LPA1 agonist, the initial model was simulated in an apo-state (150 ns) to generate intermediate or active-state features, such as breaking transmembrane (TM) 3-TM6 ionic lock (TM3~TM6 (intracellular) center of mass distance > 1.2 nm) and root mean square deviation (rmsd) of TM7 NPxxY motif from the inactive state (N(7.49)PxxY(7.53) rmsd to 3V2Y > 0.05 nm) as previously observed during β2-adrenergic receptor activation23. Apo-structures were preferentially trapped in the intermediate state (TM3-TM6 distance ≈ 1.0–1.5 nm, Cα−NPxxY rmsd to 3V2Y ≈ 0.05 nm) (Fig. 1a). Three substructures were harvested from the energy basin (ΔG ≈ 0 Kj/mol, colour bar represents energy) to investigate LPA-dependent LPA1 activation. Upon superimposition of one of the three starting structures (green cartoon) on the starting coordinate (purple cartoon), movement of TM6 (R3.50) away (green arrow) from the TM3 (L6.33) relative to the starting model was observed (inset).

Bottom Line: In activated state, LPA-type agonists interact with Arg124 (R3.28), Gln125 (Q3.29), Lys294 (K7.36) and a novel N-terminal Lys39.Structurally, LPA-type agonist via Carbonyl-oxygen/Lys39 interaction facilitated the formation of a hypothetical N-terminal cap tightly packed over LPA1 heptahelical bundle.This packing may represent a key mechanism to distinguish an apo-receptor from bound LPA1.

View Article: PubMed Central - PubMed

Affiliation: 1] From the Department of Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, Japan [2] From the Center for Drug Discovery and Therapeutic Innovation, Nagasaki University, Japan.

ABSTRACT
Lysophosphatidic acid (LPA) receptor 1 (LPA1) is a member of the G protein-coupled receptors mediating the biological response to LPA species. Lack of detailed mechanism underlying LPA/LPA1 interaction has hampered the development of specific antagonists. Here, novel N-terminal Lys39 has been identified as a key residue during LPA-type agonist binding and LPA1 activation. Analysis of the molecular dynamics (MD) trajectories showed that LPA-type agonist but not VPC-32183 (antagonist) evolved structures with classical GPCR activation signatures such as reduced cytoplasmic transmembrane (TM) 3/TM6 dynamic network, ruptured ionic lock, and formation of a continuous and highly ordered internal water pathway was also observed. In activated state, LPA-type agonists interact with Arg124 (R3.28), Gln125 (Q3.29), Lys294 (K7.36) and a novel N-terminal Lys39. Site-directed mutagenesis showed complete loss of intracellular calcium mobilization in B103 cells expressing R3.28A and Lys39Ala when treated with LPA-type agonists. Structurally, LPA-type agonist via Carbonyl-oxygen/Lys39 interaction facilitated the formation of a hypothetical N-terminal cap tightly packed over LPA1 heptahelical bundle. This packing may represent a key mechanism to distinguish an apo-receptor from bound LPA1.

No MeSH data available.


Related in: MedlinePlus