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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

N-terminal Lys39 is involved in LPA-type agonist binding during LPA1.(a, i) Location of LPA18:1 (brown stick) relative to ML5 (yellow stick) in S1PR1 (purple cartoon) structure. (a, ii,iii) Location of LPA18:1 (brown stick) relative to Lys39, R3.28, Q3.29 and K7.35 (cyan stick) in the starting LPA1 (0 ns) and after 200 ns simulation respectively. (b, i–iv). Box-and-Whisker plots of the center of mass distance between the head group oxygen atoms of LPA-type agonist and designated residues. (c, upper and lower panels). Contribution of R3.28 and Lys39 to LPA-type agonist binding energy respectively. (d) Time dependent evolution of distance between ligand head-group oxygen atoms and Lys39 during MD simulations. (inset i–iii) represent representative structure of LPA and LPA1 with emphasis on the positions of LPA18:1 relative to Lys39 (green stick) during the course of the simulations. (e) Center of mass distance between N-terminal residues and the 7TM bundle (see text for more description). **Data represent last 50 ns of the initial 200 ns production phase simulation and n = 3. Line graph is smoothed over 20 dataset window.
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f2: N-terminal Lys39 is involved in LPA-type agonist binding during LPA1.(a, i) Location of LPA18:1 (brown stick) relative to ML5 (yellow stick) in S1PR1 (purple cartoon) structure. (a, ii,iii) Location of LPA18:1 (brown stick) relative to Lys39, R3.28, Q3.29 and K7.35 (cyan stick) in the starting LPA1 (0 ns) and after 200 ns simulation respectively. (b, i–iv). Box-and-Whisker plots of the center of mass distance between the head group oxygen atoms of LPA-type agonist and designated residues. (c, upper and lower panels). Contribution of R3.28 and Lys39 to LPA-type agonist binding energy respectively. (d) Time dependent evolution of distance between ligand head-group oxygen atoms and Lys39 during MD simulations. (inset i–iii) represent representative structure of LPA and LPA1 with emphasis on the positions of LPA18:1 relative to Lys39 (green stick) during the course of the simulations. (e) Center of mass distance between N-terminal residues and the 7TM bundle (see text for more description). **Data represent last 50 ns of the initial 200 ns production phase simulation and n = 3. Line graph is smoothed over 20 dataset window.

Mentions: From the observations above, it is evident that LPA1 in complex with LPA 18:1, LPA 20:4 and AGP 18:1 did show classical GPCR activation features, thus providing the basis for studying novel N-terminal residues involved in LPA1 activation. Next, the contribution of each amino acid to the free energy of ligand binding was calculated focusing on the N-terminal region and the three previously identified amino acids (R3.28, Q3.29, K7.36) as references21. The free energy profile identified Lys39 as key N-terminal amino acids contributing to ligand binding whilst validating the contributions of the reference residues (purple arrow, supplementary Fig. 1c). Next, to prove that Lys39/ligand interaction was not present in the starting structure but formed during the simulation, we showed that head-group atoms of the starting LPA-type agonists were accurately aligned with the head-group atoms of ML5 in S1PR1-bounds state and proximal to the orthosteric residues R3.28 and E3.29 (Fig. 2a, i)2226. In similar fashion, the head-group atoms were also proximal to R3.28, Q2.39 and K7.35 as predicted by Valentine et al.21 but not Lys39 in the starting LPA1 model (Fig. 2a, ii). After 200 ns simulation, a common feature in all LPA-type simulation was the presence of LPA head-group inserted between triangle-shaped electron densities of R3.28 and K7.36 and Lys39; with the phosphate group making hydrogen bond interactions with Lys39 and R3.28 (Fig. 2a, iii). Box-and-Whisker plots affirmed that oxygen atoms of LPA head group showed residence proximal to Lys39 (<1.2 nm) comparable with K7.36 (<1.0 nm) and R3.28 (<0.6) and to a lesser extent Q3.29 (~1.5 nm) (Fig. 2b, i–iv).


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)

N-terminal Lys39 is involved in LPA-type agonist binding during LPA1.(a, i) Location of LPA18:1 (brown stick) relative to ML5 (yellow stick) in S1PR1 (purple cartoon) structure. (a, ii,iii) Location of LPA18:1 (brown stick) relative to Lys39, R3.28, Q3.29 and K7.35 (cyan stick) in the starting LPA1 (0 ns) and after 200 ns simulation respectively. (b, i–iv). Box-and-Whisker plots of the center of mass distance between the head group oxygen atoms of LPA-type agonist and designated residues. (c, upper and lower panels). Contribution of R3.28 and Lys39 to LPA-type agonist binding energy respectively. (d) Time dependent evolution of distance between ligand head-group oxygen atoms and Lys39 during MD simulations. (inset i–iii) represent representative structure of LPA and LPA1 with emphasis on the positions of LPA18:1 relative to Lys39 (green stick) during the course of the simulations. (e) Center of mass distance between N-terminal residues and the 7TM bundle (see text for more description). **Data represent last 50 ns of the initial 200 ns production phase simulation and n = 3. Line graph is smoothed over 20 dataset window.
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f2: N-terminal Lys39 is involved in LPA-type agonist binding during LPA1.(a, i) Location of LPA18:1 (brown stick) relative to ML5 (yellow stick) in S1PR1 (purple cartoon) structure. (a, ii,iii) Location of LPA18:1 (brown stick) relative to Lys39, R3.28, Q3.29 and K7.35 (cyan stick) in the starting LPA1 (0 ns) and after 200 ns simulation respectively. (b, i–iv). Box-and-Whisker plots of the center of mass distance between the head group oxygen atoms of LPA-type agonist and designated residues. (c, upper and lower panels). Contribution of R3.28 and Lys39 to LPA-type agonist binding energy respectively. (d) Time dependent evolution of distance between ligand head-group oxygen atoms and Lys39 during MD simulations. (inset i–iii) represent representative structure of LPA and LPA1 with emphasis on the positions of LPA18:1 relative to Lys39 (green stick) during the course of the simulations. (e) Center of mass distance between N-terminal residues and the 7TM bundle (see text for more description). **Data represent last 50 ns of the initial 200 ns production phase simulation and n = 3. Line graph is smoothed over 20 dataset window.
Mentions: From the observations above, it is evident that LPA1 in complex with LPA 18:1, LPA 20:4 and AGP 18:1 did show classical GPCR activation features, thus providing the basis for studying novel N-terminal residues involved in LPA1 activation. Next, the contribution of each amino acid to the free energy of ligand binding was calculated focusing on the N-terminal region and the three previously identified amino acids (R3.28, Q3.29, K7.36) as references21. The free energy profile identified Lys39 as key N-terminal amino acids contributing to ligand binding whilst validating the contributions of the reference residues (purple arrow, supplementary Fig. 1c). Next, to prove that Lys39/ligand interaction was not present in the starting structure but formed during the simulation, we showed that head-group atoms of the starting LPA-type agonists were accurately aligned with the head-group atoms of ML5 in S1PR1-bounds state and proximal to the orthosteric residues R3.28 and E3.29 (Fig. 2a, i)2226. In similar fashion, the head-group atoms were also proximal to R3.28, Q2.39 and K7.35 as predicted by Valentine et al.21 but not Lys39 in the starting LPA1 model (Fig. 2a, ii). After 200 ns simulation, a common feature in all LPA-type simulation was the presence of LPA head-group inserted between triangle-shaped electron densities of R3.28 and K7.36 and Lys39; with the phosphate group making hydrogen bond interactions with Lys39 and R3.28 (Fig. 2a, iii). Box-and-Whisker plots affirmed that oxygen atoms of LPA head group showed residence proximal to Lys39 (<1.2 nm) comparable with K7.36 (<1.0 nm) and R3.28 (<0.6) and to a lesser extent Q3.29 (~1.5 nm) (Fig. 2b, i–iv).

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