38 research outputs found
Activation of G-protein-coupled receptors correlates with the formation of a continuous internal water pathway
More than 800 human GPCRs allow the selective detection of extracellular signals as diverse as photons, odorants, flavors, nucleotides, hormones, neurotransmitters - revealing GPCRs fundamental role in signal transduction. As they regulate many central physiological processes and are thus implicated in many diseases, GPCRs are among the most important targets for modern medicines. In spite of this medical importance and the recent progress in elucidating the 3D structures of various GPCRs, central questions how these receptors recognize extracellular chemical signals and transfer them across the cellular membrane to finally evoke an intracellular response are largely unresolved at a molecular level, mainly because the different steps during signal transmission are not directly accessible by experiments. In this context we are concentrating on central questions of GPCR mediated cellular signalling using molecular dynamics simulations and structural modelling. Our work revealed for the first time, in atomic detail, the entire process of transmembrane signalling of various GPCRs: we found that ligand binding induces a series of conformational changes within a GPCR which opens a gate inside the receptor for water molecules entering the internal region of the receptor and subsequently driving conformational switches within the receptor which finally led to the activation of a G protein on the intracellular side of the receptor. The discovery of these internal water channels paved the way for novel approaches in drug development.<br>Reference:<br>1. Yuan S*, Palczewski K.; Peng Q, Kolinski M, Vogel H*, Filipek S*. (2015) Ligand specificity of the ?- and ?-opioid receptors, a matter of space. Angew. Chem. Int. Edt DOI: 10.1002/anie.201501742R2<br>2. Yuan S*, Hu Z, Filipek S, Vogel H*. (2015) W2466.48 opens a gate for a continuous intrinsic water pathway during activation of the adenosine A2A receptor. Angew. Chem. Int. Edt 127(2),566-569<br>3. Yuan S*.; Filipek S.; Palczewski K.; Vogel H*. (2014) Activation of G-protein-coupled receptor correlates with the formation of a continuous internal water pathway.Nature Communication 5, 4377 DOI:10.1038/ncomms5733<br>4. Yuan S*.; Vogel H.; Filipek S*., (2013) The Role of Water Molecules and Sodium Ions in ?-opioid Receptor Activation. Angew. Chem. Int. Edt52(38) 10112-1011
Computational Methods in GPCR Mechanism Study and Drug Discovery
More than 800 human GPCRs allow the selective detection of extracellular signals as diverse as photons, odorants, flavors, nucleotides, hormones, neurotransmitters ? revealing GPCRs fundamental role in signal transduction. As they regulate many central physiological processes and are thus implicated in many diseases, GPCRs are among the most important targets for modern medicines. In spite of this medical importance and the recent progress in elucidating the 3D structures of various GPCRs, central questions how these receptors recognize extracellular chemical signals and transfer them across the cellular membrane to finally evoke an intracellular response are largely unresolved at a molecular level, mainly because the different steps during signal transmission are not directly accessible by experiments. In this context we are concentrating on central questions of GPCR mediated cellular signalling using computer based molecular dynamics simulations and structural modelling. Our work revealed for the first time, in atomic detail, the entire process of transmembrane signalling of various GPCRs: we found that ligand binding induces a series of conformational changes within a GPCR which opened a gate inside the receptor for water molecules entering the internal region of the receptor and subsequently driving conformational switches within the receptor which finally led to the activation of a G protein on the intracellular side of the receptor. With the uncovered principle, 17 out 25 newly screened compounds were proved to be active for odorant receptors. The discovery of these internal water channels paved the way for novel approaches in drug development. <br><br
Investigating Substrate Scope and Enantioselectivity of a Defluorinase by a Stereochemical Probe
The
possibility of a double Walden inversion mechanism of the fluoracetate
dehalogenase FAcD (RPA1163) has been studied by subjecting <i>rac</i>-2-fluoro-2-phenyl acetic acid to the defluorination
process. This stereochemical probe led to inversion of configuration
in a kinetic resolution with an extremely high selectivity factor
(<i>E</i> > 500), showing that the classical mechanism
involving
S<sub>N</sub>2 reaction by Asp110 pertains. The high preference for
the (<i>S</i>)-substrate is of synthetic value. Wide substrate
scope of RPA1163 in such hydrolytic kinetic resolutions can be expected
because the reaction of the even more sterically demanding <i>rac</i>-2-fluoro-2-benzyl acetic acid proceeded similarly. Substrate
acceptance and stereoselectivity were explained by extensive molecular
modeling (MM) and molecular dynamics (MD) computations. These computations
were also applied to fluoroacetic acid itself, leading to further
insights
Investigating Substrate Scope and Enantioselectivity of a Defluorinase by a Stereochemical Probe
The
possibility of a double Walden inversion mechanism of the fluoracetate
dehalogenase FAcD (RPA1163) has been studied by subjecting <i>rac</i>-2-fluoro-2-phenyl acetic acid to the defluorination
process. This stereochemical probe led to inversion of configuration
in a kinetic resolution with an extremely high selectivity factor
(<i>E</i> > 500), showing that the classical mechanism
involving
S<sub>N</sub>2 reaction by Asp110 pertains. The high preference for
the (<i>S</i>)-substrate is of synthetic value. Wide substrate
scope of RPA1163 in such hydrolytic kinetic resolutions can be expected
because the reaction of the even more sterically demanding <i>rac</i>-2-fluoro-2-benzyl acetic acid proceeded similarly. Substrate
acceptance and stereoselectivity were explained by extensive molecular
modeling (MM) and molecular dynamics (MD) computations. These computations
were also applied to fluoroacetic acid itself, leading to further
insights
Movements of transmembrane helices in S1P<sub>1</sub> receptor.
<p>(A) RMSD of S1P<sub>1</sub> TM regions during MD simulations. Apo S1P<sub>1</sub> in black, ML056/S1P<sub>1</sub> in green and cyan, and S1P/S1P<sub>1</sub> in red and blue. (B) Different states of agonist-bound receptor structure during MD simulation. The 3D plot shows distances between cytoplasmic ends of TM helices: TM7-TM3, TM3-TM6 and TM6-TM7. The central structures from each cluster are shown. The “intermediate” and “active” conformations are superimposed on the “inactive” one (in grey).</p
Water molecules at the intracellular side.
<p>(A, A′) Number of water molecules within 4 Å of the NPxxY motif at TM7. Apo S1P<sub>1</sub> in black, complex with antagonist in green, and complex with agonist in red. (B) The final structures including water molecules near NPxxY motif in Apo (on left) and agonist-bound receptor (on right). Antagonist-bound structure is similar to the Apo S1P<sub>1</sub>.</p
Water molecules in vicinity of residue D91<sup>2.50</sup> in agonist-bound receptor during MD simulation.
<p>(A) 0 ns; (B) 100 ns; (C) 700 ns. Only water molecules within 4 Ă… of residue D91<sup>2.50</sup> are shown.</p
Proposition of activation mechanism of S1P<sub>1</sub>.
<p>Binding of agonist (S1P) can lead to conformational changes of highly conserved residues W269<sup>6.48</sup> and F265<sup>6.44</sup> (step 1 and 2) forming a core of a transmission switch. Afterwards, rearrangement of centrally located residues facilitate the redirected flow of water molecules inside a receptor (step 3) which is a prerequisite for a larger motion of cytoplasmic parts of transmembrane helices (step 4).</p
Rotamer switches at S1P<sub>1</sub> extracellular region.
<p>(A) Apo S1P<sub>1</sub>, the χ<sub>1</sub> angle of Y98<sup>2.57</sup> changed at 100 ns while W269<sup>6.48</sup> and F265<sup>6.44</sup> were stable during the whole simulation; (B, B′) antagonist ML056/S1P<sub>1</sub>, the χ<sub>1</sub> angle of Y98<sup>2.57</sup> changed at 550 ns (or 300 ns in 2<sup>nd</sup> simulation); the χ<sub>2</sub> of W269<sup>6.48</sup> fluctuated in the initial 500 ns of simulation and it was stable in 2<sup>nd</sup> simulation; F265<sup>6.44</sup> was stable in both simulation with antagonist; (C, C′) agonist S1P/S1P<sub>1</sub>, the χ<sub>1</sub> angle of Y98<sup>2.57</sup> was relatively stable while both the χ<sub>2</sub> angle of W269<sup>6.48</sup> and χ<sub>1</sub> of F265<sup>6.44</sup> fluctuated considerably during the simulations. Internal water molecules are shown as pink dots. The initial structures of complexes after equilibration are shown in grey, while the final structures are shown in color. Blue dashed ellipse indicates lack of a flip of residue Y98<sup>2.57</sup> in case of complex with agonist.</p
Binding of ligands in S1P<sub>1</sub> extracellular pocket.
<p>(A) Ligand structures after equilibration: antagonist (yellow) and agonist (purple). Helices represent the crystal structure; (B) The structures of ligand-receptor complexes after 700 ns MD simulations. The antagonist-receptor structure colored in blue, while agonist-receptor structure in yellow.</p