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pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins.

Prakash P, Sayyed-Ahmad A, Gorfe AA - PLoS Comput. Biol. (2015)

Bottom Line: Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets.However, thus far the method has been applied only to soluble proteins.We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer.

View Article: PubMed Central - PubMed

Affiliation: University of Texas Health Science Center at Houston, Department of Integrative Biology and Pharmacology, Houston, Texas, United States of America.

ABSTRACT
Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation.

No MeSH data available.


Predicted binding sites in the soluble and membrane-bound G12D K-Ras.Isosurfaces of probe densities with grid free energy values ≤ -1 kcal/mol are shown in solution (green) and membrane (blue) environment. (A) Membrane binding mode 1. (B) Membrane binding mode 2. Previously characterized pockets p1 (near β1-β3/h2), p2 (between h2/h3), p3 (near h5 and loop7) and p4 (behind s1) are labeled. The last 10 ns of the current pMD-membrane runs and last 10 ns of the longest run of G12D K-Ras in solution from ref [11] were used to calculate the probe densities and grid free energies. The protein from solution was first aligned to each of mode 1 and mode 2 membrane-bound forms prior to grid-based calculations. The protein is in yellow and a portion of the inner leaflet of the bilayer is shown as iceblue lines. See ref [11] for details.
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pcbi.1004469.g004: Predicted binding sites in the soluble and membrane-bound G12D K-Ras.Isosurfaces of probe densities with grid free energy values ≤ -1 kcal/mol are shown in solution (green) and membrane (blue) environment. (A) Membrane binding mode 1. (B) Membrane binding mode 2. Previously characterized pockets p1 (near β1-β3/h2), p2 (between h2/h3), p3 (near h5 and loop7) and p4 (behind s1) are labeled. The last 10 ns of the current pMD-membrane runs and last 10 ns of the longest run of G12D K-Ras in solution from ref [11] were used to calculate the probe densities and grid free energies. The protein from solution was first aligned to each of mode 1 and mode 2 membrane-bound forms prior to grid-based calculations. The protein is in yellow and a portion of the inner leaflet of the bilayer is shown as iceblue lines. See ref [11] for details.

Mentions: In the previous pMD analysis of G12D in solution [11], we identified five druggable sites and three sub-sites (see Fig 3 in ref. [11]). A detailed comparison with known ligand binding pockets indicated that three of the predicted druggable sites overlap well with pockets p1, p2 and p3 while two of the predicted sub-sites were found to be parts of p1 and p4, respectively (see Fig 4 of ref. [11]). Allosteric pockets p1 to p4 have been previously described in detail: p1 is the binding site of ligands reported by Maurer et al [32], Sun et al [33] and Shima et al [34]; p3 is near the C-terminus of the catalytic domain where Cu2+-cylen binds [35,36]; p4 is the proposed target site of Andrographolide derivatives [38] and Zn(II)-bis(2 picolyl)amines [36]; there is no known non-covalent binder that targets p2 but covalent ligands that target this region have been reported [37]. Direct comparison of the calculated probe occupancies in membrane-bound K-Ras with experimental results is not possible because one cannot turn on in experiment non-physical repulsive interactions to prevent bilayer partitioning of isopropanol. Nonetheless, the following analysis provides a strong evidence that pMD-membrane is able to identify true drug binding sites.


pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins.

Prakash P, Sayyed-Ahmad A, Gorfe AA - PLoS Comput. Biol. (2015)

Predicted binding sites in the soluble and membrane-bound G12D K-Ras.Isosurfaces of probe densities with grid free energy values ≤ -1 kcal/mol are shown in solution (green) and membrane (blue) environment. (A) Membrane binding mode 1. (B) Membrane binding mode 2. Previously characterized pockets p1 (near β1-β3/h2), p2 (between h2/h3), p3 (near h5 and loop7) and p4 (behind s1) are labeled. The last 10 ns of the current pMD-membrane runs and last 10 ns of the longest run of G12D K-Ras in solution from ref [11] were used to calculate the probe densities and grid free energies. The protein from solution was first aligned to each of mode 1 and mode 2 membrane-bound forms prior to grid-based calculations. The protein is in yellow and a portion of the inner leaflet of the bilayer is shown as iceblue lines. See ref [11] for details.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4623977&req=5

pcbi.1004469.g004: Predicted binding sites in the soluble and membrane-bound G12D K-Ras.Isosurfaces of probe densities with grid free energy values ≤ -1 kcal/mol are shown in solution (green) and membrane (blue) environment. (A) Membrane binding mode 1. (B) Membrane binding mode 2. Previously characterized pockets p1 (near β1-β3/h2), p2 (between h2/h3), p3 (near h5 and loop7) and p4 (behind s1) are labeled. The last 10 ns of the current pMD-membrane runs and last 10 ns of the longest run of G12D K-Ras in solution from ref [11] were used to calculate the probe densities and grid free energies. The protein from solution was first aligned to each of mode 1 and mode 2 membrane-bound forms prior to grid-based calculations. The protein is in yellow and a portion of the inner leaflet of the bilayer is shown as iceblue lines. See ref [11] for details.
Mentions: In the previous pMD analysis of G12D in solution [11], we identified five druggable sites and three sub-sites (see Fig 3 in ref. [11]). A detailed comparison with known ligand binding pockets indicated that three of the predicted druggable sites overlap well with pockets p1, p2 and p3 while two of the predicted sub-sites were found to be parts of p1 and p4, respectively (see Fig 4 of ref. [11]). Allosteric pockets p1 to p4 have been previously described in detail: p1 is the binding site of ligands reported by Maurer et al [32], Sun et al [33] and Shima et al [34]; p3 is near the C-terminus of the catalytic domain where Cu2+-cylen binds [35,36]; p4 is the proposed target site of Andrographolide derivatives [38] and Zn(II)-bis(2 picolyl)amines [36]; there is no known non-covalent binder that targets p2 but covalent ligands that target this region have been reported [37]. Direct comparison of the calculated probe occupancies in membrane-bound K-Ras with experimental results is not possible because one cannot turn on in experiment non-physical repulsive interactions to prevent bilayer partitioning of isopropanol. Nonetheless, the following analysis provides a strong evidence that pMD-membrane is able to identify true drug binding sites.

Bottom Line: Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets.However, thus far the method has been applied only to soluble proteins.We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer.

View Article: PubMed Central - PubMed

Affiliation: University of Texas Health Science Center at Houston, Department of Integrative Biology and Pharmacology, Houston, Texas, United States of America.

ABSTRACT
Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation.

No MeSH data available.