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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.


Differences in probe binding between G12D and G13D K-Ras.(A) Same as Fig 6A except that ΔR is the difference in atomic probe occupancies of G12D and G13D K-Ras. Estimated sampling errors of ΔRseq for each atom was calculated using Eq 6 and plotted in the bottom. (B) Same as Fig 6B but using the ΔR values shown in Fig 7A.
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pcbi.1004469.g007: Differences in probe binding between G12D and G13D K-Ras.(A) Same as Fig 6A except that ΔR is the difference in atomic probe occupancies of G12D and G13D K-Ras. Estimated sampling errors of ΔRseq for each atom was calculated using Eq 6 and plotted in the bottom. (B) Same as Fig 6B but using the ΔR values shown in Fig 7A.

Mentions: In an unpublished study, we observed that the active sites of G12D and G13D K-Ras significantly differ. While the active site of G12D K-Ras is similar to wild type, switch 1 is open and some functionally critical residues, such as Tyr32, have re-oriented in G13D K-Ras. At the functional level, G12D and G13D K-Ras differ in intrinsic GTPase activity [74] and oncogenicity [25]. Therefore, we ran a pMD-membrane simulation on G13D starting from the conformation shown in Fig 1A (mode 1) and calculated ΔRseq = RG13D –RG12D. We found major differences in probe accessibility of the two mutants (Fig 7A), indicating that the two simulations started from the same initial configuration have drifted apart, leading to different probe binding propensities. The differences are largely confined to helices 2, 3, and 4 (Fig 7A), representing two surfaces on the 3D structure (Fig 7B). Part of the surface of helix 2 where p1 is located is more accessible in G13D than G12D whereas the region between helices 3 and 4 is significantly more probe-accessible in G12D than G13D. This is despite the fact that these regions are far away from the site of the mutation. We propose that these observations highlight potential differences in the druggability of the two mutants and thereby the possibility of isoform-specific drug leads. We find the region between helices 3 and 4 especially interesting as it might represent a potentially unique new ligand-binding site.


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

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

Differences in probe binding between G12D and G13D K-Ras.(A) Same as Fig 6A except that ΔR is the difference in atomic probe occupancies of G12D and G13D K-Ras. Estimated sampling errors of ΔRseq for each atom was calculated using Eq 6 and plotted in the bottom. (B) Same as Fig 6B but using the ΔR values shown in Fig 7A.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4623977&req=5

pcbi.1004469.g007: Differences in probe binding between G12D and G13D K-Ras.(A) Same as Fig 6A except that ΔR is the difference in atomic probe occupancies of G12D and G13D K-Ras. Estimated sampling errors of ΔRseq for each atom was calculated using Eq 6 and plotted in the bottom. (B) Same as Fig 6B but using the ΔR values shown in Fig 7A.
Mentions: In an unpublished study, we observed that the active sites of G12D and G13D K-Ras significantly differ. While the active site of G12D K-Ras is similar to wild type, switch 1 is open and some functionally critical residues, such as Tyr32, have re-oriented in G13D K-Ras. At the functional level, G12D and G13D K-Ras differ in intrinsic GTPase activity [74] and oncogenicity [25]. Therefore, we ran a pMD-membrane simulation on G13D starting from the conformation shown in Fig 1A (mode 1) and calculated ΔRseq = RG13D –RG12D. We found major differences in probe accessibility of the two mutants (Fig 7A), indicating that the two simulations started from the same initial configuration have drifted apart, leading to different probe binding propensities. The differences are largely confined to helices 2, 3, and 4 (Fig 7A), representing two surfaces on the 3D structure (Fig 7B). Part of the surface of helix 2 where p1 is located is more accessible in G13D than G12D whereas the region between helices 3 and 4 is significantly more probe-accessible in G12D than G13D. This is despite the fact that these regions are far away from the site of the mutation. We propose that these observations highlight potential differences in the druggability of the two mutants and thereby the possibility of isoform-specific drug leads. We find the region between helices 3 and 4 especially interesting as it might represent a potentially unique new ligand-binding site.

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.