An analysis of Group IVA (GIVA) phospholipase A2 (PLA2) inhibitor binding was conducted using a combination of deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD). Models of the GIVA PLA2 inhibitors pyrrophenone and the 2-oxoamide AX007 docked into the protein were designed based on deuterium exchange results, and extensive molecular dynamics simulations were run to determine protein-inhibitor contacts. The models show that both inhibitors interact with key residues that also exhibit changes in deuterium exchange upon inhibitor binding. Pyrrophenone is bound to the protein through numerous hydrophobic residues located distal from the active site, while the oxoamide is bound mainly through contacts near the active site. We also show differences in protein dynamics around the active site between the two inhibitor-bound complexes. This combination of computational and experimental methods is useful in defining more accurate inhibitor binding sites, and can be used in the generation of better inhibitors against GIVA PLA2.

Ras GTPases mediate signaling pathways in cell proliferation, development, and apoptosis. They undergo isoprenylation at a C-terminal CaaX signal (a usually represents aliphatic and X any amino acid) followed by proteolysis of aaX and carboxymethylation. In the case of H-ras, a subsequent dual palmitoylation of cysteines adjacent to the site of farnesylation produces a mature anchor for plasma membrane targeting. [1,2] Atomistic information, such as the structure of membrane-bound ras and the free energy of complex formation, are vital in research efforts geared towards designing ras-isoform-selective anticancer agents. The most common experimental techniques are not yet able to provide such information. Here we present computational results on the free energy profile for the transfer of the H-ras membrane anchor from water to a bilayer of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (1,2-DMPC) lipids. We find that there is no significant barrier for insertion, and that once a few carbon atoms of the ras lipid chains cross the membrane-water interface, the free energy displays a steeply downhill profile. Insertion into the hydrocarbon core of the ras lipids and the interfacial localization of the backbone together produce a gain in free energy of up to 30 kcal mol À1 . Additionally, using the recently reported computationally derived structures of full-length H-ras in a DMPC bilayer, [3] we explain how a small difference in free energy would enable modulation of H-ras membrane binding by the linker and the catalytic domain.Molecular dynamics (MD) simulations of the H-ras anchor (residues 180-186) were carried out using the CHARMM27 force field [4] and the program NAMD.[5]

A variety of biomolecules mediate physiological processes by inserting and reorganizing in cell membranes, and the thermodynamic forces responsible for their partitioning are of great interest. Recent experiments provided valuable data on the free energy changes associated with the transfer of individual amino acids from water to membrane. However, a complete picture of the pathways and the associated changes in energy of peptide insertion into a membrane remains elusive. To this end, computational techniques supplement the experimental data with atomic-level details and shed light on the energetics of insertion. Here, we employed the technique of umbrella sampling in a total 850 ns of all-atom molecular dynamics simulations to study the free energy profile and the pathway of insertion of a model hexapeptide consisting of a tryptophan and five leucines (WL5). The computed free energy profile of the peptide as it travels from bulk solvent through the membrane core exhibits two minima: a local minimum at the water−membrane interface or the headgroup region and a global minimum at the hydrophobic−hydrophilic interface close to the lipid glycerol region. A rather small barrier of roughly 1 kcal mol−1 exists at the membrane core, which is explained by the enhanced flexibility of the peptide when deeply inserted. Combining our results with those in the literature, we present a thermodynamic model for peptide insertion and aggregation which involves peptide aggregation upon contact with the membrane at the solvent−lipid headgroup interface.