Recognition and Reactivity in the Binding between Raf Kinase Inhibitor Protein and Its Small-Molecule Inhibitor Locostatin

Abstract

The present work is aimed to provide detail on the binding process between Raf kinase inhibitor protein (RKIP) and locostatin, the only exogenous compound known to alter the function of RKIP. Understanding the basis of RKIP inhibition for use in pharmacological applications is of considerable interest, as dysregulated RKIP expression has the potential to contribute to pathophysiological processes. Herein, we report a series of atomistic models to describe the protein-ligand recognition step and the subsequent reactivity steps. Modeling approaches include ligand docking, molecular dynamics, and quantum mechanics/molecular mechanics calculations. We expect that such a computational assay will serve to study similar complexes in which potency is associated with recognition and reactivity. Although previous data suggested a single amino acid residue (His86) to be involved in the binding of locostatin, the actual ligand conformation and the steps involved in the reactivity process remain elusive from a detailed atomistic description. We show that the first reaction step, consisting of a nucleophilic attack of the nitrogen (Nε) of His86 at the sp(2)-hybridized carbon (C2) of locostatin, presents a late transition state (almost identical to the product). The reaction is followed by a hydrogen abstraction and hydrolysis. The theoretically predicted overall rate constant (6 M(-1) s(-1)) is in a very good agreement with the experimentally determined rate constant (13 M(-1) s(-1)).

PEBP1 Wardens Ferroptosis by Enabling Lipoxygenase Generation of Lipid Death Signals

“…A small molecule, locostatin, covalently interacts with a highly conserved His86 in the PEBP1 ligand binding pocket and dissociates the scaffold protein from Raf1 kinase (Rudnitskaya et al, 2012), hence makes PEBP1 available for pro-ferroptotic interactions with 15LO. We selected low concentrations of RSL3 inducing mild ferroptosis and explored the combined effects of RSL3 plus locostatin (Figure 5).…”

An in cellulo-derived structure of PAK4 in complex with its inhibitor Inka1

“…These proteins, much similar to the JIP family of mitogen-activated protein kinase scaffold proteins, are not stand-alone kinase inhibitors, but rather form a modulatory platform essential for CDK signalling 54 . Finally, the Raf1 and GRK2 inhibitor RKIP is extensively studied and its structure known 55 , but the way by which this protein binds to kinase targets is not known. Mapping studies indicate the non-catalytic domain of Raf1 binds RKIP 56 , which differentiates it from the protein kinase inhibitors shown in Fig.…”

SSPaQ: A Subtractive Segmentation Approach for the Exhaustive Parallel Quantification of the Extent of Protein Modification at Every Possible Site

“…From this, the authors pointed to His86, among several modified residues, as the primary target site of locostatin, based on the fact that it is a highly conserved residue. Despite the fact that locostatin, as an electrophile, likely binds to deprotonated nucleophilic residues, these results were used in support of a computational simulation for locostatin binding in the anion pocket [29]. Our results show that modification in the [81-87] segment is in fact minor, and thus residues therein cannot be considered as a specific binding site.…”