CDK2 and CDK4, using the GOLD package. GOLD treats ligands as fully flexible and allows the user to assign flexibility to a limited set of receptor residues. The best-scoring docking poses for all four systems investigated show key features known from experimentally determined CDK2/inhibitor X-ray structures such as hydrogen bonding in the hinge region. FAS and CRB docking poses are characterised by two H-bonds involving the backbone NH and carbonyl of the hinge residues Leu83CDK2 and Val96CDK4. No significant difference was found in the docking scores of both compounds with both CDK2 and CDK4 (ChemScores are in the range of 29 to 31 for CDK2, and 31 to 34 for CDK4, respectively). So while the ligand docking study generates typical kinase inhibitor binding poses, it can not explain why fascaplysin preferably binds to CDK4 rather than CDK2. A key difference between the CDK2 and CDK4 poses involves the equivalent residues His95CDK4 and Phe82CDK2. In principle three different `species’ of His95CDK4 have to be considered: His95CDK4 could have a positively charged imidazole side chain and there are two uncharged species with either Nd or Ne of the imidazole ring bearing a hydrogen. We did not consider a positively charged imidazole side chain as this would unfavourab
interact with the positively charged fascaplysin. However, alternative positioning of hydrogens in His95CDK4 (Nd-H and Ne-H) was considered in the ligand docking process. ChemScores were by a small margin higher for the His95CDK4Nd-H/fascaplysin complex (33.4 compared to 31.3 for His95CDK4Ne-H/fascaplysin complex) indicating a slight preference for the side-chain conformation in which the Nd-hydrogen of the imidazol ring forms an additional H-bond to the carbonyl of FAS and CRB, respectively. This conformation is different from the His95 conformation found in the experimentally determined CDK4 structures, but such a conformational change could occur upon ligand binding, when the alternative His95CDK4 side chain conformation is stabilised by the interaction with the inhibitor (Figure 4A). The idea of His95 as a key player for CDK4 specificity is supported by the notion that CDK6 also has a histidine residue in the equivalent position. Any energetic contribution of the additional His95-Nd H-bond to the free energy of binding should also feature in CDK6, and indeed the IC50 of CDK6/fascaplysin is, while being ,8 times higher than CDK4/fascaplysin, still ,100 times lower than CDK2/fascaplysin. However, there is a problem with this notion, as if correct, the interaction in question should occur for most inhibitors, essentially for any ligand that forms a H-bond with the backbone NH of Val96CDK4. If His95CDK4 was indeed the key to the observed fascaplysin CDK4 specificity we would expect this to be rather generic feature, rendering most CDK inhibitors more specific for CDK4 as CDK2. This is however not the case and hence it is unlikely that the difference between His95CDK4 and Phe82CDK2 can account fully for the differential binding of fascaplysin. The inaccuracy of docking scoring functions for estimating free energies of binding is a major short coming of typical ligand docking approaches [83?5]. To obtain more accurately calculated values for free energies of binding thermodynamic integration was used. A key feature of fascaplysin is its positive charge. Docking scoring functions are limited in accounting for long-range electrostatic interactions; Thermodynamic Integration however describes long-range electrostatic interactions more accurately as the Particle Mesh Ewald method for calculating electrostatic energy terms also incorporates orientation polarisation effects (conformational response to charge). The Thermodynamic Integration approach was used to specifically address the role of charge as a determinant of CDK4 inhibitor selectivity comparing the charge stabilisation in CDK2/CRB-.CDK2/FAS and His95 NeH CDK4/CRB-.CDK4/FAS complexes [40,41]. To better account for protein flexibility in response to inhibitor binding a series of six 5 ns molecular dynamics simulation was performed. The comparison between runs with all four inhibitor-protein complexes, FAS and CRB as inhibitors, and CDK2 and CDK4 (both His95 conformers) as receptors, allows the investigation of conformational change in response to changes of charge of inhibitors.
Molecular dynamics simulations
Before endeavouring on TI runs the systems corresponding to the l = 0 and l = 1 endpoints, i.e., CDK2/fascaplysin and CDK2/ carbofascaplysin, CDK4-His95CDK4-Ne-H/carbofascaplysin and CDK4-His95CDK4-Ne-H/fascaplysin, and for comparison CDK4His95CDK4-Nd-H/fascaplysin and CDK4-His95CDK4-Nd-H/carbofascaplysin were tested for stability (Figure 5). The average rmsd (compared to the energy minimised starting structure) over 5 ns ?CDK2 simulations is less than 2 A for both runs, the average rmsd for the respective CDK4 simulations is slightly higher. This higher value is not unexpected as the CDK4 structure used for simulations is the `hybrid model’ as described in the materials
