| Molecular simulations of the ligands and receptors of the insulin family were conducted. First, phenol dissociation mechanisms in the pharmaceutical storage form of insulin, R6 insulin hexamer, are studied using random acceleration molecular dynamics (RAMD) simulations. Three major escape pathways for phenols are observed. Several residues that directly participate in escape events serve to resolve ambiguities from previous NMR experiments. Potentials of mean force (PMFs) along the reaction coordinate for each pathway are further resolved using steered molecular dynamics (SMD) simulations. The results for binding free energy, DeltaF, agree reasonably well within the range of known experimental and previous simulation magnitudes of this quantity. Structural analysis and energetic barriers for each pathway suggest a plausible preferred mechanism of phenolic exchange that differs from previous mechanisms, but is consistent with RAMD results. Several weakly-bound metastable states, supported by experimental evidence, are also observed. These results give detailed insights into the dissolution kinetics of insulin hexamers.;Next, the flexibility mechanisms of the ectodomains of the insulin receptor (IR) and the type-1 insulin-like growth factor receptor (IGF1R) are studied. Both receptors are observed to display asymmetric mechanisms of flexibility due to interdomain and intersubunit conformational fluctuations that do not alter their quaternary structures. Subtle variations in intersubunit buried surface areas of both receptors are observed to coincide well with these conformational fluctuations, resulting in one easily-accessible ligand binding pocket with the other blocked. Further, Monte Carlo (MC) docking simulations are used to construct all-atom structural models of the ligand/receptor complexes which are consistent with a variety of known experimental data, and provide detailed structural bases for ligand-recognition in the IR family.;Finally, a large-scale conformational change in the activation-loop (A-loop) of the insulin receptor kinase domain (IRKD) is studied using temperature-accelerated molecular dynamics (TAMD). The A-loop is observed to form a transient three-turn helical conformation during TAMD, which facilitates the conserved DFG-motif (D1150, F1151, and G1152) to switch from a "D-out/F-in" to "D-in/F-out" conformation, thereby burying F 1151 underneath alphaC-helix, and simultaneously presenting D 1150 for ATP binding. These transient helical conformations of the A-loop have been observed in the crystal structures of related kinase families, and may be useful for the design of novel inhibitors targeting IRKD. |
