The inhibitor kappa B kinase-beta (IKK-beta) phosphorylates the NF-kappa B inhibitor protein I kappa B leading to the translocation of the transcription factor NF-kappa B to the nucleus. The transcription factor NF-kappa B and consequently IKK-beta are central to signal transduction pathways of mammalian cells. The purpose of this research was to develop a 3D structural model of the IKK-beta kinase domain with its ATP cofactor and investigate its dynamics and ligand binding potential. Through a combination of comparative modelling and simulated heating/annealing molecular dynamics (SAMD) simulation in explicit water the model accuracy could be substantially improved compared to comparative modelling on its own as shown by model validation measures. The structure revealed the details of ATP/Mg2+ binding indicating hydrophobic interactions with the adenine base and a significant contribution of Mg2+ as a bridge between ATP phosphate groups and negatively charged side chains. The molecular dynamics trajectories of the ATP-bound and free enzyme showed two conformations in each case, which contributed to the majority of the trajectory. The ATP-free enzyme revealed a novel binding site distant from the ATP binding site that was not encountered in the ATP bound enzyme. Based on the overall structural flexibility, it is suggested that a truncated version of the kinase domain from Ala 14 to Leu265 should be subjected to crystallisation trials. The 3D structure of this enzyme will enable rational design of new ligands and analysis of protein-protein interactions. Furthermore, our results may provide a new impetus for wet-lab based structural investigation focussing on a truncated kinase domain. (C) 2011 Elsevier Inc. All rights reserved.