| Summary: | Computer simulation techniques have long proven to be useful in studies of biomolecular systems by describing molecular processes at different levels of resolution and playing an important role in chemistry, biology and physics. In this work, several computational techniques – standard and enhanced sampling MD simulations, multi-conformational pKa calculations and binding free energy calculations – are applied to study the mechanism of action of two important enzymes: a C-type cytochrome c oxidase (cbb3) and an E3 ubiquitin ligase (Parkin).Cbb3 is a C-type terminal oxidase responsible for catalyzing the final step of aerobic respiration (namely the reduction of oxygen to water) and coupling this redox reaction to the active translocation of protons across the membrane, a process that is essential for ATP synthesis. It is mainly present in proteobacteria, and the fact that it is the only terminal oxidase expressed in several clinically relevant human pathogens makes it a potential drug target. The limited data available for cbb3, as well as the overall complexity of terminal oxidases in general, are the main reasons why the functioning mechanism of cbb3 oxidase is still poorly understood. The aim of this study is through a combination of several simulation methods to identify and fully characterise proton pathways in cbb3, which are essential for the translocation of chemical and pumped protons to the active site and proton-loading site, respectively. We explain the effect of previously reported mutations and put forward a proposal for the redox-driven proton pumping mechanism. Our results contribute to a better understanding of the cbb3 mechanism and provide ideas for further experimental and computational studies. Parkin is a RING-in-between-RING (RBR) E3 ubiquitin ligase which is involved in the neurodegenerative disorder Parkinson’s disease. All available X-ray structures captured Parkin in the inactive (inhibited) form. Recent biochemical and structural data suggested that several distinct factors are involved in activation of Parkin, but the mutual effect of these factors or the detailed sequence of steps occurring on the molecular scale during activation are still poorly understood. In this study, we use molecular simulation methods to examine some of the existing proposals about the activation mechanism of Parkin. The structural effects of Parkin phosphorylation and removal of its inhibitory domain are addressed. Additionally, the effect of several mutations which are thought to promote the displacement/detachment of the inhibitory domain and render Parkin active are also investigated. Our findings provide atomic-level insights into the role of various factors in the stability and conformational dynamics of Parkin.
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