“Enhancing investigations of structure, function, and inhibition at the protein-membrane interface”
Abstract:
Peripheral membrane proteins (PMPs) are water-soluble proteins that bind reversibly to membranes to perform function. Central to a variety of biological and disease processes, PMP functions are diverse, critical to life, and represent promising yet underutilized therapeutic targets. Despite their importance the functional, membrane-bound state of PMPs is typically poorly understood. Due to their relatively small size, the membrane protein revolution ushered in by cryo-EM has largely left PMPs behind. Detailed structural study is often constrained to the nonfunctional, water-solubilized state. While protein NMR is a promising method, a larger toolbox must be developed to improve the evaluation of PMPs in their membrane-bound state.
We have recently developed membrane-mimicking reverse micelles (mmRMs) as NMR-compatible models to house PMPs in their functional state. Use of mmRMs have been applied to a variety of human PMPs including glutathione peroxidase 4 (GPx4) and fatty acid binding protein 4 (FABP4). For these proteins, certain investigations of their structure, function, and interactions have been improved compared to other membrane models. New insight into high-resolution biological and disease processes gained here has revealed inhibitor design strategies for PMPs. We have applied traditional, aqueous state fragment-based inhibitor design (FBID) methods to the membrane anchoring p47phox-PX domain with promising results. However, for GPx4 and other similar PMPs, targeting the relevant membrane-bound state represents a greater challenge. Fragment screening is highly compatible with mmRMs, allowing GPx4 to be assessed in its functional state. A variety of fragments are revealed to bind in the protein-membrane interface, representing a pool of potential inhibitor building-blocks. Results have also revealed fundamental properties of fragments that bind within the protein-membrane interface, a relatively new mode of inhibition. The addition of mmRMs to the PMP toolbox promises to open new avenues of exploration an inhibition to improve our understanding of this essential category of proteins.