Exploring the effects of macromolecular crowding on the conformation and NuMA binding of 4.1G-CTD

Abstract

The C-terminal domain of protein 4.1G is an intrinsically disordered region that forms a fuzzy complex with the disordered C-terminus of the nuclear mitotic apparatus (NuMA) protein, playing a crucial role in regulating spindle orientation during cell division. However, how these interactions adapt to the physiologically crowded environments of the cell remains unclear. In this study, we employ multiple biophysical techniques to systematically investigate the effects of macromolecular crowding on the structure, conformational dynamics, and binding capacity of 4.1G-CTD. Our findings reveal that crowding induces structural compaction in 4.1G-CTD, while preserving its intrinsically disordered characteristics, despite crowder-specific modulation of transient secondary structures. Moreover, crowding consistently enhances the binding affinity of 4.1G-CTD for NuMA and accelerates the kinetics of their association; however, the extent of these effects varies significantly among different types of crowders. Collectively, our results demonstrate that intracellular crowding fine-tunes the functionality of intrinsically disordered proteins (IDPs) by exerting complex influences on their conformational landscapes as well as the thermodynamics and kinetics of their interactions.