Uncovering the Structural Basis for Mitochondrial Calcium Uniporter Dominant Negative Beta Subunit (MCUb) Function

Megan L. Noble, The University of Western Ontario

Abstract

Mitochondrial calcium (Ca²⁺) uptake is regulated by the mitochondrial Ca²⁺ uniporter (MCU), a tetrameric channel that is regulated by interactions with several accessory proteins, including MCU dominant negative beta subunit (MCUb). MCUb inhibits Ca²⁺ uptake by assembling into the MCU complex and is incapable of forming a functional Ca²⁺ channel. The MCU amino (N)-terminal domain plays an essential role in controlling MCU structure and function and contains cation binding sites that, when bound by Ca²⁺ and magnesium (Mg²⁺), cause decreased MCU assembly and reduced mitochondrial Ca²⁺ uptake. MCU and MCUb contain high sequence and topological similarities, suggesting the MCUb N-terminal domain may similarly play an important regulatory role in how MCUb functions. Thus, I aimed to characterize the biophysical and structural properties of the MCUb N-terminal domain and characterize MCUb:MCU N-terminal domain interactions. Here, I report that MCUb N-terminal domain exhibits α-helical and β-sheet structure, with a remarkably high thermal stability, both of which are highly dependent on protein concentration. The MCU:MCUb N-terminal domains interact with ~nM affinity. The interaction causes increased secondary structure and thermal stability and induces large structural perturbations in both proteins. Together, these data suggest MCUb tightly interacts with MCU via the N-terminus, which may be a critical regulatory event in the dominant negative inhibition of MCU.