Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Physiology and Pharmacology

Supervisor

Stathopulos, Peter B.

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.

Summary for Lay Audience

Regulated mitochondrial calcium (Ca2+) levels are essential to produce energy, regulate cell death pathways and control Ca2+ levels within the cell. Ca2+ uptake into the mitochondrial matrix is largely controlled by the mitochondrial Ca2+ uniporter (MCU) complex, which forms a pore through the inner mitochondrial wall. The MCU complex is composed of the MCU pore and several regulatory proteins to aid in tight regulation of the channel. MCU dominant negative beta subunit (MCUb) is one these MCU regulators, and it can insert into the pore leading to decreased Ca2+ uptake. A region of the MCU protein, called the amino (N)-terminal domain, has regulatory sites that are essential in controlling MCU structure and function. For example, the binding of Ca2+ and magnesium (Mg2+) to the MCU N-terminal domain causes decreased MCU assembly and reduced mitochondrial Ca2+ uptake. Despite the topological and sequence similarities between MCU and MCUb, MCUb cannot form a functional Ca2+ channel. However, given the similarities between MCU and MCUb, the MCUb N-terminal region may also play an important regulatory role in MCUb function. In this thesis, I have characterized the biophysical and structural properties of the MCUb N-terminal domain, revealed a tight interaction exists between the MCU and MCUb N-terminal domains, and identified that these interactions alter the biophysical and structural properties of both proteins. Overall, my research reveals the first insights into the potential molecular and structural mechanisms of MCU regulation by MCUb.

Available for download on Monday, June 26, 2023

Share

COinS