The goal of this proposal is to combine functional mutagenesis, biochemistry, and structural biology, to understand the molecular architecture of the mitochondrial Ca2+ uniporter. Early biochemical studies demonstrated that isolated mitochondria could transport and buffer huge amounts of Ca2+ across their inner membrane via a highly selective, Ru360-sensitive channel called the ?uniporter?. Uptake of Ca2+ via the uniporter is known to activate the TCA cycle, while its overload leads to cell death. Although the uniporter has been studied extensively for over 50 years, its molecular identity remained elusive until our group utilized comparative genomics to discover its molecular components. In humans, the minimal genetic elements required for uniporter current are MCU (the pore forming protein) and EMRE (a single-pass membrane protein). We have recently shown that Dictyostelium discoideum harbors a simpler uniporter, requiring only one component (DdMCU, the homolog of MCU), which is necessary and sufficient for uniporter activity and complements the requirement for MCU or EMRE in human knockout cells. At present, the molecular basis for the uniporter?s selectivity, mechanistic basis for inhibition by Ru360, and why the animal uniporter requires EMRE are unclear. Recently, we have solved the NMR structure of the channel-forming region of MCU using a N-Terminal Domain (NTD) truncated but fully functional construct from C. elegans, named cMCU-?NTD. The structure, which represents a new architecture for Ca2+ channel, provides the much-needed framework for addressing the above questions.
Aims 1 and 2 will use DdMCU, which is active by itself, to address the open state of MCU, whereas Aims 3 and 4 will focus on animal MCU and EMRE, to address the mechanism of how EMRE activates the MCU channel. Specifically, in Aim 1, we will perform systematic but hypothesis- driven mutagenesis of DdMCU to identify key residues important for the function and pharmacologic properties of this channel.
In Aim 2, we will determine the structure of DdMCU using independent structural approaches including crystallography, EM, and NMR.
In Aim 3, we will investigate the biochemistry and topology of EMRE and perform systematic mutagenesis to identify protein regions critical for MCU-EMRE interaction. Finally, in Aim 4, we will investigate Ca2+ and Ru360 binding to MCU using the cMCU-?NTD NMR system, and perform biochemical and structural characterization of MCU-EMRE interaction, including solving the high resolution structure of EMRE alone in lipid bilayer. The proposed studies will yield deep insights into the molecular architecture and mechanisms of the uniporter that promise to have implications for a range of human diseases.

Public Health Relevance

Uptake of calcium by mitochondria via the mitochondrial calcium uniporter (MCU) is known to activate the TCA cycle, while its overload leads to cell death. Recent studies show that misregulated activity of MCU leads to cardiomyopathy and abdominal aorta rupture, while modulation of MCU function may help to protect against autophagy or cell death. Our goal is to determine the molecular architecture of this newly discovered supramolecular complex, for understanding its function, regulation, and inhibition, and the results will be extremely valuable for understanding disease mutations associated with MCU as well as for developing new therapeutics targeting this protein complex.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL130143-02
Application #
9412870
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Wong, Renee P
Project Start
2017-02-01
Project End
2021-01-31
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Harvard Medical School
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Cao, Chan; Wang, Shuqing; Cui, Tanxing et al. (2017) Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter. Proc Natl Acad Sci U S A 114:E2846-E2851