The Mitochondrial Ca(2+) Uniporter: Structure, Function, and Pharmacology.

Mitochondrial Ca(2+) uptake is crucial for an array of cellular functions while an imbalance can elicit cell death. In this chapter, we briefly reviewed the various modes of mitochondrial Ca(2+) uptake and our current understanding of mitochondrial Ca(2+) homeostasis in regards to cell physiology and pathophysiology. Further, this chapter focuses on the molecular identities, intracellular regulators as well as the pharmacology of mitochondrial Ca(2+) uniporter complex

Maintaining and breaking symmetry in homomeric coiled-coil assemblies

Higher order coiled coils with five or more helices can form α-helical barrels. Here the authors show that placing β-branched aliphatic residues along the lumen yields stable and open α-helical barrels, which is of interest for the rational design of functional proteins; whereas, the absence of β-branched side chains leads to unusual low-symmetry α-helical bundles

Analysis and Prediction of the Metabolic Stability of Proteins Based on Their Sequential Features, Subcellular Locations and Interaction Networks

The metabolic stability is a very important idiosyncracy of proteins that is related to their global flexibility, intramolecular fluctuations, various internal dynamic processes, as well as many marvelous biological functions. Determination of protein's metabolic stability would provide us with useful information for in-depth understanding of the dynamic action mechanisms of proteins. Although several experimental methods have been developed to measure protein's metabolic stability, they are time-consuming and more expensive. Reported in this paper is a computational method, which is featured by (1) integrating various properties of proteins, such as biochemical and physicochemical properties, subcellular locations, network properties and protein complex property, (2) using the mRMR (Maximum Relevance & Minimum Redundancy) principle and the IFS (Incremental Feature Selection) procedure to optimize the prediction engine, and (3) being able to identify proteins among the four types: “short”, “medium”, “long”, and “extra-long” half-life spans. It was revealed through our analysis that the following seven characters played major roles in determining the stability of proteins: (1) KEGG enrichment scores of the protein and its neighbors in network, (2) subcellular locations, (3) polarity, (4) amino acids composition, (5) hydrophobicity, (6) secondary structure propensity, and (7) the number of protein complexes the protein involved. It was observed that there was an intriguing correlation between the predicted metabolic stability of some proteins and the real half-life of the drugs designed to target them. These findings might provide useful insights for designing protein-stability-relevant drugs. The computational method can also be used as a large-scale tool for annotating the metabolic stability for the avalanche of protein sequences generated in the post-genomic age

Architecture of the Mitochondrial Calcium Uniporter

Mitochondria from multiple, eukaryotic clades uptake and buffer large amounts of calcium (Ca2+) via an inner membrane transporter called the uniporter. Early studies demonstrated that this transport requires a mitochondrial membrane potential and that the uniporter is itself Ca2+ activated, and blocked by ruthenium red or Ru3601. Later, electrophysiological studies demonstrated that the uniporter is an ion channel with remarkably high conductance and selectivity2. Ca2+ entry into mitochondria is also known to activate the TCA cycle and appears to be critical for matching ATP production in mitochondria with its cytosolic demand3. MCU (mitochondrial calcium uniporter) is the pore forming and Ca2+ conducting subunit of the uniporter, but its primary sequence does not resemble any calcium channel known to date. Here, we report the structure of the core region of MCU, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer with the second transmembrane helix forming a hydrophilic pore across the membrane. The channel assembly represents a new solution of ion channel architecture and is stabilized by a coiled coil motif protruding in the mitochondrial matrix. The critical DxxE motif forms the pore entrance featuring two carboxylate rings, which appear to be the selectivity filter based on the ring dimensions and functional mutagenesis. To our knowledge, this is one of the largest structures characterized by NMR, which provides a structural blueprint for understanding the function of this channel

Elastic coupling of integral membrane protein stability to lipid bilayer forces

It has been traditionally difficult to measure the thermodynamic stability of membrane proteins because fully reversible protocols for complete folding these proteins were not available. Knowledge of the thermodynamic stability of membrane proteins is desirable not only from a fundamental theoretical standpoint, but is also of enormous practical interest for the rational design of membrane proteins and for optimizing conditions for their structure determination by crystallography or NMR. Here, we describe the design of a fully reversible system to study equilibrium folding of the outer membrane protein A from Escherichia coli in lipid bilayers. Folding is shown to be two-state under appropriate conditions permitting data analysis with a classical folding model developed for soluble proteins. The resulting free energy and m value, i.e., a measure of cooperativity, of unfolding are [Formula: see text] and m = 1.1 kcal/mol M(–1), respectively, in a reference bilayer composed of palmitoyl-oleoyl-phosphatidylcholine (C(16:0)C(18:1)PC) and palmitoyloleoyl-phosphatidylglycerol (C(16:0)C(18:1)PG). These values are strong functions of the lipid bilayer environment. By systematic variation of lipid headgroup and chain composition, we show that elastic bilayer forces such as curvature stress and hydrophobic mismatch modulate the free energy and cooperativity of folding of this and perhaps many other membrane proteins

The structural topology of wild-type phospholamban in oriented lipid bilayers using 15N solid-state NMR spectroscopy

For the first time, 15N solid-state NMR experiments were conducted on wild-type phospholamban (WT-PLB) embedded inside mechanically oriented phospholipid bilayers to investigate the topology of its cytoplasmic and transmembrane domains. 15N solid-state NMR spectra of site-specific 15N-labeled WT-PLB indicate that the transmembrane domain has a tilt angle of 13° ± 6° with respect to the POPC (1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine) bilayer normal and that the cytoplasmic domain of WT-PLB lies on the surface of the phospholipid bilayers. Comparable results were obtained from site-specific 15N-labeled WT-PLB embedded inside DOPC/DOPE (1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) mechanically oriented phospholipids' bilayers. The new NMR data support a pinwheel geometry of WT-PLB, but disagree with a bellflower structure in micelles, and indicate that the orientation of the cytoplasmic domain of the WT-PLB is similar to that reported for the monomeric AFA-PLB mutant