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Mitochondria from many eukaryotic clades take up large amounts of calcium (Ca(2+)) via an inner membrane transporter called the uniporter. Transport by the uniporter is membrane potential dependent and sensitive to ruthenium red or its derivative Ru360 (ref. 1). Electrophysiological studies have shown that the uniporter is an ion channel with remarkably high conductance and selectivity. Ca(2+) entry into mitochondria is also known to activate the tricarboxylic acid cycle and seems to be crucial for matching the production of ATP in mitochondria with its cytosolic demand. Mitochondrial calcium uniporter (MCU) is the pore-forming and Ca(2+)-conducting subunit of the uniporter holocomplex, but its primary sequence does not resemble any calcium channel studied to date. Here we report the structure of the pore domain of MCU from Caenorhabditis elegans, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer in which the second transmembrane helix forms 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 into the mitochondrial matrix. The critical DXXE motif forms the pore entrance, which features two carboxylate rings; based on the ring dimensions and functional mutagenesis, these rings appear to form the selectivity filter. To our knowledge, this is one of the largest membrane protein structures characterized by NMR, and provides a structural blueprint for understanding the function of this channel.

Membrane channels, transporters and receptors constitute essential means for cells to maintain homeostasis and communicate with the surroundings. Investigation of their molecular architecture and the dynamic process of transporting substrate or transmitting signals across the membrane barrier has been one of the frontiers in biomedical research. The past decade has seen numerous successes in the use of X-ray or electron crystallography in determining atomic-resolution structures of membrane proteins, and in some cases, even snapshots of different physiological states of the same protein have been obtained. But there are also many cases in which long-standing efforts to crystallize proteins have yet to succeed. Therefore we have practical needs for developing complementary biophysical tools such as NMR spectroscopy and electron microscopy for tackling these systems. This paper provides a number of key examples where the utility of solution NMR was pivotal in providing structural and functional information on ion channels and transporters.

Contraction and relaxation of heart muscle cells is regulated by cycling of calcium between cytoplasm and sarcoplasmic reticulum. Human phospholamban (PLN), expressed in the sarcoplasmic reticulum membrane as a 30-kDa homopentamer, controls cellular calcium levels by a mechanism that depends on its phosphorylation. Since PLN was discovered ≈30 years ago, extensive studies have aimed to explain how it influences calcium pumps and to determine whether it acts as an ion channel. We have determined by solution NMR methods the atomic resolution structure of an unphosphorylated PLN pentamer in dodecylphosphocholine micelles. The unusual bellflower-like assembly is held together by leucine/isoleucine zipper motifs along the membrane-spanning helices. The structure reveals a channel-forming architecture that could allow passage of small ions. The central pore gradually widens toward the cytoplasmic end as the transmembrane helices twist around each other and bend outward. The dynamic N-terminal amphipathic helices point away from the membrane, perhaps facilitating recognition and inhibition of the calcium pump.

Human phospholamban (PLN), a 30 kDa homopentamer in the sarcoplasmic reticulum (SR) membrane, controls the magnitude of heart muscle contraction and relaxation by regulating the calcium pumping activity of the SR Ca(2+)-ATPase (SERCA). When PLN is not phosphorylated, it binds and inhibits SERCA. Phosphorylation of PLN at S16 or T17 releases such inhibitory effect. It remains a matter of debate whether phosphorylation perturbs the structure of PLN, which in turn affects its interaction with SERCA. Here we examine by NMR spectroscopy the structure and dynamics of PLN pentamer with a physiologically relevant, phosphorylation-mimicking mutation, S16E. Based on extensive NMR data, including NOEs, dipolar couplings, and solvent exchange of backbone amides, we conclude that the phosphorylation-mimicking mutation does not perturb the pentamer structure. However, (15)N R(1) and R(2) relaxation rates and (15)N((1)H) NOEs suggest subtle differences in the dynamics of the extramembrane portion of the protein.

Prokaryotic diacylglycerol kinase (DAGK) functions as a homotrimer of 13 kDa subunits, each of which has three transmembrane segments. This enzyme is conditionally essential to some bacteria and serves as a model system for studies of membrane protein biocatalysis, stability, folding, and misfolding. In this work, the detailed topology and secondary structure of DAGK's N-terminus up through the loop following the first transmembrane domain were probed by NMR spectroscopy. Secondary structure was mapped by measuring 13C NMR chemical shifts. Residue-to-residue topology was probed by measuring 19F NMR relaxation rates for site-specifically labeled samples in the presence and absence of polar and hydrophobic paramagnetic probes. Most of DAGK's N-terminal cytoplasmic and first transmembrane segments are alpha-helical. The first and second transmembrane helices are separated by a short loop from residues 48 to 52. The first transmembrane segment extends from residues 32 to 48. Most of the N-terminal cytoplasmic domain lies near the interface but does not extend deeply into the membrane. Finally, catalytic activities measured for the single cysteine mutants before and after chemical labeling suggest that the N-terminal cytoplasmic domain likely contains a number of critical active site residues. The results, therefore, suggest that DAGK's active site lies very near to the water/bilayer interface.

Backbone nuclear magnetic resonance (NMR) assignments were achieved for diacylglycerol kinase (DAGK) in detergent micelles. DAGK is a homotrimeric integral membrane protein comprised of 121 residue subunits, each having three transmembrane segments. Assignments were made using TROSY-based pulse sequences. DAGK was found to be an almost exclusively helical protein. This work points to the feasibility of both solving the structure of DAGK using solution NMR methods and using NMR as a primary tool in structural studies of other helical integral membrane proteins of similar size and complexity.

Membrane protein misfolding is related to the etiology of many diseases, but is poorly understood, particularly from a structural standpoint. This study focuses upon misfolding of a mutant form of diacylglycerol kinase (s-DAGK), a 40 kDa homotrimeric protein having nine transmembrane segments. Preparations of s-DAGK sometimes contain a kinetically trapped misfolded population, as evidenced by lower-than-expected enzyme activity (with no accompanying change in substrate K(m)) and by the appearance of a second band in electrophoresis gels. Misfolding of s-DAGK may take place during cellular overexpression, but can also be reproduced using the purified enzyme. TROSY NMR spectra of s-DAGK as a 100 kDa complex with detergent micelles exhibit a single additional set of resonances from the misfolded form, indicating a single misfolded conformational state. The relative intensities of these extra resonances correlate with the percent reduction in enzyme activity below the maximum observed for fully folded s-DAGK. Misfolded s-DAGK exhibits a modest difference in its far-UV CD spectrum compared to the folded enzyme, consistent with a small degree of variance in secondary structural content between the two forms. However, differences in NMR chemical shift dispersion and temperature-dependent line widths exhibited by folded and misfolded s-DAGK support the notion that they represent very different structural states. Cross-linking experiments indicate that both the correctly folded enzyme and the kinetically trapped misfolded form are homotrimers. This work appears to represent the first documentation of conformationally specific misfolding of an integral membrane protein.