The plasma membrane protein Orai forms the pore of the CRAC channel (calcium release-activated calcium channel) and generates sustained cytosolic calcium signals when triggered by depletion of calcium from the endoplasmic reticulum. The crystal structure of Orai from Drosophila melanogaster, determined at 3.35 angstrom resolution, reveals that the calcium channel is comprised of a hexameric assembly of Orai subunits arranged around a central ion pore. The pore traverses the membrane and extends 20 angstroms into the cytosol. A ring of glutamates on its extracellular side forms the selectivity filter. A basic region near the intracellular side can bind anions that may stabilize the closed state. The architecture of the channel differs markedly from other ion channels and gives insight into the principles of selective calcium permeation and gating.

The transient receptor potential melastatin 8 (TRPM8) ion channel is the primary detector of environmental cold and an important target for treating pathological cold hypersensitivity. Here, we present cryo–electron microscopy structures of TRPM8 in ligand-free, antagonist-bound, or calcium-bound forms, revealing how robust conformational changes give rise to two nonconducting states, closed and desensitized. We describe a malleable ligand-binding pocket that accommodates drugs of diverse chemical structures, and we delineate the ion permeation pathway, including the contribution of lipids to pore architecture. Furthermore, we show that direct calcium binding mediates stimulus-evoked desensitization, clarifying this important mechanism of sensory adaptation. We observe large rearrangements within the S4-S5 linker that reposition the S1-S4 and pore domains relative to the TRP helix, leading us to propose a distinct model for modulation of TRPM8 and possibly other TRP channels.

The paralogous endoribonucleases, RNase E and RNase G, play major roles in intracellular RNA metabolism in Escherichia coli and related organisms. To assay the relative importance of the principal RNA binding sites identified by crystallographic analysis, we introduced mutations into the 5-sensor, the S1 domain, and the Mg ؉2 /Mn ؉2 binding sites. The RNase E/G family of bacterial endoribonucleases is widely distributed among bacteria (1). Both RNase E and RNase G are expressed in Escherichia coli. RNase E was first characterized as an essential processing enzyme required for the maturation of 5 S rRNA 2 (2, 3). It is now known also to be involved in processing the 5Ј-spacer region of 16 S rRNA (4), most tRNA precursors (5, 6), transfer messenger RNA (7), and in the metabolism of many small regulatory RNAs (8, 9). It is also responsible for catalyzing the initial cleavage in the degradation of most mRNAs (10, 11). Furthermore, RNase E is part of a larger complex, the RNA degradosome (12-14). In contrast, RNase G appears to play a more limited role in RNA metabolism. It is responsible for the formation of the mature 5Ј terminus of 16 S rRNA (4, 15) and participates in the degradation of a limited set of mRNAs (16,17). It is not essential, however. Although both enzymes prefer single-stranded substrates, neither displays stringent sequence specificity (18 -20). However, both enzymes are 5Ј-end-dependent; i.e. their activity is stimulated, both in vivo and in vitro by a 5Ј-monophosphorylated terminus on their substrates (21-26). To explain this observation, it was postulated that a 5Ј-phosphate binding pocket exists on the surface of these enzymes (24). This idea has been substantially verified by the crystal structure of the catalytic domain of RNase E in complex with a substrate analog (27). These authors showed that RNase E contains a 5Ј-sensor domain that can interact specifically with a 5Ј-monophosphorylated substrate via contacts with Gly-124, Val-128, Arg-169, and Thr-170 (27).Several investigations have identified potential RNA binding surfaces on RNase E in addition to the 5Ј-sensor, including an arginine-rich region (28 -30) and the S1 domain (31, 32). In addition, the active (catalytic) site itself must contribute to substrate binding. The arginine-rich region, however, lies outside the minimal N-terminal domain of RNase E that is sufficient for enzymatic activity (28 -30). Several residues in the S1 domain could contribute to RNA binding, but only three, Phe-57, Phe-67, and Lys-112 provide obvious contacts to the substrate (27). Thus, it is not clear to what extent the 5Ј-sensor contributes to substrate binding. Indeed, it has been suggested that interaction of RNase E or G with a 5Ј-monophosphorylated substrate increases these enzymes' V max , effectively providing activation of these enzymes (25). Because a crystal structure was not available at the time this work was initiated, we examined instead the role of two types of conserved amino acid residue lying between the S1 domain and residue 400 in RNa...

The maturation of Ras GTPases, and ~200 other cellular CaaX proteins, involves three enzymatic steps: addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine (C) in the C-terminal CaaX motif, proteolytic cleavage of the aaX residues, and methylation of the exposed prenylcysteine residue at its terminal carboxylate1. This final step is catalyzed by isoprenylcysteine carboxyl methyltransferase (ICMT), a eukaryotic-specific integral membrane enzyme of the endoplasmic reticulum (ER)2. ICMT is the only cellular enzyme known to methylate prenylcysteine substrates; methylation is important for their biological functions, including the membrane localisations and subsequent activities of Ras1, prelamin A3, and Rab4. ICMT inhibition has potential for combating progeria3 and cancer5–8. Here we present an X-ray structure of ICMT, at 2.3 Å resolution, in complex with its cofactor, an ordered lipid molecule and a monobody inhibitor. The active site spans cytosolic and membrane-exposed regions, indicating distinct entry routes for its cytosolic methyl donor, S-adenosyl-L-methionine (AdoMet), and for prenylcysteine substrates, which are associated with the ER membrane. The structure suggests how ICMT overcomes the topographical challenge and unfavourable energetics of bringing two reactants that have different cellular localisations together in a membrane environment – a relatively uncharacterized, but defining feature of many integral membrane enzymes.