Infection with the hepatitis C virus (HCV) has a huge impact on global health putting more than 170 million people at risk of developing severe liver disease. The HCV encoded p7 ion channel is essential for the production of infectious viruses. Despite a growing body of functional data, little is known about the 3-dimensional (3D) structure of the channel. Here, we present the 3D structure of a full-length viroporin, the detergent-solubilized hexameric 42 kDa form of the HCV p7 ion channel, as determined by single-particle electron microscopy using the random conical tilting approach. The reconstruction of such a small protein complex was made possible by a combination of high-contrast staining, the symmetry, and the distinct structural features of the channel. The orientation of the p7 monomers within the density was established using immunolabeling with N and C termini specific F ab fragments. The density map at a resolution of Ϸ16 Å reveals a flower-shaped protein architecture with protruding petals oriented toward the ER lumen. This broadest part of the channel presents a comparatively large surface area providing potential interaction sites for cellular and virally encoded ER resident proteins. membrane protein ͉ viroporin ͉ single particle analysis ͉ random conical tilt reconstruction T he hepatitis C virus (HCV) poses a major global health problem. It puts more than 170 million people worldwide at risk of developing liver cirrhosis and hepatocellular carcinoma. HCV comprises 6 different genotypes and is one of the fastest mutating viruses known to man. There is no vaccine available, and treatment options are genotype-specific, prone to viral escape mutations, and inadequate.The HCV p7 ion channel is a more recent addition to the growing list of potential drug targets encoded by HCV, reflecting the urgent need for a therapeutic approach. p7 is critical for the release of infectious virions in vitro (1, 2) and in vivo (3). It is not involved in HCV RNA replication (4, 5), but is required for late steps of viral particle assembly (2) and potentially cell entry (6). However, the prerequisite incorporation of p7 into budding virions has not been demonstrated. p7 belongs to the viroporins, small virally encoded proteins with at least 1 membrane-spanning helix that oligomerize to form channels or pores that modify the permeability of the cell membrane to ions and other small molecules (7). In planar lipid bilayers, p7 monomers oligomerize to form cation-selective ion channels that can be specifically inhibited by long alkylchain iminosugars, amiloride, and amantadine derivatives, with varying reported efficacies (6,(8)(9)(10)(11)(12)(13)(14)(15). Each HCV p7 monomer consists of 63 aa, most of which are hydrophobic and possibly contain endoplasmic reticulum (ER) retention signals (16-18). Computational secondary structure predictions suggest that the monomers contain 2 transmembrane spanning helices connected by a short basic loop (19,20). The loop is assumed to face the cytoplasm, with the N and C termini facing...

Using extensive atomistic simulations, we show that there is a single experimentally accessible parameter--the sterol tilt--that can be used to determine a sterol's capability to induce order, and thus to promote, e.g., formation of lipid rafts. The observations also facilitate designing new effective sterols.

SUR1 is an ATP-binding cassette (ABC) transporter with a novel function. In contrast to other ABC proteins, it serves as the regulatory subunit of an ion channel. The ATP-sensitive (K ATP ) channel is an octameric complex of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits, and it links cell metabolism to electrical activity in many cell types. ATPase activity at the nucleotidebinding domains of SUR results in an increase in K ATP channel open probability. Conversely, ATP binding to Kir6.2 closes the channel. Metabolic regulation is achieved by the balance between these two opposing effects. Precisely how SUR1 talks to Kir6.2 remains unclear, but recent studies have identified some residues and domains that are involved in both physical and functional interactions between the two proteins. The importance of these interactions is exemplified by the fact that impaired regulation of Kir6.2 by SUR1 results in human disease, with loss-of-function SUR1 mutations causing congenital hyperinsulinism and gain-of-function SUR1 mutations leading to neonatal diabetes. This paper reviews recent data on the regulation of Kir6.2 by SUR1 and considers the molecular mechanisms by which SUR1 mutations produce disease.

The effects of cholesterol (Chol) on phospholipid bilayers include ordering of the fatty acyl chains, condensing of the lipids in the bilayer plane, and promotion of the liquid-ordered phase. These effects depend on the type of phospholipids in the bilayer and are determined by the nature of the underlying molecular interactions. As for Chol, it has been shown to interact more favorably with sphingomyelin than with most phosphatidylcholines, which in given circumstances leads to formation of lateral domains. However, the exact origin and nature of Chol-phospholipid interactions have recently been subjects of speculation. We examine interactions between Chol, palmitoylsphingomyelin (PSM) and palmitoyl-oleoyl-phosphatidylcholine (POPC) in hydrated lipid bilayers by extensive atom-scale molecular dynamics simulations. We employ a tailored lipid configuration: Individual PSM and Chol monomers, as well as PSM-Chol dimers, are embedded in a POPC lipid bilayer in the liquid crystalline phase. Such a setup allows direct comparison of dimeric and monomeric PSMs and Chol, which ultimately shows how the small differences in PSM and POPC structure can lead to profoundly different interactions with Chol. Our analysis shows that direct hydrogen bonding between PSM and Chol does not provide an adequate explanation for their putative specific interaction. Rather, a combination of charge-pairing, hydrophobic, and van der Waals interactions leads to a lower tilt in PSM neighboring Chol than in Chol with only POPC neighbors. This implies improved Chol-induced ordering of PSM's chains over POPC's chains. These findings are discussed in the context of the hydrophobic mismatch concept suggested recently.