Jussi Aittoniemi

The 3-dimensional structure of a hepatitis C virus p7 ion channel by electron microscopy

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 Fab fragments. The density map at a resolution of ≈16 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.

Review. SUR1: a unique ATP-binding cassette protein that functions as an ion channel regulator.

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 (KATP) 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 nucleotide-binding domains of SUR results in an increase in KATP 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.

Asymmetric switching in a homodimeric ABC transporter: a simulation study.

ABC transporters are a large family of membrane proteins involved in a variety of cellular processes, including multidrug and tumor resistance and ion channel regulation. Advances in the structural and functional understanding of ABC transporters have revealed that hydrolysis at the two canonical nucleotide-binding sites (NBSs) is co-operative and non-simultaneous. A conserved core architecture of bacterial and eukaryotic ABC exporters has been established, as exemplified by the crystal structure of the homodimeric multidrug exporter Sav1866. Currently, it is unclear how sequential ATP hydrolysis arises in a symmetric homodimeric transporter, since it implies at least transient asymmetry at the NBSs. We show by molecular dynamics simulation that the initially symmetric structure of Sav1866 readily undergoes asymmetric transitions at its NBSs in a pre-hydrolytic nucleotide configuration. MgATP-binding residues and a network of charged residues at the dimer interface are shown to form a sequence of putative molecular switches that allow ATP hydrolysis only at one NBS. We extend our findings to eukaryotic ABC exporters which often consist of two non-identical half-transporters, frequently with degeneracy substitutions at one of their two NBSs. Interestingly, many residues involved in asymmetric conformational switching in Sav1866 are substituted in degenerate eukaryotic NBS. This finding strengthens recent suggestions that the interplay of a consensus and a degenerate NBS in eukaroytic ABC proteins pre-determines the sequence of hydrolysis at the two NBSs.

Increased ATPase activity produced by mutations at arginine-1380 in nucleotide-binding domain 2 of ABCC8 causes neonatal diabetes

Gain-of-function mutations in the genes encoding the ATP-sensitive potassium (KATP) channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) are a common cause of neonatal diabetes mellitus. Here we investigate the molecular mechanism by which two heterozygous mutations in the second nucleotide-binding domain (NBD2) of SUR1 (R1380L and R1380C) separately cause neonatal diabetes. SUR1 is a channel regulator that modulates the gating of the pore formed by Kir6.2. KATP channel activity is inhibited by ATP binding to Kir6.2 but is stimulated by MgADP binding, or by MgATP binding and hydrolysis, at the NBDs of SUR1. Functional analysis of purified NBD2 showed that each mutation enhances MgATP hydrolysis by purified isolated fusion proteins of maltose-binding protein and NBD2. Inhibition of ATP hydrolysis by MgADP was unaffected by mutation of R1380, but inhibition by beryllium fluoride (which traps the ATPase cycle in the prehydrolytic state) was reduced. MgADP-dependent activation of KATP channel activity was unaffected. These data suggest that the R1380L and R1380C mutations enhance the off-rate of Pi, thereby enhancing the hydrolytic rate. Molecular modeling studies supported this idea. Because mutant channels were inhibited less strongly by MgATP, this would increase KATP currents in pancreatic beta cells, thus reducing insulin secretion and producing diabetes.

A mutation (R826W) in nucleotide‐binding domain 1 of ABCC8 reduces ATPase activity and causes transient neonatal diabetes

Activating mutations in the pore‐forming Kir6.2 (KCNJ11) and regulatory sulphonylurea receptor SUR1 (ABCC8) subunits of the KATP channel are a common cause of transient neonatal diabetes mellitus (TNDM). We identified a new TNDM mutation (R826W) in the first nucleotide‐binding domain (NBD1) of SUR1. The mutation was found in a region that heterodimerizes with NBD2 to form catalytic site 2. Functional analysis showed that this mutation decreases MgATP hydrolysis by purified maltose‐binding protein MBP–NBD1 fusion proteins. Inhibition of ATP hydrolysis by MgADP or BeF was not changed. The results indicate that the ATPase cycle lingers in the post‐hydrolytic MgADP·Pi‐bound state, which is associated with channel activation. The extent of MgADP‐dependent activation of KATP channel activity was unaffected by the R826W mutation, but the time course of deactivation was slowed. Channel inhibition by MgATP was reduced, leading to an increase in resting whole‐cell currents. In pancreatic beta cells, this would lead to less insulin secretion and thereby diabetes.

Tetrameric structure of SUR2B revealed by electron microscopy of oriented single particles.

The ATP‐sensitive potassium (KATP) channel is a hetero‐octameric complex that links cell metabolism to membrane electrical activity in many cells, thereby controlling physiological functions such as insulin release, muscle contraction and neuronal activity. It consists of four pore‐forming Kir6.2 and four regulatory sulfonylurea receptor (SUR) subunits. SUR2B serves as the regulatory subunit in smooth muscle and some neurones. An integrative approach, combining electron microscopy and homology modelling, has been used to obtain information on the structure of this large (megadalton) membrane protein complex. Single‐particle electron microscopy of purified SUR2B tethered to a lipid monolayer revealed that it assembles as a tetramer of four SUR2B subunits surrounding a central hole. In the absence of an X‐ray structure, a homology model for SUR2B based on the X‐ray structure of the related ABC transporter Sav1866 was used to fit the experimental images. The model indicates that the central hole can readily accommodate the transmembrane domains of the Kir tetramer, suggests a location for the first transmembrane domains of SUR2B (which are absent in Sav1866) and suggests the relative orientation of the SUR and Kir6.2 subunits.

A universally conserved residue in the SUR1 subunit of the KATP channel is essential for translating nucleotide binding at SUR1 into channel opening.

•  The sulphonylurea receptor (SUR1) subunit of the ATP‐sensitive potassium (KATP) channel is a member of the ATP‐binding cassette (ABC) protein family. Binding of MgADP to nucleotide‐binding domain 2 (NBD2) is critical for channel activation. •  We identified a residue in the SUR1 subunit of the KATP channel that is essential for translating nucleotide binding to SUR1 into activation of the channel pore. •  The ability of ATP to block KATP channel activity by binding to the Kir6.2 subunit of the channel was also altered by the mutation. This effect was dependent on the integrity of the NBDs of SUR1. This suggests SUR1 also modulates nucleotide inhibition of the channel at Kir6.2. •  G1401 in SUR1 is one of 23 residues which are conserved throughout all ABC transporter proteins suggesting that it may have a universal role in coupling substrate binding to protein function.