Changes in Channel Trafficking and Protein Stability Caused by LQT2 Mutations in the PAS Domain of the HERG Channel

Inherited human long-QT2 syndrome (LQTS) results from mutations in the gene encoding the HERG channel. Several LQT2-associated mutations have been mapped to the amino terminal cytoplasmic Per-Arnt-Sim (PAS) domain of the HERG1a channel subunit. Here we have characterized the trafficking properties of some LQT2-associated PAS domain mutants and analyzed rescue of the trafficking mutants by low temperature (27°C) or by the pore blocker drug E4031. We show that the LQT2-associated mutations in the PAS domain of the HERG channel display molecular properties that are distinct from the properties of LQT2-associated mutations in the trans-membrane region. Unlike the latter, many of the tested PAS domain LQT2-associated mutations do not result in trafficking deficiency of the channel. Moreover, the majority of the PAS domain mutations that cause trafficking deficiencies are not rescued by a pore blocking drug. We have also explored the in vitro folding stability properties of isolated mutant PAS domain proteins using a thermal unfolding fluorescence assay and a chemical unfolding assay

The structure of the KtrAB potassium transporter

In bacteria, archaea, fungi and plants the Trk, Ktr and HKT ion transporters are key components of osmotic regulation, pH homeostasis and resistance to drought and high salinity. These ion transporters are functionally diverse: they can function as Na+ or K+ channels and possibly as cation/K+ symporters. They are closely related to potassium channels both at the level of the membrane protein and at the level of the cytosolic regulatory domains. Here we describe the crystal structure of a Ktr K+ transporter, the KtrAB complex from Bacillus subtilis. The structure shows the dimeric membrane protein KtrB assembled with a cytosolic octameric KtrA ring bound to ATP, an activating ligand. A comparison between the structure of KtrAB-ATP and the structures of the isolated full-length KtrA protein with ATP or ADP reveals a ligand-dependent conformational change in the octameric ring, raising new ideas about the mechanism of activation in these transporters.We are grateful for access to ID14-1/ID14-4/ID-29 at ESRF (through the Portuguese BAG), PXII at SLS, XRD1 at ELETTRA and PROXIMA1 at SOLEIL and thank the respective support staff. A.S. was supported by FEBS (Long term fellowship). This work was funded by EMBO (Installation grant), by FEDER funds through the Operational Competitiveness Program-COMPETE and by National Funds through FCT-Fundacao para a Ciencia e a Tecnologia under the projects FCOMP-01-0124-FEDER-022718 (PEst-C/SAU/LA0002/2011), FCOMP-01-0124-FEDER-009028 (PTDC/BIA-PRO/099861/2008) and FCOMP-01-0124-FEDER-010781 (PTDC/QUI-BIQ/105342/2008). We also thank G. Gabant and M. Cadene at the 'Plateforme de Spectrometrie de Masse' at CBM, CNRS, Orleans for mass spectrometry analysis, and C. Harley for critical reading of the manuscript

MouR controls the expression of the Listeria monocytogenes Agr system and mediates virulence

The foodborne pathogen Listeria monocytogenes (Lm) causes invasive infection in susceptible ani- mals and humans. To survive and proliferate within hosts, this facultative intracellular pathogen tightly coordinates the expression of a complex regulatory network that controls the expression of virulence fac- tors. Here, we identified and characterized MouR, a novel virulence regulator of Lm. Through RNA-seq transcriptomic analysis, we determined the MouR regulon and demonstrated how MouR positively con- trols the expression of the Agr quorum sensing sys- tem (agrBDCA) of Lm. The MouR three-dimensional structure revealed a dimeric DNA-binding transcrip- tion factor belonging to the VanR class of the GntR superfamily of regulatory proteins. We also showed that by directly binding to the agr promoter region, MouR ultimately modulates chitinase activity and biofilm formation. Importantly, we demonstrated by in vitro cell invasion assays and in vivo mice infec- tions the role of MouR in Lm virulence.Peer reviewe

Structural Properties of PAS Domains from the KCNH Potassium Channels

<div><p>KCNH channels form an important family of voltage gated potassium channels. These channels include a N-terminal Per-Arnt-Sim (PAS) domain with unknown function. In other proteins PAS domains are implicated in cellular responses to environmental queues through small molecule binding or involvement in signaling cascades. To better understand their role we characterized the structural properties of several channel PAS domains. We determined high resolution structures of PAS domains from the mouse EAG (mEAG), drosophila ELK (dELK) and human ERG (hERG) channels and also of the hERG domain without the first nine amino acids. We analyzed these structures for features connected to ligand binding and signaling in other PAS domains. In particular, we have found cavities in the hERG and mEAG structures that share similarities with the ligand binding sites from other PAS domains. These cavities are lined by polar and apolar chemical groups and display potential flexibility in their volume. We have also found that the hydrophobic patch on the domain β-sheet is a conserved feature and appears to drive the formation of protein-protein contacts. In addition, the structures of the dELK domain and of the truncated hERG domain revealed the presence of N-terminal helices. These helices are equivalent to the helix described in the hERG NMR structures and are known to be important for channel function. Overall, these channel domains retain many of the PAS domain characteristics known to be important for cell signaling.</p> </div

Cavity in the PAS domain of hERG channel.

<p>a) Cartoon representation of high resolution structure of PAS domain from hERG channel. Cavity is shown as wireframe surrounded by several residues shown in stick. Arrow indicates residue H70 at the entrance to cavity. b) PAS domain structure of phototropin Phot-LOV1 (PDB code 1N9N). Flavin mononucleotide, shown as red stick, is bound within binding site cavity, shown as wireframe. c) Detailed view on hERG PAS domain cavity. The molecular surface of the domain is shown as wireframe and extends to the domain cavity. Water molecules in cavity are shown as red spheres. Some of the residues lining cavity are shown and labeled. Residues that in other channel PAS domain are substituted by polar residues are shown in gold. d) Polar and apolar chemical groups lining the cavity of the hERG PAS domain are shown. Wireframe delimiting cavity is colored according to the atoms that compose the cavity lining, red for oxygen, blue for nitrogen and white for carbon; the polar face (predominantly colored red and blue) of the cavity is towards the reader and the apolar face is at the opposite side.</p

Superposition of three (in red, yellow and cyan Cα trace) of the NMR model structures of the hERG PAS domain (PDB code 1L0W).

<p>Cavities detected in the three models are shown as red, yellow or cyan wireframe.</p

Crystal contacts formed by hydrophobic patch.

<p>a) Lattice packing of two molecules of hERG PAS domain. The molecules are related by 2 fold crystallographic axis indicated by arrow. b) Packing of two molecules in the asymmetric unit of the mEAG PAS domain. The two molecules have a ∼2-fold relation that is roughly perpendicular to page. In both panels the two molecules on the right are shown in a similar orientation. Residues from the hydrophobic patches are shown as stick.</p