Ulrich Zachariae

Toward a Consensus Model of the hERG Potassium Channel

Malfunction of hERG potassium channels, due to inherited mutations or inhibition by drugs, can cause long QT syndrome, which can lead to life‐threatening arrhythmias. A three‐dimensional structure of hERG is a prerequisite to understand the molecular basis of hERG malfunction. To achieve a consensus model, we carried out an extensive analysis of hERG models based on various alignments of helix S5. We analyzed seven models using a combination of conventional geometry/packing/normality validation methods as well as molecular dynamics simulations and molecular docking. A synthetic test set with the X‐ray crystal structure of Kv1.2 with artificially shifted S5 sequences modeled into the structure served as a reference case. We docked the known hERG inhibitors (+)‐cisapride, (S)‐terfenadine, and MK‐499 into the hERG models and simulation snapshots. None of the single analyses unambiguously identified a preferred model, but the combination of all three revealed that there is only one model that fulfils all quality criteria. This model is confirmed by a recent mutation scanning experiment (P. Ju, G. Pages, R. P. Riek, P. C. Chen, A. M. Torres, P. S. Bansal, S. Kuyucak, P. W. Kuchel, J. I. Vandenberg, J. Biol. Chem. 2009, 284, 1000–1008). 1 We expect the modeled structure to be useful as a basis both for computational studies of channel function and kinetics as well as the design of experiments.

Mechanisms of Anion Conduction by Coupled Glutamate Transporters

Excitatory amino acid transporters (EAATs) are essential for terminating glutamatergic synaptic transmission. They are not only coupled glutamate/Na(+)/H(+)/K(+) transporters but also function as anion-selective channels. EAAT anion channels regulate neuronal excitability, and gain-of-function mutations in these proteins result in ataxia and epilepsy. We have combined molecular dynamics simulations with fluorescence spectroscopy of the prokaryotic homolog GltPh and patch-clamp recordings of mammalian EAATs to determine how these transporters conduct anions. Whereas outward- and inward-facing GltPh conformations are nonconductive, lateral movement of the glutamate transport domain from intermediate transporter conformations results in formation of an anion-selective conduction pathway. Fluorescence quenching of inserted tryptophan residues indicated the entry of anions into this pathway, and mutations of homologous pore-forming residues had analogous effects on GltPh simulations and EAAT2/EAAT4 measurements of single-channel currents and anion/cation selectivities. These findings provide a mechanistic framework of how neurotransmitter transporters can operate as anion-selective and ligand-gated ion channels.

Position of Transmembrane Helix 6 Determines Receptor G Protein Coupling Specificity

G protein coupled receptors (GPCRs) transmit extracellular signals into the cell by binding and activating different intracellular signaling proteins, such as G proteins (Gαβγ, families Gi, Gs, Gq, G12/13) or arrestins. To address the issue of Gs vs Gi coupling specificity, we carried out molecular dynamics simulations of lipid-embedded active β2-adrenoceptor (β2AR*) in complex with C-terminal peptides derived from the key interaction site of Gα (GαCT) as surrogate of Gαβγ. We find that GiαCT and GsαCT exploit distinct cytoplasmic receptor conformations that coexist in the uncomplexed β2AR*. The slim GiαCT stabilizes a β2AR* conformation, not accessible to the bulkier GsαCT, which requires a larger TM6 outward tilt for binding. Our results suggest that the TM6 conformational heterogeneity regulates the catalytic activity of β2AR* toward Gi or Gs.

Side Chain Flexibilities in the Human Ether-a-go-go Related Gene Potassium Channel (hERG) Together with Matched-Pair Binding Studies Suggest a New Binding Mode for Channel Blockers

The cardiac hERG K(+) channel constitutes a long-standing and expensive antitarget for the drug industry. From a study of the flexibility of hERG around its internal binding cavity, we have developed a new structural model of drug binding to hERG, which involves binding orthogonal to the pore channel and therefore can exploit the up to 4-fold symmetry of the tetrameric channel. This binding site has a base formed by four tyrosine side chains that complement reported ligand-based pharmacophores. The model is able to rationalize reduced hERG potency in matched molecular pair studies and suggests design guidelines to optimize against hERG not relying simply on lipophilicity reduction. The binding model also suggests a molecular mechanism for the link between high-affinity hERG binding and C-type inactivation.

A Molecular Switch Driving Inactivation in the Cardiac K+ Channel hERG

K+ channels control transmembrane action potentials by gating open or closed in response to external stimuli. Inactivation gating, involving a conformational change at the K+ selectivity filter, has recently been recognized as a major K+ channel regulatory mechanism. In the K+ channel hERG, inactivation controls the length of the human cardiac action potential. Mutations impairing hERG inactivation cause life-threatening cardiac arrhythmia, which also occur as undesired side effects of drugs. In this paper, we report atomistic molecular dynamics simulations, complemented by mutational and electrophysiological studies, which suggest that the selectivity filter adopts a collapsed conformation in the inactivated state of hERG. The selectivity filter is gated by an intricate hydrogen bond network around residues S620 and N629. Mutations of this hydrogen bond network are shown to cause inactivation deficiency in electrophysiological measurements. In addition, drug-related conformational changes around the central cavity and pore helix provide a functional mechanism for newly discovered hERG activators.

High-resolution experimental and computational electrophysiology reveals weak β-lactam binding events in the porin PorB

The permeation of most antibiotics through the outer membrane of Gram-negative bacteria occurs through porin channels. To design drugs with increased activity against Gram-negative bacteria in the face of the antibiotic resistance crisis, the strict constraints on the physicochemical properties of the permeants imposed by these channels must be better understood. Here we show that a combination of high-resolution electrophysiology, new noise-filtering analysis protocols and atomistic biomolecular simulations reveals weak binding events between the β-lactam antibiotic ampicillin and the porin PorB from the pathogenic bacterium Neisseria meningitidis. In particular, an asymmetry often seen in the electrophysiological characteristics of ligand-bound channels is utilised to characterise the binding site and molecular interactions in detail, based on the principles of electro-osmotic flow through the channel. Our results provide a rationale for the determinants that govern the binding and permeation of zwitterionic antibiotics in anion-selective porin channels.

The lipid environment determines the activity of the E. coli ammonium transporter, AmtB.

The movement of ammonium across biological membranes is a fundamental process in all living organisms and is mediated by the ubiquitous Amt/Mep/Rh family of transporters. Recent structural analysis and coupled mass spectrometry studies have shown that the Escherichia coli ammonium transporter, AmtB, specifically binds phosphatidylglycerol (PG). Upon PG binding, several residues of AmtB undergo a small conformational change, which stabilizes the protein against unfolding. However, no studies have so far been conducted to explore if PG binding to AmtB has functional consequences. Here, we used an in vitro experimental assay with purified components together with molecular dynamics simulations to characterise the relation between PG binding and AmtB activity. Firstly, our results indicate that the function of Amt in archaebacteria and eubacteria may differ. Secondly, we show that PG is an essential cofactor for AmtB activity and that in the absence of PG AmtB cannot complete the full translocation cycle. Furthermore, our simulations reveal previously undiscovered PG binding sites on the intracellular side of the lipid bilayer between the AmtB subunits. The possible molecular mechanisms explaining the functional role of PG are discussed.

Lipid Protein Interactions and Dynamical Properties of VDAC 1 channel

VDAC-1 (Voltage Dependent Anion Channel) is one of the main components of the outer mitochondrial membrane. It is responsible for the transport of ATP and other anions, and it is involved in apoptosis and cancer [1]. The X-ray and NMR structures [2-4] showed VDAC as a 19 beta-barrel structure with an N-terminal alpha-helix bound to its interior. Mutations of E73 facing the membrane could be associated with local perturbations of the surrounding lipids. Remarkably, these changes are coupled to the intrinsic VDAC dynamics. To address this we measured the the local average thickness of the membrane and several structural properties of several E73 mutants of VDAC-1. Solution NMR in LDAO and molecular dynamics of VDAC-1 inserted in DPMC phospholipid patches were carried out. Mutation or chemical modification of E73 strongly reduces the micro- to millisecond dynamics in solution. The results show the main distortions of the membrane are located around E73. The major fluctuations of VDAC barrel were found to be correlated with the charge of E73. The motion amplitude described as PCA eigenvectors show structural deformations of VDAC mainly around E73X. The motions correspond to changes of the whole beta-barrel structure and align with the position of E73X. These results help to understand the intrinsic dynamic of VDAC and its possible interaction mechanism with lipid membranes.[1] Zaid et al., Cell Death Differ. 12, 751 (2005)[2] Hiller et al., Science 321, 1208 (2008)[3] Ujwal et al., PNAS 105, 17742 (2008)[4] Bayrhuber et al., PNAS 105, 15370 (2008)

Computational Electrophysiology on Vdac-1 reveals Mechanism of Anion Flux

The voltage-gated anion channel VDAC-1 in mitochondrial outer membranes is the principal passageway between mitochondria and the cytoplasm, transferring ATP, the major source of chemical energy in cells. It is also implicated in rupture of the mitochondrial membrane during mitochondria-dependent cell apoptosis.We used our newly developed method, computational electrophysiology, capable of modelling detailed ion transfer processes under steady flux conditions in molecular dynamics simulations, to study VDAC-1 embedded in lipid bilayers. In particular, we investigated how changes in transmembrane voltage, alterations in membrane state, functionally important mutations, and the intrinsic dynamics of VDAC-1 impact on channel current and how it is related to the surrounding bilayer.We found that the beta-barrel structure of VDAC-1 is unexpectedly dynamic, owing to the complete lack of a hydrophobic core, and undergoes large elliptic deformations. Strikingly, the N-terminal helix proves to be the most rigid section of the protein and confers stability to the global structure. Its removal results in greatly enhanced fluctuations and can give rise to a partial collapse of the barrel, especially if coupled to induced shock waves in the membrane. In electrophysiology simulations, the partially collapsed states exhibit subconductance behavior and altered ion selectivity.