Steffen Wolf

Proton transfer via a transient linear water-molecule chain in a membrane protein

High-resolution protein ground-state structures of proton pumps and channels have revealed internal protein-bound water molecules. Their possible active involvement in protein function has recently come into focus. An illustration of the formation of a protonated protein-bound water cluster that is actively involved in proton transfer was described for the membrane protein bacteriorhodopsin (bR) [Garczarek F, Gerwert K (2006) Nature 439:109–112]. Here we show through a combination of time-resolved FTIR spectroscopy and molecular dynamics simulations that three protein-bound water molecules are rearranged by a protein conformational change that resulted in a transient Grotthuss-type proton-transfer chain extending through a hydrophobic protein region of bR. This transient linear water chain facilitates proton transfer at an intermediate conformation only, thereby directing proton transfer within the protein. The rearrangement of protein-bound water molecules that we describe, from inactive positions in the ground state to an active chain in an intermediate state, appears to be energetically favored relative to transient incorporation of water molecules from the bulk. Our discovery provides insight into proton-transfer mechanisms through hydrophobic core regions of ubiquitous membrane spanning proteins such as G-protein coupled receptors or cytochrome C oxidases.

Prediction of a Ligand‐Binding Niche within a Human Olfactory Receptor by Combining Site‐Directed Mutagenesis with Dynamic Homology Modeling

template. However, most odorants are highly flexible, soassessment of the ligand/protein dynamics might be of crucialimportance in understanding ligand recognition by ORs. Tobetter understand receptor activation, we thus searched for adynamic ligand–protein interaction pattern instead of analyz-ingligand-bindingin staticmodels. Therefore,indifference toother flexible GPCR ligand pocket analysis approaches,

Structural basis of slow activation gating in the cardiac I Ks channel complex.

Accessory β-subunits of the KCNE gene family modulate the function of various cation channel α-subunits by the formation of heteromultimers. Among the most dramatic changes of biophysical properties of a voltage-gated channel by KCNEs are the effects of KCNE1 on KCNQ1 channels. KCNQ1 and KCNE1 are believed to form nativeIKs channels. Here, we characterize molecular determinants of KCNE1 interaction with KCNQ1 channels by scanning mutagenesis, double mutant cycle analysis, and molecular dynamics simulations. Our findings suggest that KCNE1 binds to the outer face of the KCNQ1 channel pore domain, modifies interactions between voltage sensor, S4-S5 linker and the pore domain, leading to structural modifications of the selectivity filter and voltage sensor domain. Molecular dynamics simulations suggest a stable interaction of the KCNE1 transmembrane α-helix with the pore domain S5/S6 and part of the voltage sensor domain S4 of KCNQ1 in a putative pre-open channel state. Formation of this state may induce slow activation gating, the pivotal characteristic of native cardiac IKs channels. This new KCNQ1-KCNE1 model may become useful for dynamic modeling of disease-associated mutant IKs channels.

In Channelrhodopsin-2 Glu-90 Is Crucial for Ion Selectivity and Is Deprotonated during the Photocycle

Background: Channelrhodopsin-2 is a light-gated ion channel extensively used in optogenetics. Results: Glu-90 is deprotonated in the open state and is crucial for ion selectivity. Conclusion: Protonation change of Glu-90 is part of the opening/closing of the conductive pore, and the functional protein unit is assumed to be the monomer. Significance: Understanding the gating mechanism is necessary for optimizing this optogenetic tool. The light-activated microbial ion channel channelrhodopsin-2 (ChR2) is a powerful tool to study cellular processes with high spatiotemporal resolution in the emerging field of optogenetics. To customize the channel properties for optogenetic experiments, a detailed understanding of its molecular reaction mechanism is essential. Here, Glu-90, a key residue involved in the gating and selectivity mechanism of the ion channel is characterized in detail. The deprotonation of Glu-90 during the photocycle is elucidated by time-resolved FTIR spectroscopy, which seems to be part of the opening mechanism of the conductive pore. Furthermore, Glu-90 is crucial to ion selectivity as also revealed by mutation of this residue combined with voltage clamp experiments. By dynamic homology modeling, we further hypothesized that the conductive pore is flanked by Glu-90 and located between helices A, B, C, and G.

The role of protein-bound water molecules in microbial rhodopsins ☆

Protein-bound internal water molecules are essential features of the structure and function of microbial rhodopsins. Besides structural stabilization, they act as proton conductors and even proton storage sites. Currently, the most understood model system exhibiting such features is bacteriorhodopsin (bR). During the last 20 years, the importance of water molecules for proton transport has been revealed through this protein. It has been shown that water molecules are as essential as amino acids for proton transport and biological function. In this review, we present an overview of the historical development of this research on bR. We furthermore summarize the recently discovered protein-bound water features associated with proton transport. Specifically, we discuss a pentameric water/amino acid arrangement close to the protonated Schiff base as central proton-binding site, a protonated water cluster as proton storage site at the proton-release site, and a transient linear water chain at the proton uptake site. We highlight how protein conformational changes reposition or reorient internal water molecules, thereby guiding proton transport. Last, we compare the water positions in bR with those in other microbial rhodopsins to elucidate how protein-bound water molecules guide the function of microbial rhodopsins. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.

Directional Proton Transfer in Membrane Proteins Achieved through Protonated Protein-Bound Water Molecules: A Proton Diode†

The key function of energy-transducing membrane proteins is the creation of a proton gradient by directional proton transfer. The role of protein-bound water molecules herein is not fully understood, as X-ray diffraction analysis has resolved the positions of oxygen, but not of hydrogen atoms in such protein–water complexes. Here we show, now timeresolved at atomic resolution, how a membrane protein achieves directional proton transfer via protein-bound water molecules in contrast to random proton transfer in liquid water. A combination of X-ray structure analysis, timeresolved FTIR spectroscopy, and molecular dynamics (MD) simulations elucidates how directionality is achieved. Using the proton-pump bacteriorhodopsin as the paradigm, we show how controlled conformational changes of few amino acid residues rearrange preordered water molecules and induce directional proton transfer. This mechanism is analogous to an electronic diode: a “proton diode”. According to the chemiosmotic theory, the creation of a proton gradient in photosynthesis and oxidative phosphorylation by means of directional proton transfer is the key step for energy transduction in living cells. ATPases use this proton gradient to produce ATP, the fuel for life. In contrast to this directional mechanism in proteins, proton transfer in liquid water is random. Bacteriorhodopsin (bR), a protein that belongs to the microbial rhodopsin family, achieves this directional proton transfer by a light-driven protonpumping mechanism. Like other microbial rhodopsins, bR exhibits a structural motif of seven transmembrane a-helices and a retinal chromophore covalently bound to a lysine through a protonated Schiff base. The light-induced retinal isomerization from all-trans in the ground state (BR) to the 13-cis conformer drives bR through a photocycle with intermediates named J, K, L, M, N, and O in order of their appearance. During the L to M transition, the protonated Schiff base (C=NH), the central proton-binding site, deprotonates and protonates its counterion Asp85 (step 1 in Figure 1a). Protonation of Asp85 breaks its salt bridge to Arg82, which then moves towards Glu194/Glu204 (step 2). The orientation of Arg82 depends on the protonation state of Asp85. The arginine movement destabilizes a protonated water cluster between Arg82, Glu194, and Glu204 (step 3 in Figure 1a), and a proton is released to the bulk. However, the detailed nature of the proton-release group is still under debate. QM/MM simulations of the protonrelease group propose a shared proton between Glu194 and Glu204, a Zundel cation with two water molecules (H5O2 ), or an asymmetric Eigen cation of four water molecules (H9O4 ). From time-resolved FTIR experiments with site-directed mutations around the protonated water cluster and H/D-exchange experiments we have concluded that the proton-release group forms a protonated water cluster, most likely an asymmetric Eigen ion as shown in Figure 1a in purple. Glu194 and Glu204 are clearly deprotonated in the bR ground state. This experimental result was recently confirmed by L renz-Fonfr a et al. Nevertheless, the exact nature of the protonated water cluster and the release mechanism has still to be determined. Here, we used X-ray structure analysis to determine the positions of the water oxygen atoms and FTIR difference spectroscopy to determine the dynamics of the corresponding water hydrogen atoms. The proton release to the bulk in the L [*] Dr. S. Wolf, E. Freier, Dr. M. Potschies, Prof. Dr. E. Hofmann, Prof. Dr. K. Gerwert Lehrstuhl f r Biophysik, Ruhr-University Bochum Universit tsstrasse 150, 44780 Bochum (Germany) Fax: (+49)234-321-4238 E-mail: gerwert@bph.rub.de Homepage: http://www.bph.rub.de Dr. S. Wolf, Prof. Dr. K. Gerwert Department of Biophysics CAS–Max-Planck Partner Institute for Computational Biology Shanghai Institutes for Biological Sciences 320 Yue Yang Road, 200031 Shanghai (P.R. China) [] Current address: Lehrstuhl f r Biophysik, Universit t Konstanz (Germany) [] These authors contributed equally to this work.

Simulations of a G protein-coupled receptor homology model predict dynamic features and a ligand binding site

A computational approach to predict structures of rhodopsin‐like G protein‐coupled receptors (GPCRs) is presented and evaluated by comparison to the X‐ray structural models. By combining sequence alignment, the rhodopsin crystal structure, and point mutation data on the β2 adrenoreceptor (b2ar), we predict a (−)‐epinephrine‐bound computational model of the β2 adrenoreceptor. The model is evaluated by molecular dynamics simulations and by comparison with the recent X‐ray structures of b2ar. The overall correspondence between the predicted and the X‐ray structural model is high. Especially the prediction of the ligand binding site is accurate. This shows that the proposed dynamic homology modelling approach can be used to create reasonable models for the understanding of structure and dynamics of other rhodopsin‐like GPCRs.

Sequence, Structure and Ligand Binding Evolution of Rhodopsin-Like G Protein-Coupled Receptors: A Crystal Structure-Based Phylogenetic Analysis

G protein-coupled receptors (GPCRs) form the largest family of membrane receptors in the human genome. Advances in membrane protein crystallization so far resulted in the determination of 24 receptors available as high-resolution atomic structures. We performed the first phylogenetic analysis of GPCRs based on the available set of GPCR structures. We present a new phylogenetic tree of known human rhodopsin-like GPCR sequences based on this structure set. We can distinguish the three separate classes of small-ligand binding GPCRs, peptide binding GPCRs, and olfactory receptors. Analyzing different structural subdomains, we found that small molecule binding receptors most likely have evolved from peptide receptor precursors, with a rhodopsin/S1PR1 ancestor, most likely an ancestral opsin, forming the link between both classes. A light-activated receptor therefore seems to be the origin of the small molecule hormone receptors of the central nervous system. We find hints for a common evolutionary path of both ligand binding site and central sodium/water binding site. Surprisingly, opioid receptors exhibit both a binding cavity and a central sodium/water binding site similar to the one of biogenic amine receptors instead of peptide receptors, making them seemingly prone to bind small molecule ligands, e.g. opiates. Our results give new insights into the relationship and the pharmacological properties of rhodopsin-like GPCRs.

How Does a Membrane Protein Achieve a Vectorial Proton Transfer Via Water Molecules

We present a detailed mechanism for the proton transfer from a protein-bound protonated water cluster to the bulk water directed by protein side chains in the membrane protein bacteriorhodopsin. We use a combined approach of time-resolved Fourier transform infrared spectroscopy, molecular dynamics simulations, and X-ray structure analysis to elucidate the functional role of a hydrogen bond between Ser193 and Glu204. These two residues seal the internal protonated water cluster from the bulk water and the protein surface. During the photocycle of bacteriorhodopsin, a transient protonation of Glu204 leads to a breaking of this hydrogen bond. This breaking opens the gate to the extracellular bulk water, leading to a subsequent proton release from the protonated water cluster. We show in detail how the protein achieves vectorial proton transfer via protonated water clusters in contrast to random proton transfer in liquid water.

The structure of active opsin as a basis for identification of GPCR agonists by dynamic homology modelling and virtual screening assays

Most of the currently available G protein‐coupled receptor (GPCR) crystal structures represent an inactive receptor state, which has been considered to be suitable only for the discovery of antagonists and inverse agonists in structure‐based computational ligand screening. Using the β2‐adrenergic receptor (B2AR) as a model system, we show that a dynamic homology model based on an “active” opsin structure without further incorporation of experimental data performs better than the crystal structure of the inactive B2AR in finding agonists over antagonists/inverse agonists. Such “active‐like state” dynamic homology models can therefore be used to selectively identify GPCR agonists in in silico ligand libraries.