Christian Rickert

Light‐Induced Movement of the Transmembrane Helix B in Channelrhodopsin‐2

In the past decade, its versatile usage has established channelrhodopsin-2 (ChR2) as the most prominent optogenetic tool. The precise spatio-temporal control of the activity of a neuron by light allows noninvasive in vitro or in vivo investigations of neural circuits. The key is the heterologous expression of ChR2, a member of the type-I rhodopsins, that enters a photocycle upon light activation starting with the isomerization around the C13–C14 bond of its chromophore retinal. The intermediates of the photocycle are linked to a closed and an open state of an ion pore. Cation flux during the open state depolarizes the cell membrane thereby triggering neuronal action potentials. A coupling of isomerization with structural alterations is wellknown for bacteriorhodopsin (bR) and sensory rhodopsin II (SRII) that eventually leads to movement of the cytoplasmic part of transmembrane helices (TMHs) F and G. Conformational changes are also anticipated for ChR2 propagating from the chromophore to the putative cation pore. According to a homology model of ChR2 and the closed state structure of a chimera (C1C2) of ChR1 (TMHs A to E) and ChR2 (TMHs F and G), the pore comprises residues from TMHs A, B, C, and G, including a ChR characteristic patch of glutamate residues in TMH B. In this study, we followed the relative (re-)arrangements of TMHs B and F by EPR spectroscopy (double electron electron resonance, DEER) on spin-labeled mutants retaining wild-type (WT) character in ion conductance and photocycle kinetics. We could monitor a conformational change at TMH B upon light activation. Because of the unique channel properties of ChR2 and the major structural deviations in TMH A and B compared to other rhodopsins, the movement of TMH B might be a key element for channel opening. A monomeric form of ChR2 is unknown and a cysteinefree mutant shows very low light-induced currents in electrophysiological experiments and insufficient expression in yeast cells. ChR2 cysteine mutant screening led to two constructs. The first mutant has six cysteines replaced and three remaining: C34SC36SC87SC179LC183LC259L (Mut3C). The second mutant has seven cysteines replaced and only two remaining: C34SC36SC87SC179LC183LC208AC259L (Mut2C) (Figure 1A). Spin labeling of Mut3C with native cysteines C79, C128, and C208 using (1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl) methanethiosulfonate spin label (MTSSL) allows the study of interspin distances between TMHs B and TMHs F. Mut2C with native cysteines C79 and C128 reduces the analysis to interspin distances between two TMHs B sites, C79R1 and C79R1’, in the ChR2 dimer (R1 denotes the spin-labeled side chain). We found C128 to be inaccessible for MTSSL. Light-induced currents recorded from oocytes expressing WT ChR2, Mut3C, and Mut2C (Figure 1B) show typical inward rectification. All currents densities are normalized to the WT value at 60 mV (Figure 1C). Mut3C shows a 14% reduced amplitude. A further decrease to 25% of the WT ChR2 value is found for Mut2C. The effect of the C208A mutation is also observed in a triple point mutation (C34SC36SC208A). Here, the C208A mutation shows a 28 or 12 % reduction compared to WT ChR2 or C34SC36S ChR2 (Figure 1C), respectively. We cannot discriminate between a lower expression level and a functional defect in the C208A mutant. Amino acids other than alanine reduce the current densities even more. The sensitivity of position 208 is unclear as there is no obvious interaction site for the cysteine side chain in the chimera structure. The mutation of C79 to alanine (C34SC36SC79A) leads to a reduced current amplitude of about 52% of the WT ChR2 value (Figure 1 C). As for C208, other mutations at position 79 (to Leu, Thr, or Ser) provoke strong current density reductions. Replacing C79 and C208 together (C34SC36SC79AC208A) creates a variant with very low current amplitude (< 10 %) comparable to that of the cysteine-less ChR2 mutant. C79 is located at the N-terminus of TMH B close to the intracellular loop A-B. This loop is not resolved in the C1C2 structure. Hence, the flexibility of the loop together with the positional sensitivity of C79 could indicate a structurally relevant factor that is needed for the open state. This view is in line with a constriction site formed by the neighboring Y70 in the closed dark state structure. Compared to WT ChR2 no significant change in the mono[*] T. Sattig, Prof. Dr. E. Bamberg, Dr. C. Bamann Abteilung f r Biophysikalische Chemie Max-Planck-Institut f r Biophysik Max-von-Laue Str. 3, 60438 Frankfurt a. M. (Germany) E-mail: christian.bamann@biophys.mpg.de C. Rickert, Prof. Dr. H.-J. Steinhoff Fachbereich Physik, Universit t Osnabr ck Barbarastr. 7, 49076 Osnabr ck (Germany) E-mail: hsteinho@uni-osnabrueck.de [] These authors contributed equally to this work.

Light-induced switching of HAMP domain conformation and dynamics revealed by time-resolved EPR spectroscopy

HAMP domains are widely abundant signaling modules. The putative mechanism of their function comprises switching between two distinct states. To unravel these conformational transitions, we apply site‐directed spin labeling and time‐resolved EPR spectroscopy to the phototactic receptor/transducer complex NpSRII/NpHtrII. We characterize the kinetic coupling of NpHtrII to NpSRII along with the activation period of the transducer and follow the transient conformational signal. The observed transient shift towards a more compact state of the HAMP domain upon light‐activation agrees with structure‐based calculations. It thereby validates the two modeled signaling states and integrates the domains dynamics into the current model.

The Signal Transfer from the Receptor NpSRII to the Transducer NpHtrII Is Not Hampered by the D75N Mutation

Sensory rhodopsin II (NpSRII) is a phototaxis receptor of Natronomonas pharaonis that performs its function in complex with its cognate transducer (NpHtrII). Upon light activation NpSRII triggers by means of NpHtrII a signal transduction chain homologous to the two component system in eubacterial chemotaxis. The D75N mutant of NpSRII, which lacks the blue-shifted M intermediate and therefore exhibits a significantly faster photocycle compared to the wild-type, mediates normal phototaxis responses demonstrating that deprotonation of the Schiff base is not a prerequisite for transducer activation. Using site-directed spin labeling and time resolved electron paramagnetic-resonance spectroscopy, we show that the mechanism revealed for activation of the wild-type complex, namely an outward tilt motion of the cytoplasmic part of the receptor helix F and a concomitant rotation of the transmembrane transducer helix TM2, is also valid for the D75N variant. Apparently, the D75N mutation shifts the ground state conformation of NpSRII-D75N and its cognate transducer into the direction of the signaling state.

Clustering and Dynamics of Phototransducer Signaling Domains Revealed by Site-Directed Spin Labeling Electron Paramagnetic Resonance on SRII/HtrII in Membranes and Nanodiscs

In halophilic archaea the photophobic response is mediated by the membrane-embedded 2:2 photoreceptor/-transducer complex SRII/HtrII, the latter being homologous to the bacterial chemoreceptors. Both systems bias the rotation direction of the flagellar motor via a two-component system coupled to an extended cytoplasmic signaling domain formed by a four helical antiparallel coiled-coil structure. For signal propagation by the HAMP domains connecting the transmembrane and cytoplasmic domains, it was suggested that a two-state thermodynamic equilibrium found for the first HAMP domain in NpSRII/NpHtrII is shifted upon activation, yet signal propagation along the coiled-coil transducer remains largely elusive, including the activation mechanism of the coupled kinase CheA. We investigated the dynamic and structural properties of the cytoplasmic tip domain of NpHtrII in terms of signal transduction and putative oligomerization using site-directed spin labeling electron paramagnetic resonance spectroscopy. We show that the cytoplasmic tip domain of NpHtrII is engaged in a two-state equilibrium between a dynamic and a compact conformation like what was found for the first HAMP domain, thus strengthening the assumption that dynamics are the language of signal transfer. Interspin distance measurements in membranes and on isolated 2:2 photoreceptor/transducer complexes in nanolipoprotein particles provide evidence that archaeal photoreceptor/-transducer complexes analogous to chemoreceptors form trimers-of-dimers or higher-order assemblies even in the absence of the cytoplasmic components CheA and CheW, underlining conservation of the overall mechanistic principles underlying archaeal phototaxis and bacterial chemotaxis systems. Furthermore, our results revealed a significant influence of the NpHtrII signaling domain on the NpSRII photocycle kinetics, providing evidence for a conformational coupling of SRII and HtrII in these complexes.

Domain movements during CCA-addition: a new function for motif C in the catalytic core of the human tRNA nucleotidyltransferases.

CCA-adding enzymes are highly specific RNA polymerases that synthesize and maintain the sequence CCA at the tRNA 3′-end. This nucleotide triplet is a prerequisite for tRNAs to be aminoacylated and to participate in protein biosynthesis. During CCA-addition, a set of highly conserved motifs in the catalytic core of these enzymes is responsible for accurate sequential nucleotide incorporation. In the nucleotide binding pocket, three amino acid residues form Watson-Crick-like base pairs to the incoming CTP and ATP. A reorientation of these templating amino acids switches the enzymes specificity from CTP to ATP recognition. However, the mechanism underlying this essential structural rearrangement is not understood. Here, we show that motif C, whose actual function has not been identified yet, contributes to the switch in nucleotide specificity during polymerization. Biochemical characterization as well as EPR spectroscopy measurements of the human enzyme reveal that mutating the highly conserved amino acid position D139 in this motif interferes with AMP incorporation and affects interdomain movements in the enzyme. We propose a model of action, where motif C forms a flexible spring element modulating the relative orientation of the enzymes head and body domains to accommodate the growing 3′-end of the tRNA. Furthermore, these conformational transitions initiate the rearranging of the templating amino acids to switch the specificity of the nucleotide binding pocket from CTP to ATP during CCA-synthesis.

Two-Dimensional Trap for Ultrasensitive Quantification of Transient Protein Interactions

We present an ultrasensitive technique for quantitative protein-protein interaction analysis in a two-dimensional format based on phase-separated, micropatterned membranes. Interactions between proteins captured to lipid probes via an affinity tag trigger partitioning into the liquid-ordered phase, which is readily quantified by fluorescence imaging. Based on a calibration with well-defined low-affinity protein-protein interactions, equilibrium dissociation constants >1 mM were quantified. Direct capturing of proteins from mammalian cell lysates enabled us to detect homo- and heterodimerization of signal transducer and activator of transcription proteins. Using the epidermal growth factor receptor (EGFR) as a model system, quantification of low-affinity interactions between different receptor domains contributing to EGFR dimerization was achieved. By exploitation of specific features of the membrane-based assay, the regulation of EGFR dimerization by lipids was demonstrated.

Light-Induced Switching of HAMP Domain Conformation and Dynamics Revealed by Time-Resolved EPR Spectroscopy

In microbial photo- and chemotaxis a two-component signaling cascade mediates a regulated response of the flagellar motor to environmental conditions. Upon activation, photo- and chemoreceptors transfer a signal across the plasma membrane to activate the histidine kinase CheA. Successive regulation of the CheY-phosphorylation level controls the flagellar motor.In Natronomonas pharaonis a sensory rhodopsin II - transducer complex (SRII/HtrII) mediates negative phototaxis. As the initial signal, a light-induced outward movement of receptor helix F leads to a conformational change of transducer helix TM2, which in turn propagates the signal to the adjacent HAMP domain.For the HAMP domain, a widely abundant signaling module, several mechanisms were suggested, all comprising two distinct conformational states which we previously observed by two-component cw-EPR spectra at ambient temperatures.Here, we trace the conformational signal and its propagation throughout the elongated transducer.(1) We applied cw- and pulse-EPR spectroscopy in conjunction with nitroxide spin labeling. We follow transient changes by time-resolved cw-EPR spectroscopy and compare the resulting spectral changes to simulated EPR difference spectra revealing a shift in the thermodynamic equilibrium between the two states. Structure-based calculations of the expected spectral differences shows agreement with a shift towards a more compact state of the HAMP domain.To extend the current signaling models to the whole complex, a trimer of NpSRII/NpHtrII dimers, we carried out molecular dynamics simulations and observed differences between the deactivated and activated complex, ultimately leading to a signaling model that can now be tested experimentally.[1] Klose, D. et al., FEBS Lett. (2014) http://dx.doi.org/10.1016/j.febslet.2014.09.012 (in press)