Taras Balandin

Crystal structure of a light-driven sodium pump

Recently, the first known light-driven sodium pumps, from the microbial rhodopsin family, were discovered. We have solved the structure of one of them, Krokinobacter eikastus rhodopsin 2 (KR2), in the monomeric blue state and in two pentameric red states, at resolutions of 1.45 Å and 2.2 and 2.8 Å, respectively. The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface, that the ion-release cavity is capped by a unique N-terminal α-helix and that the ion-uptake cavity is unexpectedly large and open to the surface. Obstruction of the cavity with the mutation G263F imparts KR2 with the ability to pump potassium. These results pave the way for the understanding and rational design of cation pumps with new specific properties valuable for optogenetics.

Barnase as a New Therapeutic Agent Triggering Apoptosis in Human Cancer Cells

Background RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells. Methodology/Principal Findings In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC50) ranging from 0.2 to 13 µM and to leukemia cell lines with IC50 values ranging from 2.4 to 82 µM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC50 = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3. Conclusions/Significance These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells.

Mechanism of transmembrane signaling by sensor histidine kinases

Bacterial sensing mechanism revealed Escherichia coli use a transmembrane sensor protein to sense nitrate in their external environment and initiate a biochemical response. Gushchin et al. compared crystal structures of portions of the NarQ receptor that included the transmembrane helices in ligand-bound or unbound states. The structures suggest a signaling mechanism by which piston- and lever-like movements are transmitted to response regulator proteins within the cell. Such two-component systems are very common in bacteria and, if better understood, might provide targets for antimicrobial therapies. Science, this issue p. eaah6345 Crystal structures show how sensing of nitrate occurs in bacteria. INTRODUCTION Microorganisms obtain most of the information about their environments through membrane-associated signaling systems. One of the most abundant classes of membrane receptors, present in all domains of life, is sensor histidine kinases, members of two-component signaling systems (TCSs). Tens of thousands of TCSs are known. Many of these systems are essential for cell growth, survival, or pathogenicity and consequently can be targeted to reduce virulence. Several large families of transmembrane (TM) TCS receptors are known: (i) sensor kinases, which generally possess a periplasmic, membrane, or intracellular sensor module; a transmembrane domain; often one or more intracellular signal transduction domains such as HAMP, PAS, or GAF; and an intracellular autokinase module (DHp and CA domains), which phosphorylates the response regulator protein; (ii) chemoreceptors, which also possess the sensor module and the TM domain but lack the kinase domain and control a separate kinase protein (CheA) via a kinase control module; and (iii) phototaxis systems, which are similar to chemotaxis systems except that the sensor module—a light receptor sensory rhodopsin—is a separate protein. RATIONALE Despite the wealth of biochemical data, the structural mechanisms of transmembrane signaling by TCS sensors are poorly understood at the atomic level. In particular, high-resolution structures of the TM segments connected to the adjacent domains are lacking. Deciphering of the signaling-associated conformational changes would shed light on the details of long-range transmembrane signal transduction and might help in the development of novel classes of antimicrobials targeting TCSs. RESULTS We used the in meso crystallization approach and single-wavelength anomalous dispersion to determine the crystal structures, at resolutions of up to 1.9 Å, of a fragment of Escherichia coli nitrate/nitrite sensor histidine kinase NarQ that contains the sensor, TM, and HAMP domains in a symmetric ligand-free apo state and in symmetric and asymmetric ligand-bound holo-S and holo-A states. In all of the structures, the TM domain is an antiparallel four-stranded coiled coil (CC) consisting of nine CC layers. The sensor domain is connected to the TM domain through continuous α-helical linkers that are partially disrupted in the holo state. The intracellular HAMP domain is connected to the TM helices via flexible proline junctions and robust hydrogen bonds conserved in all signaling states. The structures reveal the mechanism of transmembrane signal transduction in NarQ and show that binding of ligand induces displacement of the sensor domain helices by ~0.5 to 1 Å. This displacement translates into rearrangements and ~2.5 Å pistonlike shifts of transmembrane helices and is later converted, via leverlike motions of the HAMP domain protomers, into 7 Å shifts of the output helices and changes of the CC helical phase. The structures also demonstrate that the signaling-associated conformational changes in the TM domain do not need to be symmetric. CONCLUSION The determined structures of the transmembrane and membrane-proximal domains of the nitrate/nitrite receptor NarQ in ligand-free and ligand-bound forms present a template for studies of other TCS receptors, establish the importance of the pistonlike displacements of the TM helices for TM signal transduction, and highlight the role of the HAMP domain as an amplifier and converter of a piston-like displacement into helical rotation. Overall, the results show how a mechanistic signal is generated and amplified while being transduced through the protein over distances of 100 Å or more. Because membrane-associated TCSs are ubiquitous in microorganisms and are central for bacterial sensing, we believe that our results will help to elucidate a broad range of cellular processes such as basic metabolism, sporulation, quorum sensing, and virulence. They may also provide insights useful for the development of novel antimicrobial treatments targeting TCSs. The structures of histidine kinase NarQ in ligand-free and ligand-bound forms. The structures reveal rearrangement of transmembrane α helices during signal transduction and show that pistonlike shifts of the transmembrane helices result in leverlike motions of the HAMP domain protomers. One of the major and essential classes of transmembrane (TM) receptors, present in all domains of life, is sensor histidine kinases, parts of two-component signaling systems (TCSs). The structural mechanisms of TM signaling by these sensors are poorly understood. We present crystal structures of the periplasmic sensor domain, the TM domain, and the cytoplasmic HAMP domain of the Escherichia coli nitrate/nitrite sensor histidine kinase NarQ in the ligand-bound and mutated ligand-free states. The structures reveal that the ligand binding induces rearrangements and pistonlike shifts of TM helices. The HAMP domain protomers undergo leverlike motions and convert these pistonlike motions into helical rotations. Our findings provide the structural framework for complete understanding of TM TCS signaling and for development of antimicrobial treatments targeting TCSs.

Fluorescent immunolabeling of cancer cells by quantum dots and antibody scFv fragment

Semiconductor quantum dots (QDs) coupled with cancer-specific targeting ligands are new promising agents for fluorescent visualization of cancer cells. Human epidermal growth factor receptor 2/neu (HER2/neu), overexpressed on the surface of many cancer cells, is an important target for cancer diagnostics. Antibody scFv fragments as a targeting agent for direct delivery of fluorophores offer significant advantages over full-size antibodies due to their small size, lower cross-reactivity, and immunogenicity. We have used quantum dots linked to anti-HER2/neu 4D5 scFv antibody to label HER2/neu-overexpressing live cells. Labeling of target cells was shown to have high brightness, photostability, and specificity. The results indicate that construction based on quantum dots and scFv antibody can be successfully used for cancer cell visualization.

Structural insights into ion conduction by channelrhodopsin 2.

The inner workings of an optogenetic tool Channelrhodopsins are membrane channel proteins whose gating is controlled by light. In their native setting, they allow green algae to move in response to light. Their expression in neurons allows precise control of neural activity, an approach known as optogenetics. Volkov et al. describe the high-resolution structure of channelrhodopsin 2, the most widely used optogenetics tool, as well as the structure of a mutant with a longer open-state lifetime (see the Perspective by Gerwert). Light activation perturbs an intricate hydrogen-bonding network to open the channel. The structures provide a basis for designing better optogenetic tools. Science, this issue p. 10.1126/science.eaan8862; see also p. 1000 Channelrhodopsin has an intricate hydrogen-bonding network that is perturbed by light activation, resulting in channel opening. INTRODUCTION Ion channels are integral membrane proteins that upon stimulation modulate the flow of ions across the cell or organelle membrane. The resulting electrical signals are involved in biological functions such as electrochemical transmission and information processing in neurons. Channelrhodopsins (ChRs) appear to be unusual channels. They belong to the large family of microbial rhodopsins, seven-helical transmembrane proteins containing retinal as chromophore. Photon absorption initiates retinal isomerization resulting in a photocycle, with different spectroscopically distinguishable intermediates, thereby controlling the opening and closing of the channel. In 2003, it was demonstrated that light-induced currents by heterologously expressed ChR2 can be used to change a host’s membrane potential. The concept was further applied to precisely control muscle and neural activity by using light-induced depolarization to trigger an action potential in neurons expressing ChR2. This optogenetic approach with ChR2 and other ChRs has been widely used for remote control of neural cells in culture and in living animals with high spatiotemporal resolution. It is also used in biomedical studies aimed to cure severe diseases. RATIONALE Despite the wealth of biochemical and biophysical data, a high-resolution structure and structural mechanisms of a native ChR2 (and other ChRs) have not yet been known. A step forward was the structure of a chimera (C1C2). However, recent electrophysiological and Fourier transform infrared data showed that C1C2 exhibits light-induced responses that are functionally and mechanistically different from ChR2. Given that ChR2 is the most frequently used tool in optogenetics, a high-resolution structure of ChR2 is of high importance. Deciphering the structure of the native channel would shed light on how the light-induced changes at the retinal Schiff base (RSB) are linked to the channel operation and may make engineering of enhanced optogenetic tools more efficient. RESULTS We expressed ChR2 in LEXSY and used in the meso crystallization approach to determine the crystal structure of the wild-type ChR2 and C128T slow mutant at 2.4 and 2.7 Å, respectively (C, cysteine; T, threonine). Two different dark-state conformations of ChR2 in the two protomers in the asymmetric unit were resolved. The overall structure alignment of the protomers does not show a visible difference in backbone conformation. However, the conformation of some amino acids and the position of water molecules are not the same. The dimerization is strong and provided mainly through the interaction of helices 3 and 4 and the N termini. In addition, the protomers are connected with a disulfide bond, C34/C36′. In both protomers, we identified ion conduction pathway comprising four cavities [extracellular cavity 1 (EC1), EC2, intracellular cavity 1 (IC1), and IC2] that are separated by three gates [extracellular gate (ECG), central gate (CG), and intracellular gate (ICG)] (figure, panel A). Arginines R120 and R268 are the cores of ECG and ICG, respectively, in all ChRs. The Schiff base is hydrogen-bond–connected to E123 and D253 amino acids (E, glutamic acid; D, aspartic acid) and is a key part of the CG that is further connected with two other gates through an extended H-bond network mediated by numerous water molecules (figure, panel B). The DC gate is separate from the gates in the channel pathway and is bridged by hydrogen bonds through the water molecule w5. Hydrogen bonding of the DC pair (C128 and D156) has two important consequences. It stabilizes helices 3 and 4 and provides connection from D156, a possible proton donor, to the RSB. The presence of the hydrogen bonds provides structural insights into how the DC gate controls ChR2 gating lifetime. CONCLUSION The determined structures of ChR2 and its C128T mutant present the molecular basis for the understanding of ChR functioning. They provide insights into mechanisms of channel opening and closing. A plausible scenario is that the disruption of the H-bonds between E123 and D253 and the Schiff base and the protonation of D253 upon retinal isomerization trigger rearrangements in the extended hydrogen-bonded networks, stabilizing the ECG and CG and also rearranging the H-bonding network in the cavities. Upon retinal isomerization, these two gates are opened and the network is broken. This leads to the reorientation of helix 2. Additional changes in helices 6 and 7 induced by the isomerization could help with opening the ICG and channel pore formation. General structure presentation of ChR2. (A) Four cavities and three gates forming the channel pore. (B) Extended hydrogen-bond network. The DC gate is shown in the red ellipse. The black arrows and gray horizontal lines show the putative ion pathway and position of hydrophobic/hydrophilic boundaries, respectively. The light-gated ion channel channelrhodopsin 2 (ChR2) from Chlamydomonas reinhardtii is a major optogenetic tool. Photon absorption starts a well-characterized photocycle, but the structural basis for the regulation of channel opening remains unclear. We present high-resolution structures of ChR2 and the C128T mutant, which has a markedly increased open-state lifetime. The structure reveals two cavities on the intracellular side and two cavities on the extracellular side. They are connected by extended hydrogen-bonding networks involving water molecules and side-chain residues. Central is the retinal Schiff base that controls and synchronizes three gates that separate the cavities. Separate from this network is the DC gate that comprises a water-mediated bond between C128 and D156 and interacts directly with the retinal Schiff base. Comparison with the C128T structure reveals a direct connection of the DC gate to the central gate and suggests how the gating mechanism is affected by subtle tuning of the Schiff base’s interactions.

Ground state structure of D75N mutant of sensory rhodopsin II in complex with its cognate transducer

The complex of sensory rhodopsin II (NpSRII) with its cognate transducer (NpHtrII) mediates negative phototaxis in halobacteria Natronomonas pharaonis. Upon light activation NpSRII triggers, by means of NpHtrII, a signal transduction chain homologous to the two component system in eubacterial chemotaxis. Here we report on the crystal structure of the ground state of the mutant NpSRII-D75N/NpHtrII complex in the space group I212121. Mutations of this aspartic acid in light-driven proton pumps dramatically modify or/and inhibit protein functions. However, in vivo studies show that the similar D75N mutation retains functionality of the NpSRII/NpHtrII complex. The structure provides the molecular basis for the explanation of the unexpected observation that the wild and the mutant complexes display identical physiological response on light excitation.

Nanoparticle Surface-Enhanced Raman Scattering of Bacteriorhodopsin Stabilized by Amphipol A8-35

Surface-enhanced Raman spectroscopy (SERS) has developed dramatically since its discovery in the 1970s, because of its power as an analytical tool for selective sensing of molecules adsorbed onto noble metal nanoparticles (NPs) and nanostructures, including at the single-molecule (SM) level. Despite the high importance of membrane proteins (MPs), SERS application to MPs has not really been studied, due to the great handling difficulties resulting from the amphiphilic nature of MPs. The ability of amphipols (APols) to trap MPs and keep them soluble, stable, and functional opens up onto highly interesting applications for SERS studies, possibly at the SM level. This seems to be feasible since single APol-trapped MPs can fit into gaps between noble metal NPs, or in other gap-containing SERS substrates, whereby the enhancement of Raman scattering signal may be sufficient for SM sensitivity. The goal of the present study is to give a proof of concept of SERS with APol-stabilized MPs, using bacteriorhodopsin (BR) as a model. BR trapped by APol A8-35 remains functional even after partial drying at a low humidity. A dried mixture of silver Lee–Meisel colloid NPs and BR/A8-35 complexes give rise to SERS with an average enhancement factor in excess of 102. SERS spectra resemble non-SERS spectra of a dried sample of BR/APol complexes.

New Insights on Signal Propagation by Sensory Rhodopsin II/Transducer Complex.

The complex of two membrane proteins, sensory rhodopsin II (NpSRII) with its cognate transducer (NpHtrII), mediates negative phototaxis in halobacteria N. pharaonis. Upon light activation NpSRII triggers a signal transduction chain homologous to the two-component system in eubacterial chemotaxis. Here we report on crystal structures of the ground and active M-state of the complex in the space group I212121. We demonstrate that the relative orientation of symmetrical parts of the dimer is parallel (“U”-shaped) contrary to the gusset-like (“V”-shaped) form of the previously reported structures of the NpSRII/NpHtrII complex in the space group P21212, although the structures of the monomers taken individually are nearly the same. Computer modeling of the HAMP domain in the obtained “V”- and “U”-shaped structures revealed that only the “U”-shaped conformation allows for tight interactions of the receptor with the HAMP domain. This is in line with existing data and supports biological relevance of the “U” shape in the ground state. We suggest that the “V”-shaped structure may correspond to the active state of the complex and transition from the “U” to the “V”-shape of the receptor-transducer complex can be involved in signal transduction from the receptor to the signaling domain of NpHtrII.

Crystal Structure of Escherichia coli-Expressed Haloarcula marismortui Bacteriorhodopsin I in the Trimeric Form.

Bacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps. Here, we present the crystal structure of a new member of the family, Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant, at the resolution of 2.5 Å. While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps, its proton release region is extended and contains additional water molecules. The proteins fold is reinforced by three novel inter-helical hydrogen bonds, two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins. Despite the expression in Escherichia coli and consequent absence of native lipids, the protein assembles as a trimer in crystals. The unique extended loop between the helices D and E of HmBRI makes contacts with the adjacent protomer and appears to stabilize the interface. Many lipidic hydrophobic tail groups are discernible in the membrane region, and their positions are similar to those of archaeal isoprenoid lipids in the crystals of other proton pumps, isolated from native or native-like sources. All these features might explain the HmBRI properties and establish the protein as a novel model for the microbial rhodopsin proton pumping studies.

An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin.

Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å.