Ivan Yu. Gushchin

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.

Structural insights into the proton pumping by unusual proteorhodopsin from nonmarine bacteria

Light-driven proton pumps are present in many organisms. Here, we present a high-resolution structure of a proteorhodopsin from a permafrost bacterium, Exiguobacterium sibiricum rhodopsin (ESR). Contrary to the proton pumps of known structure, ESR possesses three unique features. First, ESRs proton donor is a lysine side chain that is situated very close to the bulk solvent. Second, the α-helical structure in the middle of the helix F is replaced by 310- and π-helix–like elements that are stabilized by the Trp-154 and Asn-224 side chains. This feature is characteristic for the proteorhodopsin family of proteins. Third, the proton release region is connected to the bulk solvent by a chain of water molecules already in the ground state. Despite these peculiarities, the positions of water molecule and amino acid side chains in the immediate Schiff base vicinity are very well conserved. These features make ESR a very unusual proton pump. The presented structure sheds light on the large family of proteorhodopsins, for which structural information was not available previously.

High-resolution structure of a membrane protein transferred from amphipol to a lipidic mesophase.

Amphipols (APols) have become important tools for the stabilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs solubilized in APols would be of high importance for structural biology. However, despite considerable efforts, it is still not clear whether MP/APol complexes can form well-ordered crystals suitable for X-ray crystallography. In the present work, we show that an APol-trapped MP can be crystallized in meso. Bacteriorhodopsin (BR) trapped by APol A8-35 was mixed with a lipidic mesophase, and crystallization was induced by adding a precipitant. The crystals diffract beyond 2xa0Å. The structure of BR was solved to 2xa0Å and found to be indistinguishable from previous structures obtained after transfer from detergent solutions. We suggest the proposed protocol of in meso crystallization to be generally applicable to APol-trapped MPs.

Active state of sensory rhodopsin II: structural determinants for signal transfer and proton pumping.

The molecular mechanism of transmembrane signal transduction is still a pertinent question in cellular biology. Generally, a receptor can transfer an external signal via its cytoplasmic surface, as found for G-protein-coupled receptors such as rhodopsin, or via the membrane domain, such as that in sensory rhodopsin II (SRII) in complex with its transducer, HtrII. In the absence of HtrII, SRII functions as a proton pump. Here, we report on the crystal structure of the active state of uncomplexed SRII from Natronomonas pharaonis, NpSRII. The problem with a dramatic loss of diffraction quality upon loading of the active state was overcome by growing better crystals and by reducing the occupancy of the state. The conformational changes in the region comprising helices F and G are similar to those observed for the NpSRII-transducer complex but are much more pronounced. The meaning of these differences for the understanding of proton pumping and signal transduction by NpSRII is discussed.

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.

Low-dose X-ray radiation induces structural alterations in proteins

X-ray-radiation-induced alterations to protein structures are still a severe problem in macromolecular crystallography. One way to avoid the influence of radiation damage is to reduce the X-ray dose absorbed by the crystal during data collection. However, here it is demonstrated using the example of the membrane protein bacteriorhodopsin (bR) that even a low dose of less than 0.06u2005MGy may induce structural alterations in proteins. This dose is about 500 times smaller than the experimental dose limit which should ideally not be exceeded per data set (i.e. 30u2005MGy) and 20 times smaller than previously detected specific radiation damage at the bR active site. To date, it is the lowest dose at which radiation modification of a protein structure has been described. Complementary use was made of high-resolution X-ray crystallography and online microspectrophotometry to quantitatively study low-dose X-ray-induced changes. It is shown that structural changes of the protein correlate with the spectroscopically observed formation of the so-called bR orange species. Evidence is provided for structural modifications taking place at the protein active site that should be taken into account in crystallographic studies which aim to elucidate the molecular mechanisms of bR function.

X-ray structure of a CDP-alcohol phosphatidyltransferase membrane enzyme and insights into its catalytic mechanism

Phospholipids have major roles in the structure and function of all cell membranes. Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life. They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol. Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain (IPCT) fused with a membrane CDP-alcohol phosphotransferase domain (DIPPS) at 2.65u2009Å resolution. The bifunctional protein dimerizes through the DIPPS domains, each comprising six transmembrane α-helices. The active site cavity is hydrophilic and widely open to the cytoplasm with a magnesium ion surrounded by four highly conserved aspartate residues from helices TM2 and TM3. We show that magnesium is essential for the enzymatic activity and is involved in catalysis. Substrates docking is validated by mutagenesis studies, and a structure-based catalytic mechanism is proposed.

Two Distinct States of the HAMP Domain from Sensory Rhodopsin Transducer Observed in Unbiased Molecular Dynamics Simulations

HAMP domain is a ubiquitous module of bacterial and archaeal two-component signaling systems. Considerable progress has been made recently in studies of its structure and conformational changes. However, the mechanism of signal transduction through the HAMP domain is not clear. It remains a question whether all the HAMPs have the same mechanism of action and what are the differences between the domains from different protein families. Here, we present the results of unbiased molecular dynamics simulations of the HAMP domain from the archaeal phototaxis signal transducer NpHtrII. Two distinct conformational states of the HAMP domain are observed, that differ in relative position of the helices AS1 and AS2. The longitudinal shift is roughly equal to a half of an α-helix turn, although sometimes it reaches one full turn. The states are closely related to the position of bulky hydrophobic aminoacids at the HAMP domain core. The observed features are in good agreement with recent experimental results and allow us to propose that the states detected in the simulations are the resting state and the signaling state of the NpHtrII HAMP domain. To the best of our knowledge, this is the first observation of the same HAMP domain in different conformations. The simulations also underline the difference between AMBER ff99-SB-ILDN and CHARMM22-CMAP forcefields, as the former favors the resting state and the latter favors the signaling state.

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.

Role of the HAMP domain region of sensory rhodopsin transducers in signal transduction.

Archaea are able to sense light via the complexes of sensory rhodopsins I and II and their corresponding chemoreceptor-like transducers HtrI and HtrII. Though generation of the signal has been studied in detail, the mechanism of its propagation to the cytoplasm remains obscured. The cytoplasmic part of the transducer consists of adaptation and kinase activity modulating regions, connected to transmembrane helices via two HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, phosphatases) domains. The inter-HAMP region of Natronomonas pharaonis HtrII (NpHtrII) was found to be α-helical [Hayashi, K., et al. (2007) Biochemistry 46, 14380-14390]. We studied the inter-HAMP regions of NpHtrII and other phototactic signal transducers by means of molecular dynamics. Their structure is found to be a bistable asymmetric coiled coil, in which the protomers are longitudinally shifted by ~1.3 Å. The free energy penalty for the symmetric structure is estimated to be 1.2-1.5 kcal/mol depending on the molarity of the solvent. Both flanking HAMP domains are mechanistically coupled to the inter-HAMP region and are asymmetric. The longitudinal shift in the inter-HAMP region is coupled with the in-plane displacement of the cytoplasmic part by 8.6 Å relative to the transmembrane part. The established properties suggest that (1) the signal may be transduced through the inter-HAMP domain switching and (2) the inter-HAMP region may allow cytoplasmic parts of the transducers to come sufficiently close to each other to form oligomers.