Andrii Ishchenko

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin–arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography

Angiotensin II type 1 receptor (AT(1)R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT(1)R blockers (ARBs), the structural basis for AT(1)R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high-quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT(1)R in complex with its selective antagonist ZD7155 at 2.9-Å resolution. The AT(1)R-ZD7155 complex structure revealed key structural features of AT(1)R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT(1)R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT(1)R structure-function relationship and structure-based drug design.

Structural basis for bifunctional peptide recognition at human δ-opioid receptor

Bifunctional μ- and δ-opioid receptor (OR) ligands are potential therapeutic alternatives, with diminished side effects, to alkaloid opiate analgesics. We solved the structure of human δ-OR bound to the bifunctional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. The observed receptor-peptide interactions are critical for understanding of the pharmacological profiles of opioid peptides and for development of improved analgesics.

Preparation of microcrystals in lipidic cubic phase for serial femtosecond crystallography

We have recently established a procedure for serial femtosecond crystallography (SFX) in lipidic cubic phase (LCP) for protein structure determination at X-ray free-electron lasers (XFELs). LCP-SFX uses the gel-like LCP as a matrix for growth and delivery of membrane protein microcrystals for crystallographic data collection. LCP is a liquid-crystalline mesophase composed of lipids and water. It provides a membrane-mimicking environment that stabilizes membrane proteins and supports their crystallization. Here we describe detailed procedures for the preparation and characterization of microcrystals for LCP-SFX applications. The advantages of LCP-SFX over traditional crystallographic methods include the capability of collecting room-temperature high-resolution data with minimal effects of radiation damage from sub-10-μm crystals of membrane and soluble proteins that are difficult to crystallize, while eliminating the need for crystal harvesting and cryo-cooling. Compared with SFX methods for microcrystals in solution using liquid injectors, LCP-SFX reduces protein consumption by 2–3 orders of magnitude for data collection at currently available XFELs. The whole procedure typically takes 3–5 d, including the time required for the crystals to grow.

Structural basis for selectivity and diversity in angiotensin II receptors

The angiotensin II receptors AT1R and AT2R serve as key components of the renin–angiotensin–aldosterone system. AT1R has a central role in the regulation of blood pressure, but the function of AT2R is unclear and it has a variety of reported effects. To identify the mechanisms that underlie the differences in function and ligand selectivity between these receptors, here we report crystal structures of human AT2R bound to an AT2R-selective ligand and to an AT1R/AT2R dual ligand, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins or β-arrestins, in agreement with the lack of signalling responses in standard cellular assays. Structure–activity relationship, docking and mutagenesis studies revealed the crucial interactions for ligand binding and selectivity. Our results thus provide insights into the structural basis of the distinct functions of the angiotensin receptors, and may guide the design of new selective ligands.

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.

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.

Native phasing of x-ray free-electron laser data for a G protein–coupled receptor

Anomalous signal from sulfur atoms present in most proteins was used for de novo phasing of XFEL data and solving a GPCR structure. Serial femtosecond crystallography (SFX) takes advantage of extremely bright and ultrashort pulses produced by x-ray free-electron lasers (XFELs), allowing for the collection of high-resolution diffraction intensities from micrometer-sized crystals at room temperature with minimal radiation damage, using the principle of “diffraction-before-destruction.” However, de novo structure factor phase determination using XFELs has been difficult so far. We demonstrate the ability to solve the crystallographic phase problem for SFX data collected with an XFEL using the anomalous signal from native sulfur atoms, leading to a bias-free room temperature structure of the human A2A adenosine receptor at 1.9 Å resolution. The advancement was made possible by recent improvements in SFX data analysis and the design of injectors and delivery media for streaming hydrated microcrystals. This general method should accelerate structural studies of novel difficult-to-crystallize macromolecules and their complexes.

Serial femtosecond crystallography of soluble proteins in lipidic cubic phase.

A new approach of using lipidic cubic phase as a carrier matrix for delivering soluble protein microcrystals for serial crystallography helps to dramatically reduce protein consumption. The structures of two soluble test proteins have been determined by this method using less than 0.1 mg of each protein.

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.