Ultrafast structural changes direct the first molecular events of vision.

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.

Femtosecond-to-millisecond structural changes in a light-driven sodium pump.

Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.

Ligand channel in pharmacologically stabilized rhodopsin.

In the degenerative eye disease retinitis pigmentosa (RP), protein misfolding leads to fatal consequences for cell metabolism and rod and cone cell survival. To stop disease progression, a therapeutic approach focuses on stabilizing inherited protein mutants of the G protein-coupled receptor (GPCR) rhodopsin using pharmacological chaperones (PC) that improve receptor folding and trafficking. In this study, we discovered stabilizing nonretinal small molecules by virtual and thermofluor screening and determined the crystal structure of pharmacologically stabilized opsin at 2.4 Å resolution using one of the stabilizing hits (S-RS1). Chemical modification of S-RS1 and further structural analysis revealed the core binding motif of this class of rhodopsin stabilizers bound at the orthosteric binding site. Furthermore, previously unobserved conformational changes are visible at the intradiscal side of the seven-transmembrane helix bundle. A hallmark of this conformation is an open channel connecting the ligand binding site with the membrane and the intradiscal lumen of rod outer segments. Sufficient in size, the passage permits the exchange of hydrophobic ligands such as retinal. The results broaden our understanding of rhodopsin's conformational flexibility and enable therapeutic drug intervention against rhodopsin-related retinitis pigmentosa.

Hydroxylamine Complexes of Cytochrome c': Influence of Heme Iron Redox State on Kinetic and Spectroscopic Properties.

Hydroxylamine (NH2OH or HA) is a redox-active nitrogen oxide that occurs as a toxic intermediate in the oxidation of ammonium by nitrifying and methanotrophic bacteria. Within ammonium containing environments, HA is generated by ammonia monooxygenase (nitrifiers) or methane monooxygenase (methanotrophs). Subsequent oxidation of HA is catalyzed by heme proteins, including cytochromes P460 and multiheme hydroxylamine oxidoreductases, the former contributing to emissions of N2O, an ozone-depleting greenhouse gas. A heme-HA complex is also a proposed intermediate in the reduction of nitrite to ammonia by cytochrome c nitrite reductase. Despite the importance of heme-HA complexes within the biogeochemical nitrogen cycle, fundamental aspects of their coordination chemistry remain unknown, including the effect of the Fe redox state on heme-HA affinity, kinetics, and spectroscopy. Using stopped-flow UV-vis and resonance Raman spectroscopy, we investigated HA complexes of the L16G distal pocket variant of Alcaligenes xylosoxidans cytochrome c'-α (L16G AxCP-α), a pentacoordinate c-type cytochrome that we show binds HA in its Fe(III) (Kd ∼ 2.5 mM) and Fe(II) (Kd = 0.0345 mM) states. The ∼70-fold higher HA affinity of the Fe(II) state is due mostly to its lower koff value (0.0994 s-1 vs 11 s-1), whereas kon values for Fe(II) (2880 M-1 s-1) and Fe(III) (4300 M-1 s-1) redox states are relatively similar. A comparison of the HA and imidazole affinities of L16G AxCP-α was also used to predict the influence of Fe redox state on HA binding to other proteins. Although HA complexes of L16G AxCP-α decompose via redox reactions, the lifetime of the Fe(II)HA complex was prolonged in the presence of excess reductant. Spectroscopic parameters determined for the Fe(II)HA complex include the N-O stretching vibration of the NH2OH ligand, ν(N-O) = 906 cm-1. Overall, the kinetic trends and spectroscopic benchmarks from this study provide a foundation for future investigations of heme-HA reaction mechanisms.

Distinguishing Nitro vs Nitrito Coordination in Cytochrome c' Using Vibrational Spectroscopy and Density Functional Theory.

Nitrite coordination to heme cofactors is a key step in the anaerobic production of the signaling molecule nitric oxide (NO). An ambidentate ligand, nitrite has the potential to coordinate via the N- (nitro) or O- (nitrito) atoms in a manner that can direct its reactivity. Distinguishing nitro vs nitrito coordination, along with the influence of the surrounding protein, is therefore of particular interest. In this study, we probed Fe(III) heme-nitrite coordination in Alcaligenes xylosoxidans cytochrome c' (AXCP), an NO carrier that excludes anions in its native state but that readily binds nitrite (Kd ∼ 0.5 mM) following a distal Leu16 → Gly mutation to remove distal steric constraints. Room-temperature resonance Raman spectra (407 nm excitation) identify ν(Fe-NO2), δ(ONO), and νs(NO2) nitrite ligand vibrations in solution. Illumination with 351 nm UV light results in photoconversion to {FeNO}6 and {FeNO}7 states, enabling FTIR measurements to distinguish νs(NO2) and νas(NO2) vibrations from differential spectra. Density functional theory calculations highlight the connections between heme environment, nitrite coordination mode, and vibrational properties and confirm that nitrite binds to L16G AXCP exclusively through the N atom. Efforts to obtain the nitrite complex crystal structure were hampered by photochemistry in the X-ray beam. Although low dose crystal structures could be modeled with a mixed nitrite (nitro)/H2O distal population, their photosensitivity and partial occupancy underscores the value of the vibrational approach. Overall, this study sheds light on steric determinants of heme-nitrite binding and provides vibrational benchmarks for future studies of heme protein nitrite reactions.

Conformational control of the binding of diatomic gases to cytochrome c'.

The cytochromes c' (CYTcp) are found in denitrifying, methanotrophic and photosynthetic bacteria. These proteins are able to form stable adducts with CO and NO but not with O2. The binding of NO to CYTcp currently provides the best structural model for the NO activation mechanism of soluble guanylate cyclase. Ligand binding in CYTcps has been shown to be highly dependent on residues in both the proximal and distal heme pockets. Group 1 CYTcps typically have a phenylalanine residue positioned close to the distal face of heme, while for group 2, this residue is typically leucine. We have structurally, spectroscopically and kinetically characterised the CYTcp from Shewanella frigidimarina (SFCP), a protein that has a distal phenylalanine residue and a lysine in the proximal pocket in place of the more common arginine. Each monomer of the SFCP dimer folds as a 4-alpha-helical bundle in a similar manner to CYTcps previously characterised. SFCP exhibits biphasic binding kinetics for both NO and CO as a result of the high level of steric hindrance from the aromatic side chain of residue Phe 16. The binding of distal ligands is thus controlled by the conformation of the phenylalanine ring. Only a proximal 5-coordinate NO adduct, confirmed by structural data, is observed with no detectable hexacoordinate distal NO adduct.

Hydrogen bonding of the dissociated histidine ligand is not required for formation of a proximal NO adduct in cytochrome c'.

Cytochromes c', that occur in methanotrophic, denitrifying and photosynthetic bacteria, form unusual proximal penta-coordinate NO complexes via a hexa-coordinate distal NO intermediate. Their NO binding properties are similar to those of the eukaryotic NO sensor, soluble guanylate cyclase, for which they provide a valuable structural model. Previous studies suggested that hydrogen bonding between the displaced proximal histidine (His120) ligand (following its dissociation from heme due to trans effects from the distally bound NO) and a conserved aspartate residue (Asp121) could play a key role in allowing proximal NO binding to occur. We have characterized three variants of Alcaligenes xylosoxidans cytochrome c' (AXCP) where Asp121 has been replaced by Ala, Ile and Gln, respectively. In all variants, hydrogen bonding between residue 121 and His120 is abolished yet 5-coordinate proximal NO species are still formed. Our data therefore demonstrate that the His120-Asp121 bond is not essential for proximal NO binding although it likely provides an energy minimum for the displaced His ligand. All variants have altered proximal pocket structure relative to native AXCP.

Fingerprinting redox and ligand states in haemprotein crystal structures using resonance Raman spectroscopy.

It is crucial to assign the correct redox and ligand states to crystal structures of proteins with an active redox centre to gain valid functional information and prevent the misinterpretation of structures. Single-crystal spectroscopies, particularly when applied in situ at macromolecular crystallography beamlines, allow spectroscopic investigations of redox and ligand states and the identification of reaction intermediates in protein crystals during the collection of structural data. Single-crystal resonance Raman spectroscopy was carried out in combination with macromolecular crystallography on Swiss Light Source beamline X10SA using cytochrome c' from Alcaligenes xylosoxidans. This allowed the fingerprinting and validation of different redox and ligand states, identification of vibrational modes and identification of intermediates together with monitoring of radiation-induced changes. This combined approach provides a powerful tool to obtain complementary data and correctly assign the true oxidation and ligand state(s) in redox-protein crystals.

Oligomeric properties of adeno-associated virus Rep68 reflect its multifunctionality.

The adeno-associated virus (AAV) encodes four regulatory proteins called Rep. The large AAV Rep proteins Rep68 and Rep78 are essential factors required in almost every step of the viral life cycle. Structurally, they share two domains: a modified version of the AAA(+) domain that characterizes the SF3 family of helicases and an N-terminal domain that binds DNA specifically. The combination of these two domains imparts extraordinary multifunctionality to work as initiators of DNA replication and regulators of transcription, in addition to their essential role during site-specific integration. Although most members of the SF3 family form hexameric rings in vitro, the oligomeric nature of Rep68 is unclear due to its propensity to aggregate in solution. We report here a comprehensive study to determine the oligomeric character of Rep68 using a combination of methods that includes sedimentation velocity ultracentrifugation, electron microscopy, and hydrodynamic modeling. We have determined that residue Cys151 induces Rep68 to aggregate in vitro. We show that Rep68 displays a concentration-dependent dynamic oligomeric behavior characterized by the presence of two populations: one with monomers and dimers in slow equilibrium and a second one consisting of a mixture of multiple-ring structures of seven and eight members. The presence of either ATP or ADP induces formation of larger complexes formed by the stacking of multiple rings. Taken together, our results support the idea of a Rep68 molecule that exhibits the flexible oligomeric behavior needed to perform the wide range of functions occurring during the AAV life cycle.

The interdomain linker of AAV-2 Rep68 is an integral part of its oligomerization domain: role of a conserved SF3 helicase residue in oligomerization.

The four Rep proteins of adeno-associated virus (AAV) orchestrate all aspects of its viral life cycle, including transcription regulation, DNA replication, virus assembly, and site-specific integration of the viral genome into the human chromosome 19. All Rep proteins share a central SF3 superfamily helicase domain. In other SF3 members this domain is sufficient to induce oligomerization. However, the helicase domain in AAV Rep proteins (i.e. Rep40/Rep52) as shown by its monomeric characteristic, is not able to mediate stable oligomerization. This observation led us to hypothesize the existence of an as yet undefined structural determinant that regulates Rep oligomerization. In this document, we described a detailed structural comparison between the helicase domains of AAV-2 Rep proteins and those of the other SF3 members. This analysis shows a major structural difference residing in the small oligomerization sub-domain (OD) of Rep helicase domain. In addition, secondary structure prediction of the linker connecting the helicase domain to the origin-binding domain (OBD) indicates the potential to form α-helices. We demonstrate that mutant Rep40 constructs containing different lengths of the linker are able to form dimers, and in the presence of ATP/ADP, larger oligomers. We further identified an aromatic linker residue (Y224) that is critical for oligomerization, establishing it as a conserved signature motif in SF3 helicases. Mutation of this residue critically affects oligomerization as well as completely abolishes the ability to produce infectious virus. Taken together, our data support a model where the linker residues preceding the helicase domain fold into an α-helix that becomes an integral part of the helicase domain and is critical for the oligomerization and function of Rep68/78 proteins through cooperative interaction with the OBD and helicase domains.

Watching the release of a photopharmacological drug from tubulin using time-resolved serial crystallography.

The binding and release of ligands from their protein targets is central to fundamental biological processes as well as to drug discovery. Photopharmacology introduces chemical triggers that allow the changing of ligand affinities and thus biological activity by light. Insight into the molecular mechanisms of photopharmacology is largely missing because the relevant transitions during the light-triggered reaction cannot be resolved by conventional structural biology. Using time-resolved serial crystallography at a synchrotron and X-ray free-electron laser, we capture the release of the anti-cancer compound azo-combretastatin A4 and the resulting conformational changes in tubulin. Nine structural snapshots from 1 ns to 100 ms complemented by simulations show how cis-to-trans isomerization of the azobenzene bond leads to a switch in ligand affinity, opening of an exit channel, and collapse of the binding pocket upon ligand release. The resulting global backbone rearrangements are related to the action mechanism of microtubule-destabilizing drugs.

Dynamics and mechanism of a light-driven chloride pump.

Chloride transport by microbial rhodopsins is an essential process for which molecular details such as the mechanisms that convert light energy to drive ion pumping and ensure the unidirectionality of the transport have remained elusive. We combined time-resolved serial crystallography with time-resolved spectroscopy and multiscale simulations to elucidate the molecular mechanism of a chloride-pumping rhodopsin and the structural dynamics throughout the transport cycle. We traced transient anion-binding sites, obtained evidence for how light energy is used in the pumping mechanism, and identified steric and electrostatic molecular gates ensuring unidirectional transport. An interaction with the π-electron system of the retinal supports transient chloride ion binding across a major bottleneck in the transport pathway. These results allow us to propose key mechanistic features enabling finely controlled chloride transport across the cell membrane in this light-powered chloride ion pump.

Pink-beam serial femtosecond crystallography for accurate structure-factor determination at an X-ray free-electron laser.

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables essentially radiation-damage-free macromolecular structure determination using microcrystals that are too small for synchrotron studies. However, SFX experiments often require large amounts of sample in order to collect highly redundant data where some of the many stochastic errors can be averaged out to determine accurate structure-factor amplitudes. In this work, the capability of the Swiss X-ray free-electron laser (SwissFEL) was used to generate large-bandwidth X-ray pulses [Δλ/λ = 2.2% full width at half-maximum (FWHM)], which were applied in SFX with the aim of improving the partiality of Bragg spots and thus decreasing sample consumption while maintaining the data quality. Sensitive data-quality indicators such as anomalous signal from native thaumatin micro-crystals and de novo phasing results were used to quantify the benefits of using pink X-ray pulses to obtain accurate structure-factor amplitudes. Compared with data measured using the same setup but using X-ray pulses with typical quasi-monochromatic XFEL bandwidth (Δλ/λ = 0.17% FWHM), up to fourfold reduction in the number of indexed diffraction patterns required to obtain similar data quality was achieved. This novel approach, pink-beam SFX, facilitates the yet underutilized de novo structure determination of challenging proteins at XFELs, thereby opening the door to more scientific breakthroughs.

Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography.

Conformational dynamics are essential for proteins to function. We adapted time-resolved serial crystallography developed at x-ray lasers to visualize protein motions using synchrotrons. We recorded the structural changes in the light-driven proton-pump bacteriorhodopsin over 200 milliseconds in time. The snapshot from the first 5 milliseconds after photoactivation shows structural changes associated with proton release at a quality comparable to that of previous x-ray laser experiments. From 10 to 15 milliseconds onwards, we observe large additional structural rearrangements up to 9 angstroms on the cytoplasmic side. Rotation of leucine-93 and phenylalanine-219 opens a hydrophobic barrier, leading to the formation of a water chain connecting the intracellular aspartic acid-96 with the retinal Schiff base. The formation of this proton wire recharges the membrane pump with a proton for the next cycle.

Enzyme catalysis captured using multiple structures from one crystal at varying temperatures.

High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate 'structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a 'top-hat' geometry, which was rapidly transformed to a 'side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.

Retinal isomerization in bacteriorhodopsin captured by a femtosecond x-ray laser.

Ultrafast isomerization of retinal is the primary step in photoresponsive biological functions including vision in humans and ion transport across bacterial membranes. We used an x-ray laser to study the subpicosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin. A series of structural snapshots with near-atomic spatial resolution and temporal resolution in the femtosecond regime show how the excited all-trans retinal samples conformational states within the protein binding pocket before passing through a twisted geometry and emerging in the 13-cis conformation. Our findings suggest ultrafast collective motions of aspartic acid residues and functional water molecules in the proximity of the retinal Schiff base as a key facet of this stereoselective and efficient photochemical reaction.

Recent structural insights into the function of copper nitrite reductases.

Copper nitrite reductases (CuNiR) carry out the first committed step of the denitrification pathway of the global nitrogen cycle, the reduction of nitrite (NO2-) to nitric oxide (NO). As such, they are of major agronomic and environmental importance. CuNiRs occur primarily in denitrifying soil bacteria which carry out the overall reduction of nitrate to dinitrogen. In this article, we review the insights gained into copper nitrite reductase (CuNiR) function from three dimensional structures. We particularly focus on developments over the last decade, including insights from serial femtosecond crystallography using X-ray free electron lasers (XFELs) and from the recently discovered 3-domain CuNiRs.

Photoreduction and validation of haem-ligand intermediate states in protein crystals by in situ single-crystal spectroscopy and diffraction.

Powerful synergies are available from the combination of multiple methods to study proteins in the crystalline form. Spectroscopies which probe the same region of the crystal from which X-ray crystal structures are determined can give insights into redox, ligand and spin states to complement the information gained from the electron-density maps. The correct assignment of crystal structures to the correct protein redox and ligand states is essential to avoid the misinterpretation of structural data. This is a particular concern for haem proteins, which can occupy a wide range of redox states and are exquisitely sensitive to becoming reduced by solvated electrons generated from interactions of X-rays with water molecules in the crystal. Here, single-crystal spectroscopic fingerprinting has been applied to investigate the laser photoreduction of ferric haem in cytochrome c'. Furthermore, in situ X-ray-driven generation of haem intermediates in crystals of the dye-decolourizing-type peroxidase A (DtpA) from Streptomyces lividans is described.

Active-site protein dynamics and solvent accessibility in native Achromobacter cycloclastes copper nitrite reductase.

Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (AspCAT and HisCAT) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the AspCAT protonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site 'capping residue' (IleCAT), a determinant of ligand binding, are influenced both by temperature and by the protonation state of AspCAT. A previously unobserved conformation of IleCAT is seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.

Engineering proximal vs. distal heme-NO coordination via dinitrosyl dynamics: implications for NO sensor design.

Proximal vs. distal heme-NO coordination is a novel strategy for selective gas response in heme-based NO-sensors. In the case of Alcaligenes xylosoxidans cytochrome c' (AXCP), formation of a transient distal 6cNO complex is followed by scission of the trans Fe-His bond and conversion to a proximal 5cNO product via a putative dinitrosyl species. Here we show that replacement of the AXCP distal Leu16 residue with smaller or similar sized residues (Ala, Val or Ile) traps the distal 6cNO complex, whereas Leu or Phe residues lead to a proximal 5cNO product with a transient or non-detectable distal 6cNO precursor. Crystallographic, spectroscopic, and kinetic measurements of 6cNO AXCP complexes show that increased distal steric hindrance leads to distortion of the Fe-N-O angle and flipping of the heme 7-propionate. However, it is the kinetic parameters of the distal NO ligand that determine whether 6cNO or proximal 5cNO end products are formed. Our data support a 'balance of affinities' mechanism in which proximal 5cNO coordination depends on relatively rapid release of the distal NO from the dinitrosyl precursor. This mechanism, which is applicable to other proteins that form transient dinitrosyls, represents a novel strategy for 5cNO formation that does not rely on an inherently weak Fe-His bond. Our data suggest a general means of engineering selective gas response into biologically-derived gas sensors in synthetic biology.