Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature.

Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

A subtle structural change in the distal haem pocket has a remarkable effect on tuning hydrogen peroxide reactivity in dye decolourising peroxidases from Streptomyces lividans.

Dye decolourising peroxidases (DyPs) are oxidative haem containing enzymes that can oxidise organic substrates by first reacting with hydrogen peroxide. Herein, we have focused on two DyP homologs, DtpAa and DtpA, from the soil-dwelling bacterium Streptomyces lividans. By using X-ray crystallography, stopped-flow kinetics, deuterium kinetic isotope studies and EPR spectroscopy, we show that both DyPs react with peroxide to form compound I (a FeIV[double bond, length as m-dash]O species and a porphyrin π-cation radical), via a common mechanism, but the reactivity and rate limits that define the mechanism are markedly different between the two homologs (DtpA forms compound I rapidly, no kinetic isotope effect; DtpAa 100-fold slower compound I formation and a distinct kinetic isotope effect). By determining the validated ferric X-ray structure of DtpAa and comparing it with the ferric DtpA structure, we attribute the kinetic differences to a subtle structural repositioning of the distal haem pocket Asp side chain. Through site-directed mutagenesis we show the acid-base catalyst responsible for proton-transfer to form compound I comprises a combination of a water molecule and the distal Asp. Compound I formation in the wild-type enzymes as well as their distal Asp variants is pH dependent, sharing a common ionisation equilibrium with an apparent pKa of ∼4.5-5.0. We attribute this pKa to the deprotonation/protonation of the haem bound H2O2. Our studies therefore reveal a mechanism for compound I formation in which the rate limit may be shifted from peroxide binding to proton-transfer controlled by the distal Asp position and the associated hydrogen-bonded water molecules.

Successful sample preparation for serial crystallography experiments.

Serial crystallography, at both synchrotron and X-ray free-electron laser light sources, is becoming increasingly popular. However, the tools in the majority of crystallization laboratories are focused on producing large single crystals by vapour diffusion that fit the cryo-cooled paradigm of modern synchrotron crystallography. This paper presents several case studies and some ideas and strategies on how to perform the conversion from a single crystal grown by vapour diffusion to the many thousands of micro-crystals required for modern serial crystallography grown by batch crystallization. These case studies aim to show (i) how vapour diffusion conditions can be converted into batch by optimizing the length of time crystals take to appear; (ii) how an understanding of the crystallization phase diagram can act as a guide when designing batch crystallization protocols; and (iii) an accessible methodology when attempting to scale batch conditions to larger volumes. These methods are needed to minimize the sample preparation gap between standard rotation crystallography and dedicated serial laboratories, ultimately making serial crystallography more accessible to all crystallographers.

High-throughput structures of protein-ligand complexes at room temperature using serial femtosecond crystallography.

High-throughput X-ray crystal structures of protein-ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein-ligand complexes using SFX.

Dose-resolved serial synchrotron and XFEL structures of radiation-sensitive metalloproteins.

An approach is demonstrated to obtain, in a sample- and time-efficient manner, multiple dose-resolved crystal structures from room-temperature protein microcrystals using identical fixed-target supports at both synchrotrons and X-ray free-electron lasers (XFELs). This approach allows direct comparison of dose-resolved serial synchrotron and damage-free XFEL serial femtosecond crystallography structures of radiation-sensitive proteins. Specifically, serial synchrotron structures of a heme peroxidase enzyme reveal that X-ray induced changes occur at far lower doses than those at which diffraction quality is compromised (the Garman limit), consistent with previous studies on the reduction of heme proteins by low X-ray doses. In these structures, a functionally relevant bond length is shown to vary rapidly as a function of absorbed dose, with all room-temperature synchrotron structures exhibiting linear deformation of the active site compared with the XFEL structure. It is demonstrated that extrapolation of dose-dependent synchrotron structures to zero dose can closely approximate the damage-free XFEL structure. This approach is widely applicable to any protein where the crystal structure is altered by the synchrotron X-ray beam and provides a solution to the urgent requirement to determine intact structures of such proteins in a high-throughput and accessible manner.

Correction: One fold, two functions: cytochrome P460 and cytochrome c'-ß from the methanotroph Methylococcus capsulatus (Bath).

[This corrects the article DOI: 10.1039/C8SC05210G.].

One fold, two functions: cytochrome P460 and cytochrome c'-ß from the methanotroph Methylococcus capsulatus (Bath).

Nature is adept at utilising highly similar protein folds to carry out very different functions, yet the mechanisms by which this functional divergence occurs remain poorly characterised. In certain methanotrophic bacteria, two homologous pentacoordinate c-type heme proteins have been identified: a cytochrome P460 (cyt P460) and a cytochrome c'-ß (cyt cp-ß). Cytochromes P460 are able to convert hydroxylamine to nitrous oxide (N2O), a potent greenhouse gas. This reactivity is similar to that of hydroxylamine oxidoreductase (HAO), which is a key enzyme in nitrifying and methanotrophic bacteria. Cyt P460 and HAO both have unusual protein-heme cross-links, formed by a Tyr residue in HAO and a Lys in cyt P460. In contrast, cyts cp-ß (the only known cytochromes c' with a ß-sheet fold) lack this crosslink and appears to be optimized for binding non-polar molecules (including NO and CO) without enzymatic conversion. Our bioinformatics analysis supports the proposal that cyt cp-ß may have evolved from cyt P460 via a gene duplication event. Using high-resolution X-ray crystallography, UV-visible absorption, electron paramagnetic resonance (EPR) and resonance Raman spectroscopy, we have characterized the overall protein folding and active site structures of cyt cp-ß and cyt P460 from the obligate methanotroph, Methylococcus capsulatus (Bath). These proteins display a similar ß-sheet protein fold, together with a pattern of changes to the heme pocket regions and localised tertiary structure that have converted a hydroxylamine oxidizing enzyme into a gas-binding protein. Structural comparisons provide insights relevant to enzyme redesign for synthetic enzymology and engineering of gas sensor proteins. We also show the widespread occurrence of cyts cp-ß and characterise their phylogeny.

Resolving polymorphs and radiation-driven effects in microcrystals using fixed-target serial synchrotron crystallography.

The ability to determine high-quality, artefact-free structures is a challenge in micro-crystallography, and the rapid onset of radiation damage and requirement for a high-brilliance X-ray beam mean that a multi-crystal approach is essential. However, the combination of crystal-to-crystal variation and X-ray-induced changes can make the formation of a final complete data set challenging; this is particularly true in the case of metalloproteins, where X-ray-induced changes occur rapidly and at the active site. An approach is described that allows the resolution, separation and structure determination of crystal polymorphs, and the tracking of radiation damage in microcrystals. Within the microcrystal population of copper nitrite reductase, two polymorphs with different unit-cell sizes were successfully separated to determine two independent structures, and an X-ray-driven change between these polymorphs was followed. This was achieved through the determination of multiple serial structures from microcrystals using a high-throughput high-speed fixed-target approach coupled with robust data processing.

Correction: One fold, two functions: cytochrome P460 and cytochrome c'-ß from the methanotroph Methylococcus capsulatus (Bath).

[This corrects the article DOI: 10.1039/C8SC05210G.].

Heightened Dynamics of the Oxidized Y48H Variant of Human Cytochrome c Increases Its Peroxidatic Activity.

Proteins performing multiple biochemical functions are called "moonlighting proteins" or extreme multifunctional (EMF) proteins. Mitochondrial cytochrome c is an EMF protein that binds multiple partner proteins to act as a signaling molecule, transfers electrons in the respiratory chain, and acts as a peroxidase in apoptosis. Mutations in the cytochrome c gene lead to the disease thrombocytopenia, which is accompanied by enhanced apoptotic activity. The Y48H variant arises from one such mutation and is found in the 40-57 Ω-loop, the lowest-unfolding free energy substructure of the cytochrome c fold. A 1.36 Å resolution X-ray structure of the Y48H variant reveals minimal structural changes compared to the wild-type structure, with the axial Met80 ligand coordinated to the heme iron. Despite this, the intrinsic peroxidase activity is enhanced, implying that a pentacoordinate heme state is more prevalent in the Y48H variant, corroborated through determination of a Met80 "off rate" of >125 s-1 compared to a rate of ∼6 s-1 for the wild-type protein. Heteronuclear nuclear magnetic resonance measurements with the oxidized Y48H variant reveal heightened dynamics in the 40-57 Ω-loop and the Met80-containing 71-85 Ω-loop relative to the wild-type protein, illustrating communication between these substructures. Placed into context with the G41S cytochrome c variant, also implicated in thrombocytopenia, a dynamic picture associated with this disease relative to cytochrome c is emerging whereby increasing dynamics in substructures of the cytochrome c fold serve to facilitate an increased population of the peroxidatic pentacoordinate heme state in the following order: wild type < G41S < Y48H.

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.

Interaction of Azole-Based Environmental Pollutants with the Coelomic Hemoglobin from Amphitrite ornata: A Molecular Basis for Toxicity.

The toxicities of azole pollutants that have widespread agricultural and industrial uses are either poorly understood or unknown, particularly with respect to how infaunal organisms are impacted by this class of persistent organic pollutant. To identify a molecular basis by which azole compounds may have unforeseen toxicity on marine annelids, we examine here their impact on the multifunctional dehaloperoxidase (DHP) hemoglobin from the terebellid polychaete Amphitrite ornata. Ultraviolet-visible and resonance Raman spectroscopic studies showed an increase in the six-coordinate low-spin heme population in DHP isoenzyme B upon binding of imidazole, benzotriazole, and benzimidazole (Kd values of 52, 82, and 110 µM, respectively), suggestive of their direct binding to the heme-Fe. Accordingly, atomic-resolution X-ray crystal structures, supported by computational studies, of the DHP B complexes of benzotriazole (1.14 Å), benzimidazole (1.08 Å), imidazole (1.08 Å), and indazole (1.12 Å) revealed two ligand binding motifs, one with direct ligand binding to the heme-Fe, and another in which the ligand binds in the hydrophobic distal pocket without coordinating the heme-Fe. Taken together, the results demonstrate a new mechanism by which azole pollutants can potentially disrupt hemoglobin function, thereby improving our understanding of their impact on infaunal organisms in marine and aquatic environments.