Development of crystallographic techniques and their application to several protein targets

Since its first use to solve the structure of sodium chloride in 1915 X-ray crystallography has developed significantly to become the premier technique for obtaining 3D structural information of small molecules and macromolecules alike. As the technique continues to develop and focus its attention on weak diffraction from the likes of micro-crystals and poorly packed crystals of membrane proteins and large protein complexes; as well as ultra-high resolution data and weak anomalous signal from native atoms, data quality is becoming more and more important. Data quality is particularly important in the wake of long wavelength macromolecular crystallography (MX) for phasing using anomalous signal from native sulphur and phosphorous atoms in proteins and DNA. This thesis first investigated the use of a new sample handling technique using a humidity controlled stream to preserve macromolecular crystals while excess surrounding solvent is removed (Chapter 2). Following the successful development of this technique the effects of excess surrounding solvent on data quality was assessed when collecting at standard MX X-ray wavelengths (~ 1 Å) and longer X-ray wavelengths (~ 2 Å). Datasets were collected from large populations of control and test crystals at standard and longer wavelengths to allow robust statistical methods to be applied; a practice not widely adopted in method development studies in X-ray crystallography. This made it possible to assess the small differences in data quality in the presence and absence of excess surrounding solvent. The effects of surrounding solvent at longer wavelengths appear to be protein dependent with some proteins tested showing no significant difference and others a significant decrease in data quality at longer wavelengths (Chapter 3). Originally this project aimed to use the new long wavelength in-vacuum MX beamline, I23, at Diamond Light Source UK to carry out phasing experiments using native sulphurs for structure solution. However, the considerable complexity involved in developing in-vacuum MX meant these experiments could not be carried out during the time frame of this thesis. Chapters 4 and 5 outline the production of a novel cancer protein (cancerous inhibitor of protein phosphatase 2A) and two protein targets from the Achromobacter xylosoxidans (Ax) genome intended for sulphur single wavelength anomalous dispersion phasing experiments on I23. Of these proteins the structure of Ax-α/β hydrolase was solved by conventional methods, the structure of which is discussed in Chapter 5. Of the protein crystals used in long wavelength data quality experiments in Chapter 3 the molecular biology of PA3825-EAL, a biofilm regulating protein essential to the swarming ability of Pseudomonas aeruginosa, was investigated further. The crystal structure of PA3825-EAL was solved in the resting, substrate bound and product bound states to high resolution. Comparison of the crystal structures of monomeric and dimeric PA3825-EAL with the inactive dimeric structure of MucR-EAL suggests dimerisation via helix 8 plays a role in inhibition of EAL domains. Prior to this, dimerisation was thought to be an activating factor in EAL domains. The product bound state of PA3825-EAL showed the presence of a previously unreported third metal binding site which may form an essential component of the reaction mechanism of EAL domains. Inability of MucR-EAL to incorporate this third metal due to dimerisation may explain the lack of activity despite possessing the conserved catalytic residues necessary. The fast detector technology and improvements in automated data processing software that allowed diffraction data for large populations of crystals to be collected in Chapters 2 and 3 have also been applied to development of a serial data collection technique. Of 159 datasets collected from 8 crystals of a copper nitrite reductase from Achromobacter cycloclastes, 45 datasets from a single crystal were analysed to observe the reaction mechanism using high resolution crystal structures. X-ray radiolysis initiated the reaction and high resolution data allowed the conversion of nitrite (NO2) to nitric oxide (NO) to be observed in the crystal. Other aspects of the reaction were investigated from the data series including a conserved water chain connecting the copper sites which may act as a proton wire to donate a proton and produce NO. This technique may have wide applications to the study of the reaction mechanisms of other metallo-proteins

Homogeneous batch micro-crystallization of proteins from ammonium sulfate

The emergence of X-ray free-electron lasers has led to the development of serial macromolecular crystallography techniques, making it possible to study smaller and more challenging crystal systems and to perform time-resolved studies on fast time scales. For most of these studies the desired crystal size is limited to a few micrometres, and the generation of large amounts of nanocrystals or microcrystals of defined size has become a bottleneck for the wider implementation of these techniques. Despite this, methods to reliably generate microcrystals and fine-tune their size have been poorly explored. Working with three different enzymes, L-aspartate alpha-decarboxylase, copper nitrite reductase and copper amine oxidase, the precipitating properties of ammonium sulfate were exploited to quickly transition from known vapour-diffusion conditions to reproducible, large-scale batch crystallization, circumventing the tedious determination of phase diagrams. Furthermore, the specific ammonium sulfate concentration was used to fine-tune the crystal size and size distribution. Ammonium sulfate is a common precipitant in protein crystallography, making these findings applicable to many crystallization systems to facilitate the production of large amounts of microcrystals for serial macromolecular crystallography experiments.Peer reviewe

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

Making the invisible enemy visible

Structural biology plays a crucial role in the fight against COVID-19, permitting us to ‘see’ and understand SARS-CoV-2. However, the macromolecular structures of SARS-CoV-2 proteins that were solved with great speed and urgency can contain errors that may hinder drug design. The Coronavirus Structural Task Force has been working behind the scenes to evaluate and improve these structures, making the results freely available at https://insidecorona.net/.publishe

Changes in metal coordination are required to regulate activity of bacterial phosphodiesterases, implicated in c-di-GMP regulated biofilm dispersal

Bacterial biofilms play a key role in prosthetic infection (PI) pathogenesis. Establishment of the biofilm phenotype confers the bacteria with significant tolerance to systemic antibiotics and the host immune system meaning thorough debridement and prosthesis removal often remain the only possible course of treatment. Protection of the prosthesis and dead-space management may be achieved through the use of antibiotic loaded cements and beads to release high concentrations of antibiotics at the surgical site. The antibacterial and antibiofilm efficacy of these materials is poorly understood in the context of mixed species models, such as are often encountered clinically

Three-dimensional structure of the single domain cupredoxin AcoP

Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper center coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in term of copper-binding ligands, copper center geometry, redox potential, as well as biological function. AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans . AcoP presents original features: highly resistant to acidic pH, it possesses a constrained green-type copper center of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structure of AcoP at 1.65 Å resolution unveils a typical cupredoxin fold with extended loops, never observed in previously characterized cupredoxins, that might be involved in the interaction of AcoP with its physiological partners. Moreover, the structure shows that the green color of AcoP cannot be attributed to nonclassical copper ligands, its green-colored copper center raising from a long Cu-S (Cys) bond, determined by both X-ray diffraction and EXAFS. The crystal structures of two AcoP mutants confirm that the active center of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active center rigidity due to the presence of an extensive hydrogen bond network

Fixed target serial data collection at Diamond Light Source

Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is presented with detailed step-by-step instructions, figures, and videos for smooth data collection

Spectroscopically Validated pH-dependent MSOX Movies Provide Detailed Mechanism of Copper Nitrite Reductases.

Copper nitrite reductases (CuNiRs) exhibit a strong pH dependence of their catalytic activity. Structural movies can be obtained by serially recording multiple structures (frames) from the same spot of a crystal using the MSOX serial crystallography approach. This method has been combined with on-line single crystal optical spectroscopy to capture the pH-dependent structural changes that accompany during turnover of CuNiRs from two Rhizobia species. The structural movies, initiated by the redox activation of a type-1 copper site (T1Cu) via X-ray generated photoelectrons, have been obtained for the substrate-free and substrate-bound states at low (high enzymatic activity) and high (low enzymatic activity) pH. At low pH, formation of the product nitric oxide (NO) is complete at the catalytic type-2 copper site (T2Cu) after a dose of 3 MGy (frame 5) with full bleaching of the T1Cu ligand-to-metal charge transfer (LMCT) 455 nm band (S(σ)Cys → T1Cu2+) which in itself indicates the electronic route of proton-coupled electron transfer (PCET) from T1Cu to T2Cu. In contrast at high pH, the changes in optical spectra are relatively small and the formation of NO is only observed in later frames (frame 15 in Br2DNiR, 10 MGy), consistent with the loss of PCET required for catalysis. This is accompanied by decarboxylation of the catalytic AspCAT residue, with CO2 trapped in the catalytic pocket

Making the invisible enemy visible.

Structural biology plays a crucial role in the fight against COVID-19, permitting us to ‘see’ and understand SARS-CoV-2. However, the macromolecular structures of SARS-CoV-2 proteins that were solved with great speed and urgency can contain errors that may hinder drug design. The Coronavirus Structural Task Force has been working behind the scenes to evaluate and improve these structures, making the results freely available at https://insidecorona.net/