Determining Protein Structures Using X-Ray Crystallography.

X-ray crystallography is a robust and widely used technique that facilitates the three-dimensional structure determination of proteins at an atomic scale. This methodology entails the growth of protein crystals under controlled conditions followed by their exposure to X-ray beams and the subsequent analysis of the resulting diffraction patterns via computational tools to determine the three-dimensional architecture of the protein. However, achieving high-resolution structures through X-ray crystallography can be quite challenging due to complexities associated with protein purity, crystallization efficiency, and crystal quality.In this chapter, we provide a detailed overview of the gene to structure determination pipeline used in X-ray crystallography, a crucial tool for understanding protein structures. The chapter covers the steps in protein crystallization, along with the processes of data collection, processing, structure determination, and refinement. The most commonly faced challenges throughout this procedure are also addressed. Finally, the importance of standardized protocols for reproducibility and accuracy is emphasized, as they are crucial for advancing the understanding of protein structure and function.

Structure of a ribonucleotide reductase R2 protein radical.

Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O-O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.

Crystallization of Nuclear Export Signals or Small-Molecule Inhibitors Bound to Nuclear Exporter CRM1.

The Karyopherin protein CRM1 or XPO1 is the major nuclear export receptor that regulates nuclear exit of thousands of macromolecules in the cell. CRM1 recognizes protein cargoes by binding to their 8-15 residue-long nuclear export signals (NESs). A ternary CRM1-Ran-RanBP1 complex engineered to be suitable for crystallization has enabled structure determination by X-ray crystallography of CRM1 bound to many NES peptides and small-molecule inhibitors. Here, we present a protocol for the purification of the individual proteins, formation of the ternary CRM1-Ran-RanBP1 complex and crystallization of this complex for X-ray crystallography.

Variability in the Spatial Structure of the Central Loop in Cobra Cytotoxins Revealed by X-ray Analysis and Molecular Modeling.

Cobra cytotoxins (CTs) belong to the three-fingered protein family and possess membrane activity. Here, we studied cytotoxin 13 from Naja naja cobra venom (CT13Nn). For the first time, a spatial model of CT13Nn with both "water" and "membrane" conformations of the central loop (loop-2) were determined by X-ray crystallography. The "water" conformation of the loop was frequently observed. It was similar to the structure of loop-2 of numerous CTs, determined by either NMR spectroscopy in aqueous solution, or the X-ray method. The "membrane" conformation is rare one and, to date has only been observed by NMR for a single cytotoxin 1 from N. oxiana (CT1No) in detergent micelle. Both CT13Nn and CT1No are S-type CTs. Membrane-binding of these CTs probably involves an additional step-the conformational transformation of the loop-2. To confirm this suggestion, we conducted molecular dynamics simulations of both CT1No and CT13Nn in the Highly Mimetic Membrane Model of palmitoiloleoylphosphatidylglycerol, starting with their "water" NMR models. We found that the both toxins transform their "water" conformation of loop-2 into the "membrane" one during the insertion process. This supports the hypothesis that the S-type CTs, unlike their P-type counterparts, require conformational adaptation of loop-2 during interaction with lipid membranes.

Implications of AlphaFold2 for crystallographic phasing by molecular replacement.

The AlphaFold2 results in the 14th edition of Critical Assessment of Structure Prediction (CASP14) showed that accurate (low root-mean-square deviation) in silico models of protein structure domains are on the horizon, whether or not the protein is related to known structures through high-coverage sequence similarity. As highly accurate models become available, generated by harnessing the power of correlated mutations and deep learning, one of the aspects of structural biology to be impacted will be methods of phasing in crystallography. Here, the data from CASP14 are used to explore the prospects for changes in phasing methods, and in particular to explore the prospects for molecular-replacement phasing using in silico models.

Best practices for time-resolved serial synchrotron crystallography.

With recent developments in X-ray sources, instrumentation and data-analysis tools, time-resolved crystallographic experiments, which were originally the preserve of a few expert groups, are becoming simpler and can be carried out at more radiation sources, and are thus increasingly accessible to a growing user base. However, these experiments are just that: discrete experiments, not just `data collections'. As such, careful planning and consideration of potential pitfalls is required to enable a successful experiment. Here, some of the key factors that should be considered during the planning and execution of a time-resolved structural study are outlined, with a particular focus on synchrotron-based experiments.

Structural analysis of cysteine-free Nt.BspD6 nicking endonuclease and its functional features.

Nicking endonuclease Nt.BspD6I (Nt.BspD6I) is the large subunit of the heterodimeric restriction endonuclease R.BspD6I. It recognizes the short specific DNA sequence 5´'- GAGTC and cleaves only the top strand in dsDNA at a distance of four nucleotides downstream the recognition site toward the 3´'-terminus. A mechanism of interaction of this protein with DNA is still unknown. Here we report the crystal structure of Cysteine-free Nt.BspD6I, with four cysteine residues (11, 160, 508, 578) substituted by serine, which was determined with a resolution of 1.93 Å. A comparative structural analysis showed that the substitution of cysteine residues induced marked conformational changes in the N-terminal recognition and the C-terminal cleavage domains. As a result of this changes were formed three new hydrogen bonds and the electrostatic field in these regions changed compared with wild type Nt.BspD6I. The substitution of cysteine residues did not alter the nicking function of Cysteine-free Nt.BspD6I but caused change in the activity of Cysteine-free heterodimeric restriction endonuclease R.BspD6I due to a change in the interaction between its large and small subunits. The results obtained contribute to the identification of factors influencing the interactions of subunits in the heterodimeric restriction enzyme R.BspD6I.

Structure and in vitro and in silico biological activity of zinc(II) complexes with 3,5-dichloro-salicylaldehyde.

Five Zn(II) complexes with 3,5-dichloro-salicylaldehyde (3,5-diCl-saloH) in the absence or presence of N,N'-donor co-ligands (2,2'-bipyridine, 1,10-phenanthroline, 2,9-dimethyl-1,10-phenanthroline, or 2,2'-bipyridylamine) were synthesized and formulated as [Zn(3,5-diCl-salo)2(CH3OH)2] (1) and [Zn(3,5-diCl-salo)2(N,N'-donor)] (2-5), respectively, and characterized by diverse techniques. The crystal structures of four complexes were determined by single-crystal X-ray crystallography. The ability of the compounds to scavenge 1,1-diphenyl-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radicals and to reduce H2O2 was investigated. In addition, their antimicrobial profile against two Gram-positive and two Gram-negative bacterial strains were investigated. The affinity of the complexes for calf-thymus DNA was examined by diverse techniques, and the DNA-binding constants of the complexes were determined. The cleavage ability of the complexes towards supercoiled circular pBR322 plasmid DNA was examined by agarose gel electrophoretic experiments. The binding of the complexes with bovine and human serum albumins was investigated in order to determine the corresponding binding constants and the binding subdomain. In order to explain the described in vitro activity of the compounds and possibly establish a rational approach in the mechanism of action, molecular docking studies were adopted on the crystal structure of E. coli and S. aureus DNA-gyrase, 5-lipoxygenase, and 5-lipoxygenase-activating protein.

A non-catalytic herpesviral protein reconfigures ERK-RSK signaling by targeting kinase docking systems in the host.

The Kaposi's sarcoma associated herpesvirus protein ORF45 binds the extracellular signal-regulated kinase (ERK) and the p90 Ribosomal S6 kinase (RSK). ORF45 was shown to be a kinase activator in cells but a kinase inhibitor in vitro, and its effects on the ERK-RSK complex are unknown. Here, we demonstrate that ORF45 binds ERK and RSK using optimized linear binding motifs. The crystal structure of the ORF45-ERK2 complex shows how kinase docking motifs recognize the activated form of ERK. The crystal structure of the ORF45-RSK2 complex reveals an AGC kinase docking system, for which we provide evidence that it is functional in the host. We find that ORF45 manipulates ERK-RSK signaling by favoring the formation of a complex, in which activated kinases are better protected from phosphatases and docking motif-independent RSK substrate phosphorylation is selectively up-regulated. As such, our data suggest that ORF45 interferes with the natural design of kinase docking systems in the host.

Spectroscopic and Computational Investigation of the Epoxyqueuosine Reductase QueG Reveals Intriguing Similarities with the Reductive Dehalogenase PceA.

Queuosine (Q) is a highly modified nucleoside of transfer RNA that is formed from guanosine triphosphate over the course of eight steps. The final step in this process, involving the conversion of epoxyqueuosine (oQ) to Q, is catalyzed by the enzyme QueG. A recent X-ray crystallographic study revealed that QueG possesses the same cofactors as reductive dehalogenases, including a base-off Co(II)cobalamin (Co(II)Cbl) species and two [4Fe-4S] clusters. While the initial step in the catalytic cycle of QueG likely involves the formation of a reduced Co(I)Cbl species, the mechanisms employed by this enzyme to accomplish the thermodynamically challenging reduction of base-off Co(II)Cbl to Co(I)Cbl and to convert oQ to Q remain unknown. In this study, we have used electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopies in conjunction with whole-protein quantum mechanics/molecular mechanics (QM/MM) computations to further characterize wild-type QueG and select variants. Our data indicate that the Co(II)Cbl cofactor remains five-coordinate upon substrate binding to QueG. Notably, during a QM/MM optimization of a putative QueG reaction intermediate featuring an alkyl-Co(III) species, the distance between the Co ion and coordinating C atom of oQ increased to >3.3 Å and the C-O bond of the epoxide reformed to regenerate the oQ-bound Co(I)Cbl reactant state of QueG. Thus, our computations indicate that the QueG mechanism likely involves single-electron transfer from the transient Co(I)Cbl species to oQ rather than direct Co-C bond formation, similar to the mechanism that has recently been proposed for the tetrachloroethylene reductive dehalogenase PceA.

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate.

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze ß-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate ß-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue ßLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cß and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

Atomic-resolution chemical characterization of (2x)72-kDa tryptophan synthase via four- and five-dimensional 1H-detected solid-state NMR.

NMR chemical shifts provide detailed information on the chemical properties of molecules, thereby complementing structural data from techniques like X-ray crystallography and electron microscopy. Detailed analysis of protein NMR data, however, often hinges on comprehensive, site-specific assignment of backbone resonances, which becomes a bottleneck for molecular weights beyond 40 to 45 kDa. Here, we show that assignments for the (2x)72-kDa protein tryptophan synthase (665 amino acids per asymmetric unit) can be achieved via higher-dimensional, proton-detected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atom-specific characterization of chemical properties and relaxation for the backbone and side chains, including those residues important for the catalytic turnover. Combined with first-principles calculations, the chemical shifts in the ß-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the ß-subunit from solution.

Reaction of a {Co(NO)}8 complex with superoxide: Formation of a six coordinated [CoII(NO)(O2-)] species followed by peroxynitrite intermediate.

A nitrosyl complex of cobalt(II) porphyrinate, [Co(F20TPP2-)(NO)], (F20TPPH2 = 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin) having {Co(NO)}8 configuration was synthesized and characterized by means of spectroscopic and structural analyses. Single crystal X-ray structure of the complex revealed the square pyramidal geometry around the cobalt center with a bent nitrosyl group. It reacts with superoxide (O2-) ion in CH2Cl2 at -40 °C to result in the corresponding nitrite (NO2-) complex. Involvement of a cobalt(II)-peroxynitrite intermediate is proposed in the course of the reaction. Moreover, spectroscopic studies suggested the formation of a transient six-coordinated [CoII(NO)(O2-)] species.

Low-dimensional compounds containing bioactive ligands. Part XVII: Synthesis, structural, spectral and biological properties of hybrid organic-inorganic complexes based on [PdCl4]2- with derivatives of 8-hydroxyquinolinium.

In this study, four hybrid organic-inorganic compounds (8-H2Q)2[PdCl4] (1), (H2ClQ)2[PdCl4] (2), (H2NQ)2[PdCl4] (3) and (H2MeQ)2[PdCl4]·2H2O (4) (where 8-H2Q = 8-hydroxyquinolinium, H2ClQ = 5-chloro-8-hydroxyquinolinium, H2NQ = 5-nitro-8-hydroxyquinolinium and H2MeQ = 2-methyl-8-hydroxyquinolinium) were synthesized through organic cation modulation. Single-crystal X-ray structure analysis of compounds 1 and 3 indicates that their structures are planar and consist of [PdCl4]2- anions and 8-H2Q or H2NQ cations, respectively. Both ionic components are held together through ionic interactions and hydrogen bonds forming infinite chains linked through π-π interactions to form 2D structures. Furthermore, NMR spectroscopy, UV-Vis spectroscopy, elemental analysis, and FT-IR spectroscopy were used to explore the synthesized compounds. The DNA interaction, antimicrobial activity, antiproliferative activity, and radical scavenging effect of the compounds were evaluated. The hybrid compounds and their free ligands can interact with the calf thymus DNA via an intercalation mode involving the insertion of the aromatic chromophore between the base pairs of DNA; compound 1 has the highest binding affinity. Moreover, they have high antimicrobial efficacy against the tested 14 strains of microorganisms with minimum inhibitory concentration values ranging from <1.95 to 250 µg/mL. The antiproliferative activity of the compounds was investigated against three different cancer cell lines, and their selectivity was verified on mesenchymal stem cells. Compounds 1 and 2 displayed selective and high cytotoxicity against human lung and breast cancer cells and showed moderate cytotoxicity against colon cancer cells. Accordingly, they might be auspicious candidates for future pharmacological investigations in lung and breast cancer research.

Crystal structure of the Cys-NO modified YopH tyrosine phosphatase.

Protein tyrosine phosphatases (PTPs) are key virulence factors in pathogenic bacteria, consequently, they have become important targets for new approaches against these pathogens, especially in the fight against antibiotic resistance. Among these targets of interest YopH (Yersinia outer protein H) from virulent species of Yersinia is an example. PTPs can be reversibly inhibited by nitric oxide (NO) since the oxidative modification of cysteine residues may influence the protein structure and catalytic activity. We therefore investigated the effects of NO on the structure and enzymatic activity of Yersinia enterocolitica YopH in vitro. Through phosphatase activity assays, we observe that in the presence of NO YopH activity was inhibited by 50%, and that this oxidative modification is partially reversible in the presence of DTT. Furthermore, YopH S-nitrosylation was clearly confirmed by a biotin switch assay, high resolution mass spectrometry (MS) and X-ray crystallography approaches. The crystal structure confirmed the S-nitrosylation of the catalytic cysteine residue, Cys403, while the MS data provide evidence that Cys221 and Cys234 might also be modified by NO. Interestingly, circular dichroism spectroscopy shows that the S-nitrosylation affects secondary structure of wild type YopH, though to a lesser extent on the catalytic cysteine to serine YopH mutant. The data obtained demonstrate that S-nitrosylation inhibits the catalytic activity of YopH, with effects beyond the catalytic cysteine. These findings are helpful for designing effective YopH inhibitors and potential therapeutic strategies to fight this pathogen or others that use similar mechanisms to interfere in the signal transduction pathways of their hosts.

Beef tallow injection matrix for serial crystallography.

Serial crystallography (SX) enables the visualization of the time-resolved molecular dynamics of macromolecular structures at room temperature while minimizing radiation damage. In SX experiments, the delivery of a large number of crystals into an X-ray interaction point in a serial and stable manner is key. Sample delivery using viscous medium maintains the stable injection stream at low flow rates, markedly reducing sample consumption compared with that of a liquid jet injector and is widely applied in SX experiments with low repetition rates. As the sample properties and experimental environment can affect the stability of the injection stream of a viscous medium, it is important to develop sample delivery media with various characteristics to optimize the experimental environment. In this study, a beef tallow injection matrix possessing a higher melting temperature than previously reported fat-based shortening and lard media was introduced as a sample delivery medium and applied to SX. Beef tallow was prepared by heat treating fats from cattle, followed by the removal of soluble impurities from the extract by phase separation. Beef tallow exhibited a very stable injection stream at room temperature and a flow rate of < 10 nL/min. The room-temperature structures of lysozyme and glucose isomerase embedded in beef tallow were successfully determined at 1.55 and 1.60 Å, respectively. The background scattering of beef tallow was higher than that of previously reported fat-based shortening and lard media but negligible for data processing. In conclusion, the beef tallow matrix can be employed for sample delivery in SX experiments conducted at temperatures exceeding room temperature.

Anti-inflammatory Dimeric Tetrahydroxanthones from an Endophytic Muyocopron laterale.

Twelve new dimeric tetrahydroxanthones, muyocoxanthones A-L (1-12), were isolated from the endophytic fungus, Muyocopron laterale. Their structures were characterized on the basis of the interpretation of NMR and HRESIMS data. The absolute configurations of 1-10 and 12 were unambiguously determined by ECD spectrum data and single-crystal X-ray diffraction analysis. Compounds 2, 6, and 11 showed inhibitory activity against the LPS-induced production of nitric oxide (NO) in RAW 264.7 cells with IC50 values of 5.2, 1.3, and 5.1 µM, respectively.

In vitro biological activity of copper(II) complexes with NSAIDs and nicotinamide: Characterization, DNA- and BSA-interaction study and anticancer activity.

Through the reaction of copper(II) acetate with nicotinamide (pyridine-3-carboxylic acid amide, niacinamide) and some derivatives of N-phenylanthranilic acid (fenamates), seven new mixed-ligand copper(II) compounds were isolated: [Cu(tolf-O)(tolf-O,O')nia-N)2(EtOH)] (1), [Cu(tolf-O)(tolf-O,O')(nia-N)2(MeOH)] (2), [Cu(meclf-O)(meclf-O,O')(nia-N)2(EtOH)] (3), [Cu(meclf-O)(meclf-O,O')(nia-N)2(MeOH)] (4), [Cu(meclf-O)(meclf-O,O')(nia-N)2(ACN)] (5), [Cu(mef-O)(mef-O,O')(nia-N)2(EtOH)] (6) and [Cu(mef-O)(mef-O,O')(nia-N)2(ACN)] (7) containing a molecule of relevant solvent as ligand in their primary crystal structure (tolf = tolfenamate, meclf = meclofenamate, mef = mefenamate, nia = nicotinamide, EtOH = ethanol, MeOH = methanol, ACN = acetonitrile). The structures of the complexes were determined by single-crystal X-ray analysis. The intermolecular interactions were studied by Hirshfeld surface analysis. The complexes were characterized by IR, UV-vis and EPR spectroscopy and their redox properties were determined by cyclic voltammetry. The interaction of the complexes with bovine serum albumin was studied by fluorescence emission spectroscopy and the albumin-binding constants of the compounds were calculated. The interaction of the complexes with calf-thymus DNA was monitored by diverse techniques (UV-vis spectroscopy, cyclic voltammetry, viscosity measurements) suggesting intercalation as the most possible mode of binding. DNA-competitive studies of the complexes with ethidium bromide were monitored by fluorescence emission spectroscopy. The cytotoxic effects of copper(II) complexes on lung carcinoma cells and healthy cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric technique.

Structures and Biological Activities of Polyacylated ent-Kaurane Diterpenoid Glycosides from the Aerial Parts of Inula hupehensis.

Sixteen new (1-16) and three known (17-19) polyacylated ent-kaurane diterpenoid glycosides were isolated from the aerial parts of Inula hupehensis. The planar structures of 1-16 and their relative configurations were established on the basis of extensive spectroscopic analysis. The absolute configurations of all stereogenic centers for compounds 1 and 6 were determined by single-crystal X-ray diffraction experiments, and the absolute configurations of the other new compounds were assigned by chemical degradation and experimental ECD data. Antineuroinflammatory testing of all the isolates showed that compound 5 inhibited lipopolysaccharide-induced nitric oxide production in BV-2 microglial cells with an IC50 value of 15.6 µM. In an α-glucosidase inhibitory assay, compound 13 exhibited a strong inhibitory effect with an IC50 value of 32.8 µM, whereas the IC50 value of the positive control, acarbose, was 387.8 µM.

Pinguisane Sesquiterpenoids from the Chinese Liverwort Trocholejeunea sandvicensis and Their Anti-Inflammatory Activity.

Nine new pinguisane sesquiterpenoid compounds, 1-9, including a highly oxygenated compound (1) and two amides (7 and 8), along with three known compounds (10, 11, and 12), were isolated from the Chinese liverwort Trocholejeunea sandvicensis Mizut (Lejeuneaceae). The structures of these compounds were determined by analysis of IR, UV, HRESIMS, NMR spectroscopic data, electronic circular dichroism calculations, and single-crystal X-ray diffraction analysis. Inhibitory effects against lipopolysaccharide (LPS)-induced NO production in RAW 264.7 murine macrophages indicated that the maximum inhibition rates of NO production of compounds 1, 9, and 10 were 83.15%, 83.54%, and 96.28% under the nontoxic tested concentration, respectively. Compound 9 also displayed moderate anti-inflammatory activity in vivo in a CuSO4-induced transgenic zebrafish model.