Probing the active site of Class 3 L-asparaginase by mutagenesis. I. Tinkering with the zinc coordination site of ReAV.

ReAV, the inducible Class-3 L-asparaginase from the nitrogen-fixing symbiotic bacterium Rhizobium etli, is an interesting candidate for optimizing its enzymatic potential for antileukemic applications. Since it has no structural similarity to known enzymes with this activity, it may offer completely new ways of approach. Also, as an unrelated protein, it would evade the immunological response elicited by other asparaginases. The crystal structure of ReAV revealed a uniquely assembled protein homodimer with a highly specific C135/K138/C189 zinc binding site in each subunit. It was also shown before that the Zn2+ cation at low and optimal concentration boosts the ReAV activity and improves substrate specificity, which indicates its role in substrate recognition. However, the detailed catalytic mechanism of ReAV is still unknown. In this work, we have applied site-directed mutagenesis coupled with enzymatic assays and X-ray structural analysis to elucidate the role of the residues in the zinc coordination sphere in catalysis. Almost all of the seven ReAV muteins created in this campaign lost the ability to hydrolyze L-asparagine, confirming our predictions about the significance of the selected residues in substrate hydrolysis. We were able to crystallize five of the ReAV mutants and solve their crystal structures, revealing some intriguing changes in the active site area as a result of the mutations. With alanine substitutions of Cys135 or Cys189, the zinc coordination site fell apart and the mutants were unable to bind the Zn2+ cation. Moreover, the absence of Lys138 induced atomic shifts and conformational changes of the neighboring residues from two active-site Ser-Lys tandems. Ser48 from one of the tandems, which is hypothesized to be the catalytic nucleophile, usually changes its hydration pattern in response to the mutations. Taken together, the results provide many useful clues about the catalytic mechanism of the enzyme, allowing one to cautiously postulate a possible enzymatic scenario.

Rhizobium etli has two L-asparaginases with low sequence identity but similar structure and catalytic center.

The genome of Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes two L-asparaginases, ReAIV and ReAV, that have no similarity to the well characterized enzymes of class 1 (bacterial type) and class 2 (plant type). It has been hypothesized that ReAIV and ReAV might belong to the same structural class 3 despite their low level of sequence identity. When the crystal structure of the inducible and thermolabile protein ReAV was solved, this hypothesis gained a stronger footing because the key residues of ReAV are also present in the sequence of the constitutive and thermostable ReAIV protein. High-resolution crystal structures of ReAIV now confirm that it is a class 3 L-asparaginase that is structurally similar to ReAV but with important differences. The most striking differences concern the peculiar hydration patterns of the two proteins, the presence of three internal cavities in ReAIV and the behavior of the zinc-binding site. ReAIV has a high pH optimum (9-11) and a substrate affinity of ∼1.3 mM at pH 9.0. These parameters are not suitable for the direct application of ReAIV as an antileukemic drug, although its thermal stability and lack of glutaminase activity would be of considerable advantage. The five crystal structures of ReAIV presented in this work allow a possible enzymatic scenario to be postulated in which the zinc ion coordinated in the active site is a dispensable element. The catalytic nucleophile seems to be Ser47, which is part of two Ser-Lys tandems in the active site. The structures of ReAIV presented here may provide a basis for future enzyme-engineering experiments to improve the kinetic parameters for medicinal applications.

Right-handed Z-DNA at ultrahigh resolution: a tale of two hands and the power of the crystallographic method.

The self-complementary L-d(CGCGCG)2 purine/pyrimidine hexanucleotide was crystallized in complex with the polyamine cadaverine and potassium cations. Since the oligonucleotide contained the enantiomeric 2'-deoxy-L-ribose, the Z-DNA duplex is right-handed, as confirmed by the ultrahigh-resolution crystal structure determined at 0.69 Šresolution. Although the X-ray diffraction data were collected at a very short wavelength (0.7085 Å), where the anomalous signal of the P and K atoms is very weak, the signal was sufficiently outstanding to clearly indicate the wrong hand when the structure was mistakenly solved assuming the presence of 2'-deoxy-D-ribose. The electron density clearly shows the entire cadaverinium dication, which has an occupancy of 0.53 and interacts with one Z-DNA duplex. The K+ cation, with an occupancy of 0.32, has an irregular coordination sphere that is formed by three OP atoms of two symmetry-related Z-DNA duplexes and one O5' hydroxyl O atom, and is completed by three water sites, one of which is twofold disordered. The K+ site is complemented by a partial water molecule, the hydrogen bonds of which have the same lengths as the K-O bonds. The sugar-phosphate backbone assumes two conformations, but the base pairs do not show any sign of disorder.

Structural and biophysical studies of new L-asparaginase variants: lessons from random mutagenesis of the prototypic Escherichia coli Ntn-amidohydrolase.

This work reports the results of random mutagenesis of the Escherichia coli class 2 L-asparaginase EcAIII belonging to the Ntn-hydrolase family. New variants of EcAIII were studied using structural, biophysical and bioinformatic methods. Activity tests revealed that the L-asparaginase activity is abolished in all analyzed mutants with the absence of Arg207, but some of them retained the ability to undergo the autoproteolytic maturation process. The results of spectroscopic studies and the determined crystal structures showed that the EcAIII fold is flexible enough to accept different types of mutations; however, these mutations may have a diverse impact on the thermal stability of the protein. The conclusions from the experiments are grouped into six lessons focused on (i) the adaptation of the EcAIII fold to new substitutions, (ii) the role of Arg207 in EcAIII activity, (iii) a network of residues necessary for autoprocessing, (iv) the complexity of the autoprocessing reaction, (v) the conformational changes observed in enzymatically inactive variants and (vi) the cooperativity of the EcAIII dimer subunits. Additionally, the structural requirements (pre-maturation checkpoints) that are necessary for the initiation of the autocleavage of Ntn-hydrolases have been classified. The findings reported in this work provide useful hints that should be considered before planning enzyme-engineering experiments aimed at the design of proteins for therapeutic applications. This is especially important for L-asparaginases that can be utilized in leukemia therapy, as alternative therapeutics are urgently needed to circumvent the severe side effects associated with the currently used enzymes.

Crystal structures of the elusive Rhizobium etli L-asparaginase reveal a peculiar active site.

Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes an essential L-asparaginase (ReAV) with no sequence homology to known enzymes with this activity. High-resolution crystal structures of ReAV show indeed a structurally distinct, dimeric enzyme, with some resemblance to glutaminases and ß-lactamases. However, ReAV has no glutaminase or lactamase activity, and at pH 9 its allosteric asparaginase activity is relatively high, with Km for L-Asn at 4.2 mM and kcat of 438 s-1. The active site of ReAV, deduced from structural comparisons and confirmed by mutagenesis experiments, contains a highly specific Zn2+ binding site without a catalytic role. The extensive active site includes residues with unusual chemical properties. There are two Ser-Lys tandems, all connected through a network of H-bonds to the Zn center, and three tightly bound water molecules near Ser48, which clearly indicate the catalytic nucleophile.

Crystal structure of Z-DNA in complex with the polyamine putrescine and potassium cations at ultra-high resolution.

The X-ray crystal structure of the d(CGCGCG)2/putrescine(2+)/K+ complex has been determined at 0.60 Šresolution. Stereochemical restraints were used only for the putrescinium dication, and 23 bonds and 18 angles of the Z-DNA nucleotides with dual conformation. The N atoms of the putrescine(2+) dication form three direct hydrogen bonds with the N7_G atoms of three different Z-DNA molecules, plus three water-mediated hydrogen bonds with cytosine, guanine and phosphate acceptors. A unique potassium cation was also unambiguously identified in the structure, albeit at a ∼0.5 occupation site shared with a water molecule, providing the first example of such a complex with Z-DNA. The K+ cation has coordination number of eight and an irregular coordination sphere, formed by four water molecules and four O atoms from two phosphate groups of the Z-DNA, including ligands present at fractional occupancy. The structural disorder of the Z-DNA duplex is manifested by the presence of alternate conformations along the DNA backbone. Comparison of the position and interactions of putrescine(2+) in the present structure with other ultra-high-resolution structures of Z-DNA in complexes with Mn2+ and Zn2+ ions shows that the dicationic putrescinium moiety can effectively substitute these metal ions for stabilization of Z-type DNA duplexes. Furthermore, this comparison also suggests that the spermine(4+) tetracation has a higher affinity for Z-DNA than K+.

Rapid response to emerging biomedical challenges and threats.

As part of the global mobilization to combat the present pandemic, almost 100 000 COVID-19-related papers have been published and nearly a thousand models of macromolecules encoded by SARS-CoV-2 have been deposited in the Protein Data Bank within less than a year. The avalanche of new structural data has given rise to multiple resources dedicated to assessing the correctness and quality of structural data and models. Here, an approach to evaluate the massive amounts of such data using the resource https://covid19.bioreproducibility.org is described, which offers a template that could be used in large-scale initiatives undertaken in response to future biomedical crises. Broader use of the described methodology could considerably curtail information noise and significantly improve the reproducibility of biomedical research.

Crystal structures of inhibitor complexes of M-PMV protease with visible flap loops.

Mason-Pfizer monkey virus protease (PR) was crystallized in complex with two pepstatin-based inhibitors in P1 space group. In both crystal structures, the extended flap loops that lock the inhibitor/substrate over the active site, are visible in the electron density either completely or with only small gaps, providing the first observation of the conformation of the flap loops in dimeric complex form of this retropepsin. The H-bond network in the active site (with D26N mutation) differs from that reported for the P21 crystal structures and is similar to a rarely occurring system in HIV-1 PR.

Crystallographic models of SARS-CoV-2 3CLpro: in-depth assessment of structure quality and validation.

The appearance at the end of 2019 of the new SARS-CoV-2 coronavirus led to an unprecedented response by the structural biology community, resulting in the rapid determination of many hundreds of structures of proteins encoded by the virus. As part of an effort to analyze and, if necessary, remediate these structures as deposited in the Protein Data Bank (PDB), this work presents a detailed analysis of 81 crystal structures of the main protease 3CLpro, an important target for the design of drugs against COVID-19. The structures of the unliganded enzyme and its complexes with a number of inhibitors were determined by multiple research groups using different experimental approaches and conditions; the resulting structures span 13 different polymorphs representing seven space groups. The structures of the enzyme itself, all determined by molecular replacement, are highly similar, with the exception of one polymorph with a different inter-domain orientation. However, a number of complexes with bound inhibitors were found to pose significant problems. Some of these could be traced to faulty definitions of geometrical restraints for ligands and to the general problem of a lack of such information in the PDB depositions. Several problems with ligand definition in the PDB itself were also noted. In several cases extensive corrections to the models were necessary to adhere to the evidence of the electron-density maps. Taken together, this analysis of a large number of structures of a single, medically important protein, all determined within less than a year using modern experimental tools, should be useful in future studies of other systems of high interest to the biomedical community.

Covid-19.bioreproducibility.org: A web resource for SARS-CoV-2-related structural models.

The COVID-19 pandemic has triggered numerous scientific activities aimed at understanding the SARS-CoV-2 virus and ultimately developing treatments. Structural biologists have already determined hundreds of experimental X-ray, cryo-EM, and NMR structures of proteins and nucleic acids related to this coronavirus, and this number is still growing. To help biomedical researchers, who may not necessarily be experts in structural biology, navigate through the flood of structural models, we have created an online resource, covid19.bioreproducibility.org, that aggregates expert-verified information about SARS-CoV-2-related macromolecular models. In this article, we describe this web resource along with the suite of tools and methodologies used for assessing the structures presented therein.

C60 in a peptidic cage: a case of symmetry mismatch studied by crystallography and solid-state NMR.

A supramolecular complex, formed by encapsulation of C60 fullerene in a molecular container built from two resorcin[4]arene rims zipped together by peptidic arms hydrogen bonded into a cylindrical ß-sheet, was studied by X-ray crystallography, solid-state and solution NMR, EPR spectroscopy and differential scanning calorimetry (DSC). The crystal structure, determined at 100 K, reveals that the complex occupies 422 site symmetry, which is compatible with the molecular symmetry of the container but not of the fullerene molecule, which has only 222 symmetry. The additional crystallographic symmetry leads to a complicated but discrete disorder, which could be resolved and modelled using advanced features of the existing refinement software. Solid-state NMR measurements at 184-333 K indicate that the thermal motion of C60 in this temperature range is fast but has different activation energies at different temperatures, which was attributed to a phase transition, which was confirmed by DSC. Intriguingly, the activation energy for reorientations of C60 in the solid state is very similar for the free and encaged molecules. Also, the rotational diffusion coefficients seem to be very similar or even slightly higher for the encaged fullerene compared to the free molecule. We also found that chemical shift anisotropy (CSA) is not the main relaxation mechanism for the 13C spins of C60 in the studied complex.

A new modulated crystal structure of the ANS complex of the St John's wort Hyp-1 protein with 36 protein molecules in the asymmetric unit of the supercell.

Superstructure modulation, with violation of the strict short-range periodic order of consecutive crystal unit cells, is well known in small-molecule crystallography but is rarely reported for macromolecular crystals. To date, one modulated macromolecular crystal structure has been successfully determined and refined for a pathogenesis-related class 10 protein from Hypericum perforatum (Hyp-1) crystallized as a complex with 8-anilinonaphthalene-1-sulfonate (ANS) [Sliwiak et al. (2015), Acta Cryst. D71, 829-843]. The commensurate modulation in that case was interpreted in a supercell with sevenfold expansion along c. When crystallized in the additional presence of melatonin, the Hyp-1-ANS complex formed crystals with a different pattern of structure modulation, in which the supercell shows a ninefold expansion of c, manifested in the diffraction pattern by a wave of reflection-intensity modulation with crests at l = 9n and l = 9n ± 4. Despite complicated tetartohedral twinning, the structure has been successfully determined and refined to 2.3 Šresolution using a description in a ninefold-expanded supercell, with 36 independent Hyp-1 chains and 156 ANS ligands populating the three internal (95 ligands) and five interstitial (61 ligands) binding sites. The commensurate superstructures and ligand-binding sites of the two crystal structures are compared, with a discussion of the effect of melatonin on the co-crystallization process.

Dynamer and Metallodynamer Interconversion: An Alternative View to Metal Ion Complexation.

A bifunctional molecule containing both a bidentate binding site for metal ions and an aminopyrimidine H-bond donor-acceptor site has been synthesized, and its properties, in its free and coordinated forms, have been established in solution and in the solid state by analytical and spectroscopic methods as well as by X-ray structure determinations. Structural characterization has shown that it forms a one-dimensional H-bonded polymeric assembly in the solid state, while spectroscopic measurements indicate that it also aggregates in solution. The reaction of a simple Fe(II) salt with this assembly results in the emergence of two geometrical isomers of the complex: [FeL3](BF4)2·9H2O-C1 (meridional, mer) and [FeL3]2(SiF6)(BF4)2·12H2O-C2 (facial, fac). While, complex C1 in the solid state generates a one-dimensional H-bonded polymer involving just two ligands on each Fe center, with the chirality of the complex units alternating along the polymer chain, the structure of complex C2 shows NH···N interactions seen in both the ligand and mer complex (C1) structures to be completely absent. Physicochemical properties of the free and complexed ligand differ substantially.

Ligand-centered assessment of SARS-CoV-2 drug target models in the Protein Data Bank.

A bright spot in the SARS-CoV-2 (CoV-2) coronavirus pandemic has been the immediate mobilization of the biomedical community, working to develop treatments and vaccines for COVID-19. Rational drug design against emerging threats depends on well-established methodology, mainly utilizing X-ray crystallography, to provide accurate structure models of the macromolecular drug targets and of their complexes with candidates for drug development. In the current crisis, the structural biological community has responded by presenting structure models of CoV-2 proteins and depositing them in the Protein Data Bank (PDB), usually without time embargo and before publication. Since the structures from the first-line research are produced in an accelerated mode, there is an elevated chance of mistakes and errors, with the ultimate risk of hindering, rather than speeding up, drug development. In the present work, we have used model-validation metrics and examined the electron density maps for the deposited models of CoV-2 proteins and a sample of related proteins available in the PDB as of April 1, 2020. We present these results with the aim of helping the biomedical community establish a better-validated pool of data. The proteins are divided into groups according to their structure and function. In most cases, no major corrections were necessary. However, in several cases significant revisions in the functionally sensitive area of protein-inhibitor complexes or for bound ions justified correction, re-refinement, and eventually reversioning in the PDB. The re-refined coordinate files and a tool for facilitating model comparisons are available at https://covid-19.bioreproducibility.org. DATABASE: Validated models of CoV-2 proteins are available in a dedicated, publicly accessible web service https://covid-19.bioreproducibility.org.

3D domain swapping in the TIM barrel of the α subunit of Streptococcus pneumoniae tryptophan synthase.

Tryptophan synthase catalyzes the last two steps of tryptophan biosynthesis in plants, fungi and bacteria. It consists of two protein chains, designated α and ß, encoded by trpA and trpB genes, that function as an αßßα complex. Structural and functional features of tryptophan synthase have been extensively studied, explaining the roles of individual residues in the two active sites in catalysis and allosteric regulation. TrpA serves as a model for protein-folding studies. In 1969, Jackson and Yanofsky observed that the typically monomeric TrpA forms a small population of dimers. Dimerization was postulated to take place through an exchange of structural elements of the monomeric chains, a phenomenon later termed 3D domain swapping. The structural details of the TrpA dimer have remained unknown. Here, the crystal structure of the Streptococcus pneumoniae TrpA homodimer is reported, demonstrating 3D domain swapping in a TIM-barrel fold for the first time. The N-terminal domain comprising the H0-S1-H1-S2 elements is exchanged, while the hinge region corresponds to loop L2 linking strand S2 to helix H2'. The structural elements S2 and L2 carry the catalytic residues Glu52 and Asp63. As the S2 element is part of the swapped domain, the architecture of the catalytic apparatus in the dimer is recreated from two protein chains. The homodimer interface overlaps with the α-ß interface of the tryptophan synthase αßßα heterotetramer, suggesting that the 3D domain-swapped dimer cannot form a complex with the ß subunit. In the crystal, the dimers assemble into a decamer comprising two pentameric rings.

Conformation-dependent restraints for polynucleotides: the sugar moiety.

Stereochemical restraints are commonly used to aid the refinement of macromolecular structures obtained by experimental methods at lower resolution. The standard restraint library for nucleic acids has not been updated for over two decades and needs revision. In this paper, geometrical restraints for nucleic acids sugars are derived using information from high-resolution crystal structures in the Cambridge Structural Database. In contrast to the existing restraints, this work shows that different parts of the sugar moiety form groups of covalent geometry dependent on various chemical and conformational factors, such as the type of ribose or the attached nucleobase, and ring puckering or rotamers of the glycosidic (χ) or side-chain (γ) torsion angles. Moreover, the geometry of the glycosidic link and the endocyclic ribose bond angles are functionally dependent on χ and sugar pucker amplitude (τm), respectively. The proposed restraints have been positively validated against data from the Nucleic Acid Database, compared with an ultrahigh-resolution Z-DNA structure in the Protein Data Bank, and tested by re-refining hundreds of crystal structures in the Protein Data Bank. The conformation-dependent sugar restraints presented in this work are publicly available in REFMAC, PHENIX and SHELXL format through a dedicated RestraintLib web server with an API function.

Comparison of a retroviral protease in monomeric and dimeric states.

Retroviral proteases (RPs) are of high interest owing to their crucial role in the maturation process of retroviral particles. RPs are obligatory homodimers, with a pepsin-like active site built around two aspartates (in DTG triads) that activate a water molecule, as the nucleophile, under two flap loops. Mason-Pfizer monkey virus (M-PMV) is unique among retroviruses as its protease is also stable in the monomeric form, as confirmed by an existing crystal structure of a 13 kDa variant of the protein (M-PMV PR) and its previous biochemical characterization. In the present work, two mutants of M-PMV PR, D26N and C7A/D26N/C106A, were crystallized in complex with a peptidomimetic inhibitor and one mutant (D26N) was crystallized without the inhibitor. The crystal structures were solved at resolutions of 1.6, 1.9 and 2.0 Å, respectively. At variance with the previous study, all of the new structures have the canonical dimeric form of retroviral proteases. The protomers within a dimer differ mainly in the flap-loop region, with the most extreme case observed in the apo structure, in which one flap loop is well defined while the other flap loop is not defined by electron density. The presence of the inhibitor molecules in the complex structures was assessed using polder maps, but some details of their conformations remain ambiguous. In all of the presented structures the active site contains a water molecule buried deeply between the Asn26-Thr27-Gly28 triads of the protomers. Such a water molecule is completely unique not only in retropepsins but also in aspartic proteases in general. The C7A and C106A mutations do not influence the conformation of the protein. The Cys106 residue is properly placed at the homodimer interface area for a disulfide cross-link, but the reducing conditions of the crystallization experiment prevented S-S bond formation. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Acta_Cryst_D:S2059798319011355.

Accurate geometrical restraints for Watson-Crick base pairs.

Geometrical restraints provide key structural information for the determination of biomolecular structures at lower resolution by experimental methods such as crystallography or cryo-electron microscopy. In this work, restraint targets for nucleic acids bases are derived from three different sources and compared: small-molecule crystal structures in the Cambridge Structural Database (CSD), ultrahigh-resolution structures in the Protein Data Bank (PDB) and quantum-mechanical (QM) calculations. The best parameters are those based on CSD structures. After over two decades, the standard library of Parkinson et al. [(1996), Acta Cryst. D52, 57-64] is still valid, but improvements are possible with the use of the current CSD database. The CSD-derived geometry is fully compatible with Watson-Crick base pairs, as comparisons with QM results for isolated and paired bases clearly show that the CSD targets closely correspond to proper base pairing. While the QM results are capable of distinguishing between single and paired bases, their level of accuracy is, on average, nearly two times lower than for the CSD-derived targets when gauged by root-mean-square deviations from ultrahigh-resolution structures in the PDB. Nevertheless, the accuracy of QM results appears sufficient to provide stereochemical targets for synthetic base pairs where no reliable experimental structural information is available. To enable future tests for this approach, QM calculations are provided for isocytosine, isoguanine and the iCiG base pair.

Self-Assembly and Ordering of Peptide-Based Cavitands in Water and DMSO: The Power of Hydrophobic Effects Combined with Neutral Hydrogen Bonds.

Directional self-assembly of uncharged molecules in water is a major challenge in supramolecular chemistry. Herein, it is demonstrated that peptide-based cavitands wrap around a hydrophobic core (fullerene C60 ) by a combination of the hydrophobic effect and hydrogen-bonding interactions to form highly ordered three-component complexes in water that resemble the molten-globule stage of protein folding. The complexes were characterized by DOSY NMR spectroscopy, small-angle X-ray scattering, and circular dichroism, and their structures were confirmed by X-ray crystallography. Enhancement of the CD signals by nearly one order of magnitude and increased hydrolytic stability of hydrazone bonds of the complexes relative to the nonassembled species were observed. In contrast, DMSO and DMSO/water mixtures were found to be highly disintegrative for these complexes. Interestingly, some cavitands can only be synthesized in the presence of the hydrophobic template followed by disassembly of the complexes.

Health hazards related to conidia of Cladosporium-biological air pollutants in Poland, central Europe.

The spores of Cladosporium Link. are often present in the air in high quantities and produce many allergenic proteins, which may lead to asthma. An aerobiological spore monitoring program can inform patients about the current spore concentration in air and help their physicians determine the spore dose that is harmful for a given individual. This makes it possible to develop optimized responses and propose personalized therapy for a particular sensitive patient. The aim of this study was to assess the extent of the human health hazard posed by the fungal genus Cladosporium. For the first time, we have determined the number of days on which air samples in Poland exceeded the concentrations linked to allergic responses of sensitive patients, according to thresholds established by three different groups (2800/3000/4000 spores per 1m3 of the air). The survey was conducted over three consecutive growing seasons (April-September, 2010-2012) in three cities located in different climate zones of Poland (Poznan, Lublin and Rzeszow). The average number of days exceeding 2800 spores per cubic meter (the lowest threshold) ranged from 61 (2010) through 76 (2011) to 93 (2012), though there was significant variation between cities. In each year the highest concentration of spores in the air was detected in either Poznan or Lublin, both located on large plains with intensive agriculture. We have proposed that an effective, science-based software platform to support policy-making on air quality should incorporate biological air pollutant data, such as allergenic fungal spores and pollen grains.