Publications by authors named "Mariusz Jaskolski"

160 Publications

Dr. Alexander Wlodawer-celebrating five decades of service to the structural biology community.

FEBS J 2021 07;288(14):4160-4164

Center for Structural Biology, National Cancer Institute, Frederick, MD, USA.

This 75th birthday tribute to our Editorial Board member Alexander Wlodawer recounts his decades-long service to the community of structural biology researchers. His former and current colleagues tell the story of his upbringing and education, followed by an account of his dedication to quality and rigor in crystallography and structural science. The FEBS Journal Editor-in-Chief Seamus Martin further highlights Alex's outstanding contributions to the journal's success over many years.
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http://dx.doi.org/10.1111/febs.16064DOI Listing
July 2021

Structural and biophysical aspects of l-asparaginases: a growing family with amazing diversity.

IUCrJ 2021 Jul 30;8(Pt 4):514-531. Epub 2021 Jun 30.

Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland.

l-Asparaginases have remained an intriguing research topic since their discovery ∼120 years ago, especially after their introduction in the 1960s as very efficient antileukemic drugs. In addition to bacterial asparaginases, which are still used to treat childhood leukemia, enzymes of plant and mammalian origin are now also known. They have all been structurally characterized by crystallography, in some cases at outstanding resolution. The structural data have also shed light on the mechanistic details of these deceptively simple enzymes. Yet, despite all this progress, no better therapeutic agents have been found to beat bacterial asparaginases. However, a new option might arise with the discovery of yet another type of asparaginase, those from symbiotic nitrogen-fixing Rhizobia, and with progress in the protein engineering of enzymes with desired properties. This review surveys the field of structural biology of l-asparaginases, focusing on the mechanistic aspects of the well established types and speculating about the potential of the new members of this amazingly diversified family.
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http://dx.doi.org/10.1107/S2052252521006011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8256714PMC
July 2021

A topological proof of the modified Euler characteristic based on the orbifold concept.

Acta Crystallogr A Found Adv 2021 Jul 21;77(Pt 4):317-326. Epub 2021 Jun 21.

Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznań, Poland.

The notion of the Euler characteristic of a polyhedron or tessellation has been the subject of in-depth investigations by many authors. Two previous papers worked to explain the phenomenon of the vanishing (or zeroing) of the modified Euler characteristic of a polyhedron that underlies a periodic tessellation of a space under a crystallographic space group. The present paper formally expresses this phenomenon as a theorem about the vanishing of the Euler characteristic of certain topological spaces called topological orbifolds. In this new approach, it is explained that the theorem in question follows from the fundamental properties of the orbifold Euler characteristic. As a side effect of these considerations, a theorem due to Coxeter about the vanishing Euler characteristic of a honeycomb tessellation is re-proved in a context which frees the calculations from the assumptions made by Coxeter in his proof. The abstract mathematical concepts are visualized with down-to-earth examples motivated by concrete situations illustrating wallpaper and 3D crystallographic space groups. In a way analogous to the application of the classic Euler equation to completely bounded solids, the formula proven in this paper is applicable to such important crystallographic objects as asymmetric units and Dirichlet domains.
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http://dx.doi.org/10.1107/S2053273321004320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8248890PMC
July 2021

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

Acta Crystallogr B Struct Sci Cryst Eng Mater 2021 Jun 6;77(Pt 3):331-338. Epub 2021 May 6.

Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

The X-ray crystal structure of the d(CGCGCG)/putrescine(2+)/K complex has been determined at 0.60 Å resolution. 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 Mn and Zn 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.
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http://dx.doi.org/10.1107/S2052520621002663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8182800PMC
June 2021

Rapid response to emerging biomedical challenges and threats.

IUCrJ 2021 May 26;8(Pt 3):395-407. Epub 2021 Mar 26.

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, USA.

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.
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http://dx.doi.org/10.1107/S2052252521003018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8086160PMC
May 2021

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

Protein Sci 2021 Jun 8;30(6):1258-1263. Epub 2021 Apr 8.

Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

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 P2 crystal structures and is similar to a rarely occurring system in HIV-1 PR.
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http://dx.doi.org/10.1002/pro.4072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138519PMC
June 2021

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

IUCrJ 2021 Mar 9;8(Pt 2):238-256. Epub 2021 Feb 9.

Center for Structural Biology, National Cancer Institute, Frederick, MD 21702, USA.

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 3CL, 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.
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http://dx.doi.org/10.1107/S2052252521001159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924243PMC
March 2021

Arithmetic proof of the multiplicity-weighted Euler characteristic for symmetrically arranged space-filling polyhedra.

Acta Crystallogr A Found Adv 2021 Mar 4;77(Pt 2):126-129. Epub 2021 Feb 4.

Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland.

The puzzling observation that the famous Euler's formula for three-dimensional polyhedra V - E + F = 2 or Euler characteristic χ = V - E + F - I = 1 (where V, E, F are the numbers of the bounding vertices, edges and faces, respectively, and I = 1 counts the single solid itself) when applied to space-filling solids, such as crystallographic asymmetric units or Dirichlet domains, are modified in such a way that they sum up to a value one unit smaller (i.e. to 1 or 0, respectively) is herewith given general validity. The proof provided in this paper for the modified Euler characteristic, χ = V - E + F - I = 0, is divided into two parts. First, it is demonstrated for translational lattices by using a simple argument based on parity groups of integer-indexed elements of the lattice. Next, Whitehead's theorem, about the invariance of the Euler characteristic, is used to extend the argument from the unit cell to its asymmetric unit components.
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http://dx.doi.org/10.1107/S2053273320016186DOI Listing
March 2021

Sensitized photo-oxidation of plant cytokinin-specific binding protein - Does the environment of the thioether group influence the oxidation reaction? From primary intermediates to stable products.

Free Radic Biol Med 2021 03 10;165:411-420. Epub 2021 Feb 10.

Faculty of Chemistry, Adam Mickiewicz University, And Center for Advanced Technology, Poznan, Poland.

The reactions of protein oxidation play a significant role in many biological processes, especially in diseases development. Therefore, it is important to understand, how the protein molecule behaves in the presence of oxidants. In the present work, photo-oxidation of phytohormone-binding plant protein (VrPhBP) was investigated using light and 3-carboxybenzophenone (3CB) as a sensitizer (one electron oxidant). The protein interacts with the sensitizer in the ground state forming a weak binding complex leading to the presence of bound and free 3CB in solution. The early events and transient species (such as radicals and radical ions) formed during irradiation were characterised by transient spectroscopy showing the formation of the sulphur radical cation Met>S (stabilized by (S∴N))and the tyrosyl radical TyrO on VrPhBP. Thus the 3CB excited triplet state was quenched by the Met and Tyr residues and mostly by Met (based on the deconvoluted transient absorption spectra).The presence of a Tyr side chain in the vicinity of a Met residue results in intramolecular electron transfer from Tyr to the Met>S radical cation, leading to regeneration of the thioether side chain and formation of TyrO. The presence of other side chains close to Met, such as Arg or Lys can induce the stabilization of Met>S via the formation of two-centered three-electron bonded species (S∴N). The transient species were additionally confirmed by stable product analysis. Based on SDS-PAGE, chromatography and mass spectrometry, the formation of methionine sulphoxide and Met-3CB adduct was identified together with di-Tyr cross links. On the basis of the experimental results the overall mechanism of VrPhBP photo-oxidation, from its early events to the formation of stable products, is described. In addition, a good correlation between the mechanisms of photooxidation of model compounds such as Met derivatives and peptides and those for real biological systems is emphasized.
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http://dx.doi.org/10.1016/j.freeradbiomed.2021.02.004DOI Listing
March 2021

Structural studies of human muscle FBPase.

Acta Biochim Pol 2021 Jan;68(1):5-14

1Institute of Genetics and Microbiology, Wroclaw University, Wrocław, Poland; 2Klodzko School of Medicine, Kłodzko, Poland.

Muscle fructose-1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose-1,6-bisphosphate (F1,6BP) to fructose-6-phosphate (F6P) and inorganic phosphate, regulates glucose homeostasis by controlling the glyconeogenic pathway. FBPase requires divalent cations, such as Mg2+, Mn2+, or Zn2+, for its catalytic activity; however, calcium ions inhibit the muscle isoform of FBPase by interrupting the movement of the catalytic loop. It has been shown that residue E69 in this loop plays a key role in the sensitivity of muscle FBPase towards calcium ions. The study presented here is based on five crystal structures of wild-type human muscle FBPase and its E69Q mutant in complexes with the substrate and product of the enzymatic reaction, namely F1,6BP and F6P. The ligands are bound in the active site of the studied proteins in the same manner and have excellent definition in the electron density maps. In all studied crystals, the homotetrameric enzyme assumes the same cruciform quaternary structure, with the κ angle, which describes the orientation of the upper dimer with respect to the lower dimer, of -85o. This unusual quaternary arrangement of the subunits, characteristic of the R-state of muscle FBPase, is also observed in solution by small-angle X-ray scattering (SAXS).
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http://dx.doi.org/10.18388/abp.2020_5554DOI Listing
January 2021

Serendipitous crystallization of E. coli HPII catalase, a sequel to "the tale usually not told".

Acta Biochim Pol 2021 Jan;68(1):29-31

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland.

Protein crystallographers are well aware of the trap of crystallizing E. coli proteins instead of the macromolecule of interest if heterologous recombinant protein expression in E. coli was part of the experimental pipeline. Among the well-known culprits are YodA metal-binding lipocalin (25 kDa) and YadF carbonic anhydrase (a tetramer of 25 kDa subunits). We report a novel crystal form of another such culprit, E. coli HPII catalase, which is a tetrameric protein of ~340 kDa molecular weight. HPII is likely to contaminate recombinant protein samples, co-purify, and then co-crystallize with the target proteins, especially if their masses in size exclusion chromatography are ~300-400 kDa. What makes this case more interesting but also parlous, is the fact that HPII can crystallize from very low concentrations, even well below 1 mg/mL.
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http://dx.doi.org/10.18388/abp.2020_5501DOI Listing
January 2021

Celebrating the 75th birthday of Professor Wladek Minor, one of the most accomplished Polish-American structural biologists.

Acta Biochim Pol 2021 Jan;68(1):1-4

Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC, U.S.A.

This paper describes the scientific career and accomplishments of Professor Wladek Minor, who holds an endowed chair at the University of Virginia in Charlottesville, USA. Prof. Minor is a coauthor of data processing software used by macromolecular crystallographers world-wide, as well as of structural biology servers and of a repository for raw diffraction data. He made major contributions to the validation of biostructural data, with special focus on drug design targets and reproducibility in biomedical research. He is among the most highly cited molecular biologists ever.
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http://dx.doi.org/10.18388/abp.2020_5539DOI Listing
January 2021

In-vitro anti-fungal assay and association analysis reveal a role for the PR10 gene (PmPR10-3.1) in quantitative disease resistance to white pine blister rust.

Genome 2021 Jul 19;64(7):693-704. Epub 2021 Jan 19.

United States Department of Agriculture Forest Service, Dorena Genetic Resource Center, Cottage Grove, OR, USA.

Pathogenesis-related (PR) proteins play important roles in plant defense response. However, functional investigation of PR10 genes is still limited and their physiological roles have not been conclusively characterized in biological processes of conifer trees. Here, we identified multiple novel members in the western white pine () PmPR10 family by bioinformatic mining available transcriptomic data. Phylogenetic analysis of protein sequences revealed four PR10 and two PR10-like clusters with a high synteny across different species of five-needle pines. Of 10 PmPR10 genes, was selected and expressed in The purified recombinant protein exhibited inhibitory effects on spore hyphal growth of fungal pathogens , , and by in-vitro anti-fungal analysis. Genetic variation analysis detected a total of 21 single nucleotide polymorphisms (SNPs) within in a collection of seed families. A nonsynonymous SNP (t178g) showed significant association with relative levels of quantitative disease resistance (QDR), explaining about 8.7% of phenotypic variation as the peak value across all SNPs. Our results provide valuable insight into the genetic architecture underlying QDR and imply that may function as an important component in conifer basal immunity for non-specific resistance to a wide spectrum of pathogens.
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http://dx.doi.org/10.1139/gen-2020-0080DOI Listing
July 2021

Strong interactions between Salp15 homologues from the tick I. ricinus and distinct types of the outer surface OspC protein from Borrelia.

Ticks Tick Borne Dis 2021 03 14;12(2):101630. Epub 2020 Dec 14.

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland. Electronic address:

Ticks belonging to the genus Ixodes are parasites feeding on vertebrate blood and vectors for many pathogenic microbes, including Borrelia burgdorferi sensu lato spirochetes, the causative agent of Lyme borreliosis. The tick saliva contains a mixture of bioactive molecules showing a wide range of properties for efficient engorgement. One of the most extensively studied components of tick saliva is a 15-kDa salivary gland protein (Salp15) from Ixodes scapularis. This multifunctional protein suppresses the immune response of hosts through pleiotropic action on a few crucial defense pathways. Salp15 and its homologue from I. ricinus Iric1 have been also shown to bind to Borrelia burgdorferi sensu stricto outer surface protein C (OspC) permitting the spirochetes to evade antibody-mediated killing in the human host. Further studies revealed that Salp15 and Iric1 protected B. burgdorferi s. s. and B. garinii expressing OspC against the complement system. OspC is the most variable protein on the outer surface of Borrelia, which in addition to Salp15 can also bind other ligands, such as plasminogen, fibrinogen, fibronectin or complement factor 4. So far several OspC variants produced by B. burgdorferi s. l. spirochetes were shown to be capable of binding Salp15 or its homologue, but the protection against borreliacidal antibodies has only been proven in the case of B. burgdorferi s. s. The question of Salp15 contribution to Borrelia survival during the infection has been comprehensively studied during the last decades. In contrast, the organization of the OspC-Salp15 complex has been poorly explored. This report describes the binding between three Salp15 homologues from the tick Ixodes ricinus (Iric1, Iric2 and Iric3) and OspC from four B. burgdorferi sensu lato strains in terms of the binding parameters, analyzed with two independent biophysical methods - Microscale thermophoresis (MST) and Biolayer interferometry (BLI). The results of both experiments show a binding constant at the nanomolar level, which indicates very strong interactions. While the Iric1-OspC binding has been reported before, we show in this study that also Iric2 and Iric3 are capable of OspC binding with high affinity. This observation suggests that these two Salp15 homologues might be used by B. burgdorferi s. l. in a way analogous to Iric1. A comparison of the results from the two methods let us propose that N-terminal immobilization of OspC significantly increases the affinity between the two proteins. Finally, our results indicate that the Iric binding site is located in close proximity of the OspC epitopes recognized by human antibodies, which may have important biological and medical implications.
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http://dx.doi.org/10.1016/j.ttbdis.2020.101630DOI Listing
March 2021

Properties of Cavities in Biological Structures-A Survey of the Protein Data Bank.

Front Mol Biosci 2020 6;7:591381. Epub 2020 Nov 6.

Institute of Physics, Polish Academy of Sciences, Warsaw, Poland.

We performed a PDB-wide survey of proteins to assess their cavity content, using the SPACEBALL algorithm to calculate the cavity volumes. In addition, we determined the hydropathy character of the cavities. We demonstrate that the cavities of most proteins are hydrophilic, but smaller proteins tend to have cavities with hydrophobic walls. We propose criteria for distinguishing between cavities and pockets, and single out proteins with the largest cavities.
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http://dx.doi.org/10.3389/fmolb.2020.591381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7677499PMC
November 2020

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

Acta Crystallogr B Struct Sci Cryst Eng Mater 2020 Oct 29;76(Pt 5):815-824. Epub 2020 Aug 29.

Department of Crystallography, Faculty of Chemistry, Adam Mickiewicz University, Poznan, 61-614, Poland.

A supramolecular complex, formed by encapsulation of C fullerene in a molecular container built from two resorcin[4]arene rims zipped together by peptidic arms hydrogen bonded into a cylindrical β-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 C 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 C 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 C spins of C in the studied complex.
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http://dx.doi.org/10.1107/S2052520620009944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586347PMC
October 2020

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

Protein Sci 2021 01 8;30(1):115-124. Epub 2020 Oct 8.

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, USA.

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.
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http://dx.doi.org/10.1002/pro.3959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7537053PMC
January 2021

Multiplicity-weighted Euler's formula for symmetrically arranged space-filling polyhedra.

Acta Crystallogr A Found Adv 2020 Sep 9;76(Pt 5):580-583. Epub 2020 Jul 9.

Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University and Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

The famous Euler's rule for three-dimensional polyhedra, F - E + V = 2 (F, E and V are the numbers of faces, edges and vertices, respectively), when extended to many tested cases of space-filling polyhedra such as the asymmetric unit (ASU), takes the form Fn - En + Vn = 1, where Fn, En and Vn enumerate the corresponding elements, normalized by their multiplicity, i.e. by the number of times they are repeated by the space-group symmetry. This modified formula holds for the ASUs of all 230 space groups and 17 two-dimensional planar groups as specified in the International Tables for Crystallography, and for a number of tested Dirichlet domains, suggesting that it may have a general character. The modification of the formula stems from the fact that in a symmetrical space-filling arrangement the polyhedra (such as the ASU) have incomplete bounding elements (faces, edges, vertices), since they are shared (in various degrees) with the space-filling neighbors.
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http://dx.doi.org/10.1107/S2053273320007093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459769PMC
September 2020

Structural Studies of Glutamate Dehydrogenase (Isoform 1) From , an Important Enzyme at the Branch-Point Between Carbon and Nitrogen Metabolism.

Front Plant Sci 2020 5;11:754. Epub 2020 Jun 5.

Center for Biocrystallographic Research Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland.

Glutamate dehydrogenase (GDH) releases ammonia in a reversible NAD(P)-dependent oxidative deamination of glutamate that yields 2-oxoglutarate (2OG). In current perception, GDH contributes to Glu homeostasis and plays a significant role at the junction of carbon and nitrogen assimilation pathways. GDHs are members of a superfamily of ELFV (Glu/Leu/Phe/Val) amino acid dehydrogenases and are subdivided into three subclasses, based on coenzyme specificity: NAD-specific, NAD/NADP dual-specific, and NADP-specific. We determined in this work that the mitochondrial GDH1 isozyme from is NAD-specific. Altogether, expresses three GDH isozymes (GDH1-3) targeted to mitochondria, of which GDH2 has an extra EF-hand motif and is stimulated by calcium. Our enzymatic assays of GDH1 established that its sensitivity to calcium is negligible. the GDH1-3 enzymes form homo- and heterohexamers of varied composition. We solved the crystal structure of recombinant GDH1 in the apo-form and in complex with NAD at 2.59 and 2.03 Å resolution, respectively. We demonstrate also that both in the apo form and in 1:1 complex with NAD, it forms -symmetric homohexamers. A subunit of GDH1 consists of domain I, which is involved in hexamer formation and substrate binding, and of domain II which binds coenzyme. Most of the subunits in our crystal structures, including those in NAD complex, are in open conformation, with domain II forming a large (albeit variable) angle with domain I. One of the subunits of the GDH1-NAD hexamer contains a serendipitous 2OG molecule in the active site, causing a dramatic (∼25°) closure of the domains. We provide convincing evidence that the N-terminal peptide preceding domain I is a mitochondrial targeting signal, with a predicted cleavage site for mitochondrial processing peptidase (MPP) at Leu17-Leu18 that is followed by an unexpected potassium coordination site (Ser27, Ile30). We also identified several MPD [(+/-)-2-methyl-2,4-pentanediol] binding sites with conserved sequence. Although GDH1 is insensitive to MPD in our assays, the observation of druggable sites opens a potential for non-competitive herbicide design.
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http://dx.doi.org/10.3389/fpls.2020.00754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326016PMC
June 2020

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.

Acta Crystallogr D Struct Biol 2020 Jul 17;76(Pt 7):653-667. Epub 2020 Jun 17.

Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

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 Å resolution 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.
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http://dx.doi.org/10.1107/S2059798320006841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336385PMC
July 2020

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

FEBS J 2020 09 24;287(17):3703-3718. Epub 2020 Jun 24.

Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland.

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.
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http://dx.doi.org/10.1111/febs.15366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7276724PMC
September 2020

Flexible loops of New Delhi metallo-β-lactamase modulate its activity towards different substrates.

Int J Biol Macromol 2020 Apr 28;158:104-115. Epub 2020 Apr 28.

Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland. Electronic address:

Two accessory loop regions that are present in numerous variants of New Delhi metallo-β-lactamases (NDM) are important for the enzymatic activity. The first one is a flexible loop L3 that is located near the active site and is thought to play an important role in the catalytic process. The second region, Ω loop is located close to a structural element that coordinates two essential zinc ions. Both loops are not involved in any specific interactions with a substrate. Herein, we investigated how the length and hydrophobicity of loop L3 influence the enzymatic activity of NDMs, by analyzing mutants of NDM-1 with various deletions/point mutations within the L3 loop. We also investigated NDM variants with sequence variations/artificial deletions within the Ω loop. For all these variants we determined kinetic parameters for the hydrolysis of ampicillin, imipenem, and a chromogenic cephalosporin (CENTA). None of the mutations in the L3 loop completely abolished the enzymatic activity of NDM-1. Our results suggest that various elements of the loop play different roles in the hydrolysis of different substrates and the flexibility of the loop seems necessary to fulfill the requirements imposed by various substrates. Deletions within the Ω loop usually enhanced the enzymatic activity, particularly for the hydrolysis of ampicillin and imipenem. However, the exact role of the Ω loop in the catalytic reaction remains unclear. In our kinetic tests, the NDM enzymes were inhibited in the β-lactamase reaction by the CENTA substrate. We also present the X-ray crystal structures of the NDM-1, NDM-9 and NDM-12 proteins.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.04.219DOI Listing
April 2020

On the evolution of the quality of macromolecular models in the PDB.

FEBS J 2020 07 20;287(13):2685-2698. Epub 2020 Apr 20.

Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

Crystallographic models of biological macromolecules have been ranked using the quality criteria associated with them in the Protein Data Bank (PDB). The outcomes of this quality analysis have been correlated with time and with the journals that published papers based on those models. The results show that the overall quality of PDB structures has substantially improved over the last ten years, but this period of progress was preceded by several years of stagnation or even depression. Moreover, the study shows that the historically observed negative correlation between journal impact and the quality of structural models presented therein seems to disappear as time progresses.
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http://dx.doi.org/10.1111/febs.15314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340579PMC
July 2020

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

Acta Crystallogr D Struct Biol 2020 Feb 31;76(Pt 2):166-175. Epub 2020 Jan 31.

Midwest Center for Structural Genomics, X-ray Science Division, Argonne National Laboratory, Argonne, IL 60439, USA.

Tryptophan synthase catalyzes the last two steps of tryptophan biosynthesis in plants, fungi and bacteria. It consists of two protein chains, designated α and β, encoded by trpA and trpB genes, that function as an αββα 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-β interface of the tryptophan synthase αββα heterotetramer, suggesting that the 3D domain-swapped dimer cannot form a complex with the β subunit. In the crystal, the dimers assemble into a decamer comprising two pentameric rings.
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http://dx.doi.org/10.1107/S2059798320000212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7008512PMC
February 2020

Conformation-dependent restraints for polynucleotides: the sugar moiety.

Nucleic Acids Res 2020 01;48(2):962-973

Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan 61-704, Poland.

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.
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http://dx.doi.org/10.1093/nar/gkz1122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954431PMC
January 2020

Comparison of a retroviral protease in monomeric and dimeric states.

Acta Crystallogr D Struct Biol 2019 Oct 20;75(Pt 10):904-917. Epub 2019 Sep 20.

Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland.

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.
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http://dx.doi.org/10.1107/S2059798319011355DOI Listing
October 2019

Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity.

Biochem J 2019 08 22;476(16):2297-2319. Epub 2019 Aug 22.

Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from (PPA1) and (PPA1) were cloned into a bacterial expression vector and the proteins were produced in cells and crystallized. In terms of their subunit fold, PPA1 and PPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast with the fungal counterparts, which are dimeric. Unexpectedly, the crystallized PPA1 and PPA1 proteins lack ∼30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. , self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31-Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic cleavage of a mitochondrial targeting peptide would change our perception of this signaling process.
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http://dx.doi.org/10.1042/BCJ20190427DOI Listing
August 2019

Accurate geometrical restraints for Watson-Crick base pairs.

Acta Crystallogr B Struct Sci Cryst Eng Mater 2019 Apr 27;75(Pt 2):235-245. Epub 2019 Mar 27.

Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, 61-614, Poland.

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.
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http://dx.doi.org/10.1107/S2052520619002002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6457083PMC
April 2019

Borrelia outer surface protein C is capable of human fibrinogen binding.

FEBS J 2019 06 23;286(12):2415-2428. Epub 2019 Mar 23.

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

Outer surface protein C (OspC) is one of the most abundant surface lipoproteins produced during early infection by the Borrelia spirochete, the causative agent of Lyme disease. The high sequence variability of the ospC gene results in the production of several and strongly divergent OspC types. One of the known roles of OspC is the recruitment of blood components, including complement regulators, to facilitate the bloodstream survival of Borrelia at an essential stage of host infection. Here, we identify and describe a new interaction between OspC and human fibrinogen. To test the ability of OspC to bind fibrinogen, we developed a microscale thermophoresis assay using four fluorescently labeled types of OspC. We show that OspC binds fibrinogen tightly, with nanomolar K , and that the binding depends on the OspC type. The binding assays combined with SAXS studies allowed us to map the OspC-binding site on the fibrinogen molecule. Spectrometric measurements of fibrinogen clotting in the presence of OspC indicate that OspC negatively influences the clot formation process. Taken together, our findings are consistent with the hypothesis that OspC interacts with blood protein partners to facilitate Borrelia spreading by the hematogenous route.
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http://dx.doi.org/10.1111/febs.14810DOI Listing
June 2019

Circadian oscillator proteins across the kingdoms of life: structural aspects.

BMC Biol 2019 02 18;17(1):13. Epub 2019 Feb 18.

Max-Planck-Institut für Pflanzenzüchtungsforschung, Cologne, Germany.

Circadian oscillators are networks of biochemical feedback loops that generate 24-hour rhythms in organisms from bacteria to animals. These periodic rhythms result from a complex interplay among clock components that are specific to the organism, but share molecular mechanisms across kingdoms. A full understanding of these processes requires detailed knowledge, not only of the biochemical properties of clock proteins and their interactions, but also of the three-dimensional structure of clockwork components. Posttranslational modifications and protein-protein interactions have become a recent focus, in particular the complex interactions mediated by the phosphorylation of clock proteins and the formation of multimeric protein complexes that regulate clock genes at transcriptional and translational levels. This review covers the structural aspects of circadian oscillators, and serves as a primer for this exciting realm of structural biology.
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http://dx.doi.org/10.1186/s12915-018-0623-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378743PMC
February 2019
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