A closer look at molecular mechanisms underlying inhibition of S-adenosyl-L-homocysteine hydrolase by transition metal cations.

We report biochemical and structural studies on inhibiting bacterial S-adenosyl-L-homocysteine hydrolase by transition metal cations. Our results revealed diverse molecular mechanisms of enzyme inactivation. Depending on the cation, the mechanism is based on arresting the enzyme in its closed, inactive conformation, disulfide bond formation within the active site or oxidation of the intermediate form of a cofactor.

Legume-type glutamate dehydrogenase: Structure, activity, and inhibition studies.

Glutamate dehydrogenases (GDHs) are key enzymes at the crossroads of N and C metabolism in plants. Legumes, whose N metabolism is particularly intricate, possess a unique type of GDH. This study presents an analysis of a legume-type GDH (isoform 2) from Medicago truncatula (MtGDH2). We measured MtGDH2 activity in both the Glu â†’ 2-oxoglutarate (2OG) and 2OG â†’ Glu reaction directions and obtained kinetic parameters for Glu, 2OG, NAD+, and NADH. Inhibition assays revealed that compounds possessing di- or tricarboxylates act as inhibitors of plant GDHs. Interestingly, 2,6-pyridinedicarboxylate (PYR) weakly inhibits MtGDH2 compared to Arabidopsis thaliana homologs. Furthermore, we explored tetrazole derivatives to discover 3-(1H-tetrazol-5-yl)benzoic acid (TBA) as an MtGDH2 inhibitor. The kinetic experiments are supported by six crystal structures, solved as: (i) unliganded enzyme, (ii) trapping the reaction intermediate 2-amino-2-hydroxyglutarate and NAD+, and also complexed with NAD+ and inhibitors such as (iii) citrate, (iv) PYR, (v) isophthalate, and (vi) TBA. The complex with TBA revealed a new mode of action that, in contrast to other inhibitors, prevents domain closure. This discovery points to TBA as a starting point for the development of novel GDH inhibitors to study the functions of GDH in plants and potentially boost biomass production.

Microcalorimetry as a tool in enzymatic kinetics research / Mikrokalorymetria jako narzedzie w badaniach kinetyki enzymatycznej.

Enzymes, as biocatalysts, are an important target of many therapies and are also of great industrial importance, which is why repeatable and accurate parameterization of enzymatic catalysis is very important. The most popular spectrophotometric detection method in enzymology, despite its low cost and speed, often cannot be used directly due to the inappropriate spectral properties of substrates and products. It is then necessary to use auxiliary enzymes, chemical modification of substrates or post-reaction analysis, which may increase the cost of measurement, extend its time or affect the accuracy. Isothermal titration calorimetry is a method widely used mainly for the characterization of inter-molecular interactions, however, its use in enzyme kinetics is gaining more and more recognition due to the direct measurement of the reaction rate using the universal parameter of heat, high sensitivity and low reagent consumption. This work discusses two strategies for conducting a kinetic calorimetric experiment and their applications.

Substrate Affinity Is Not Crucial for Therapeutic L-Asparaginases: Antileukemic Activity of Novel Bacterial Enzymes.

L-asparaginases are used in the treatment of acute lymphoblastic leukemia. The aim of this work was to compare the antiproliferative potential and proapoptotic properties of novel L-asparaginases from different structural classes, viz. EcAIII and KpAIII (class 2), as well as ReAIV and ReAV (class 3). The EcAII (class 1) enzyme served as a reference. The proapoptotic and antiproliferative effects were tested using four human leukemia cell models: MOLT-4, RAJI, THP-1, and HL-60. The antiproliferative assay with the MOLT-4 cell line indicated the inhibitory properties of all tested L-asparaginases. The results from the THP-1 cell models showed a similar antiproliferative effect in the presence of EcAII, EcAIII, and KpAIII. In the case of HL-60 cells, the inhibition of proliferation was observed in the presence of EcAII and KpAIII, whereas the proliferation of RAJI cells was inhibited only by EcAII. The results of the proapoptotic assays showed individual effects of the enzymes toward specific cell lines, suggesting a selective (time-dependent and dose-dependent) action of the tested L-asparaginases. We have, thus, demonstrated that novel L-asparaginases, with a lower substrate affinity than EcAII, also exhibit significant antileukemic properties in vitro, which makes them interesting new drug candidates for the treatment of hematological malignancies. For all enzymes, the kinetic parameters (Km and kcat) and thermal stability (Tm) were determined. Structural and catalytic properties of L-asparaginases from different classes are also summarized.

Antisense RNA C9orf72 hexanucleotide repeat associated with amyotrophic lateral sclerosis and frontotemporal dementia forms a triplex-like structure and binds small synthetic ligand.

The abnormal expansion of GGGGCC/GGCCCC hexanucleotide repeats (HR) in C9orf72 is associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Structural polymorphisms of HR result in the multifactorial pathomechanism of ALS/FTD. Consequently, many ongoing studies are focused at developing therapies targeting pathogenic HR RNA. One of them involves small molecules blocking sequestration of important proteins, preventing formation of toxic nuclear foci. However, rational design of potential therapeutics is hindered by limited number of structural studies of RNA-ligand complexes. We determined the crystal structure of antisense HR RNA in complex with ANP77 ligand (1.1 Šresolution) and in the free form (0.92 and 1.5 Šresolution). HR RNA folds into a triplex structure composed of four RNA chains. ANP77 interacted with two neighboring single-stranded cytosines to form pseudo-canonical base pairs by adopting sandwich-like conformation and adjusting the position of its naphthyridine units to the helical twist of the RNA. In the unliganded structure, the cytosines formed a peculiar triplex i-motif, assembled by trans C•C+ pair and a third cytosine located at the Hoogsteen edge of the C•C+ pair. These results extend our knowledge of the structural polymorphisms of HR and can be used for rational design of small molecules targeting disease-related RNAs.

Targeting imidazole-glycerol phosphate dehydratase in plants: novel approach for structural and functional studies, and inhibitor blueprinting.

The histidine biosynthetic pathway (HBP) is targeted for herbicide design with preliminary success only regarding imidazole-glycerol phosphate dehydratase (IGPD, EC 4.2.1.19), or HISN5, as referred to in plants. HISN5 catalyzes the sixth step of the HBP, in which imidazole-glycerol phosphate (IGP) is dehydrated to imidazole-acetol phosphate. In this work, we present high-resolution cryoEM and crystal structures of Medicago truncatula HISN5 (MtHISN5) in complexes with an inactive IGP diastereoisomer and with various other ligands. MtHISN5 can serve as a new model for plant HISN5 structural studies, as it enables resolving protein-ligand interactions at high (2.2 Å) resolution using cryoEM. We identified ligand-binding hotspots and characterized the features of plant HISN5 enzymes in the context of the HISN5-targeted inhibitor design. Virtual screening performed against millions of small molecules not only revealed candidate molecules but also identified linkers for fragments that were experimentally confirmed to bind. Based on experimental and computational approaches, this study provides guidelines for designing symmetric HISN5 inhibitors that can reach two neighboring active sites. Finally, we conducted analyses of sequence similarity networks revealing that plant HISN5 enzymes derive from cyanobacteria. We also adopted a new approach to measure MtHISN5 enzymatic activity using isothermal titration calorimetry and enzymatically synthesized IGP.

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.

Biochemical characterization of L-asparaginase isoforms from Rhizobium etli-the boosting effect of zinc.

L-Asparaginases, divided into three structural Classes, catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. The members of Class 3, ReAIV and ReAV, encoded in the genome of the nitrogen fixing Rhizobium etli, have the same fold, active site, and quaternary structure, despite low sequence identity. In the present work we examined the biochemical consequences of this difference. ReAIV is almost twice as efficient as ReAV in asparagine hydrolysis at 37°C, with the kinetic KM, kcat parameters (measured in optimal buffering agent) of 1.5 mM, 770 s-1 and 2.1 mM, 603 s-1, respectively. The activity of ReAIV has a temperature optimum at 45°C-55°C, whereas the activity of ReAV, after reaching its optimum at 37°C, decreases dramatically at 45°C. The activity of both isoforms is boosted by 32 or 56%, by low and optimal concentration of zinc, which is bound three times more strongly by ReAIV then by ReAV, as reflected by the KD values of 1.2 and 3.3 µM, respectively. We also demonstrate that perturbation of zinc binding by Lys→Ala point mutagenesis drastically decreases the enzyme activity but also changes the mode of response to zinc. We also examined the impact of different divalent cations on the activity, kinetics, and stability of both isoforms. It appeared that Ni2+, Cu2+, Hg2+, and Cd2+ have the potential to inhibit both isoforms in the following order (from the strongest to weakest inhibitors) Hg2+ > Cu2+ > Cd2+ > Ni2+. ReAIV is more sensitive to Cu2+ and Cd2+, while ReAV is more sensitive to Hg2+ and Ni2+, as revealed by IC50 values, melting scans, and influence on substrate specificity. Low concentration of Cd2+ improves substrate specificity of both isoforms, suggesting its role in substrate recognition. The same observation was made for Hg2+ in the case of ReAIV. The activity of the ReAV isoform is less sensitive to Cl- anions, as reflected by the IC50 value for NaCl, which is eightfold higher for ReAV relative to ReAIV. The uncovered complementary properties of the two isoforms help us better understand the inducibility of the ReAV enzyme.

Structural and functional studies of Arabidopsis thaliana glutamate dehydrogenase isoform 2 demonstrate enzyme dynamics and identify its calcium binding site.

Glutamate dehydrogenase (GDH) is an enzyme at the crossroad of plant nitrogen and carbon metabolism. GDH catalyzes the conversion of 2-oxoglutarate into glutamate (2OG → Glu), utilizing ammonia as cosubstrate and NADH as coenzyme. The GDH reaction is reversible, meaning that the NAD+-dependent reaction (Glu → 2OG) releases ammonia. In Arabidopsis thaliana, three GDH isoforms exist, AtGDH1, AtGDH2, and AtGDH3. The subject of this work is AtGDH2. Previous reports have suggested that enzymes homologous to AtGDH2 contain a calcium-binding EF-hand motif located in the coenzyme binding domain. Here, we show that while AtGDH2 indeed does bind calcium, the binding occurs elsewhere and the region predicted to be the EF-hand motif has a completely different structure. As the true calcium binding site is > 20 Å away from the active site, it seems to play a structural, rather than catalytic role. We also performed comparative kinetic characterization of AtGDH1 and AtGDH2 using spectroscopic methods and isothermal titration calorimetry, to note that the isoenzymes generally exhibit similar behavior, with calcium having only a minor effect. However, the spatial and temporal changes in the gene expression profiles of the three AtGDH genes point to AtGDH2 as the most prevalent isoform.

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.

Structural, Thermodynamic and Enzymatic Characterization of N,N-Diacetylchitobiose Deacetylase from Pyrococcus chitonophagus.

Chitin is a major source of energy and macroelements for many organisms. An important step in its degradation is the deacetylation of chitin or its fragments. Deacetylase from the extremophile Pyrococcus chitonophagus has been analyzed by X-ray crystallography, small-angle X-ray scattering, differential scanning calorimetry, isothermal titration calorimetry and NMR to determine its structure, thermodynamics and enzymatic properties. It is a hexameric, zinc-containing metalloenzyme that retains its structural integrity up to temperatures slightly exceeding 100 °C. It removes the acetyl group specifically from the non-reducing end of the sugar substrate. Its main substrate is N,N-diacetylchitobiose but it also active, at a reduced level, toward N-acetyl-d-glucosamine or a trimer of N-acetyl-d-glucosamine units. Crystallographic analysis includes the structure of the enzyme with its main substrate approaching the active site in a monodentate manner, replacing the single water molecule that is bound at the Zn2+ cation when the ligand is absent. The Zn2+ cation remains tetrahedrally coordinated, with three of its ligands provided by the protein's conserved His-Asp-His triad. The crystal structures are consistent with the reaction mechanism proceeding via an anhydride intermediate. Hydrolysis as the first step cannot be ruled out in a hydrated environment but no defined 'hydrolytic water' site can be identified in the analyzed structures.

Biochemical and structural insights into an unusual, alkali-metal-independent S-adenosyl-L-homocysteine hydrolase from Synechocystis sp. PCC 6803.

The mesophilic cyanobacterium Synechocystis sp. PCC 6803 encodes an S-adenosyl-L-homocysteine hydrolase (SAHase) of archaeal origin in its genome. SAHases are essential enzymes involved in the regulation of cellular S-adenosyl-L-methionine (SAM)-dependent methylation reactions. They are usually active as homotetramers or, less commonly, as homodimers. A SAHase subunit is composed of two major domains: a cofactor (NAD+)-binding domain and a substrate (S-adenosyl-L-homocysteine)-binding domain. These are connected by a hinge element that is also a coordination site for an alkali-metal cation that influences domain movement during the catalytic cycle. Typically, the highest activity and strongest substrate binding of bacterial SAHases are observed in the presence of K+ ions. The SAHase from Synechocystis (SynSAHase) is an exception in this respect. Enzymatic and isothermal titration calorimetry studies demonstrated that in contrast to K+-dependent SAHases, the activity and ligand binding of SynSAHase are not affected by the presence of any particular alkali ion. Moreover, in contrast to other SAHases, the cyanobacterial enzyme is in an equilibrium of two distinct oligomeric states corresponding to its dimeric and tetrameric forms in solution. To explain these phenomena, crystal structures of SynSAHase were determined for the enzyme crystallized in the presence of adenosine (a reaction byproduct or substrate) and sodium or rubidium cations. The structural data confirm that while SynSAHase shares common structural features with other SAHases, no alkali metal is coordinated by the cyanobacterial enzyme as a result of a different organization of the macromolecular environment of the site that is normally supposed to coordinate the metal cation. This inspired the generation of SynSAHase mutants that bind alkali-metal cations analogously to K+-dependent SAHases, as confirmed by crystallographic studies. Structural comparisons of the crystal structure of SynSAHase with other experimental models of SAHases suggest a possible explanation for the occurrence of the cyanobacterial enzyme in the tetrameric state. On the other hand, the reason for the existence of SynSAHase in the dimeric state in solution remains elusive.

New aspects of DNA recognition by group II WRKY transcription factor revealed by structural and functional study of AtWRKY18 DNA binding domain.

WRKY transcription factors (TFs) constitute one of the largest families of plant TFs. Based on the organization of domains and motifs, WRKY TFs are divided into three Groups (I-III). The WRKY subgroup IIa includes three representatives in A. thaliana, AtWRKY18, AtWRKY40, and AtWRKY60, that participate in biotic and abiotic stress responses. Here we present crystal structures of the DNA binding domain (DBD) of AtWRKY18 alone and in the complex with a DNA duplex containing the WRKY-recognition sequence, W-box. Subgroup IIa WRKY TFs are known to form homo and heterodimers. Our data suggest that the dimerization interface of the full-length AtWRKY18 involves contacts between the DBD subunits. DNA binding experiments and structural analysis point out novel aspects of DNA recognition by WRKY TFs. In particular, AtWRKY18-DBD preferentially binds an overlapping tandem of W-boxes accompanied by a quasi-W-box motif. The binding of DNA deforms the B-type double helix, which suggests that the DNA fragment must be prone to form a specific structure. This can explain why despite the short W-box consensus, WRKY TFs can precisely control gene expression. Finally, this first experimental structure of a Group II WRKY TF allowed us to compare Group I-III representatives.

New ligand-binding sites identified in the crystal structures of ß-lactoglobulin complexes with desipramine.

The homodimeric ß-lactoglobulin belongs to the lipocalin family of proteins that transport a wide range of hydrophobic molecules and can be modified by mutagenesis to develop specificity for novel groups of ligands. In this work, new lactoglobulin variants, FAF (I56F/L39A/M107F) and FAW (I56F/L39A/M107W), were produced and their interactions with the tricyclic drug desipramine (DSM) were studied using X-ray crystallography, calorimetry (ITC) and circular dichroism (CD). The ITC and CD data showed micromolar affinity of the mutants for DSM and interactions according to the classical one-site binding model. However, the crystal structures unambiguously showed that the FAF and FAW dimers are capable of binding DSM not only inside the ß-barrel as expected, but also at the dimer interface and at the entrance to the binding pocket. The presented high-resolution crystal structures therefore provide important evidence of the existence of alternative ligand-binding sites in the ß-lactoglobulin molecule. Analysis of the crystal structures highlighted the importance of shape complementarity for ligand recognition and selectivity. The binding sites identified in the crystal structures of the FAF-DSM and FAW-DSM complexes together with data from the existing literature are used to establish a systematic classification of the ligand-binding sites in the ß-lactoglobulin molecule.

The Study of Protein-Cyclitol Interactions.

Investigation of interactions between the target protein molecule and ligand allows for an understanding of the nature of the molecular recognition, functions, and biological activity of protein-ligand complexation. In the present work, non-specific interactions between a model protein (Bovine Serum Albumin) and four cyclitols were investigated. D-sorbitol and adonitol represent the group of linear-structure cyclitols, while shikimic acid and D-(-)-quinic acid have cyclic-structure molecules. Various analytical methods, including chromatographic analysis (HPLC-MS/MS), electrophoretic analysis (SDS-PAGE), spectroscopic analysis (spectrofluorimetry, Fourier transform infrared spectroscopy, and Raman spectroscopy), and isothermal titration calorimetry (ITC), were applied for the description of protein-cyclitol interactions. Additionally, computational calculations were performed to predict the possible binding places. Kinetic studies allowed us to clarify interaction mechanisms that may take place during BSA and cyclitol interaction. The results allow us, among other things, to evaluate the impact of the cyclitol's structure on the character of its interactions with the protein.

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.

Structural and mechanistic insights into the bifunctional HISN2 enzyme catalyzing the second and third steps of histidine biosynthesis in plants.

The second and third steps of the histidine biosynthetic pathway (HBP) in plants are catalyzed by a bifunctional enzyme-HISN2. The enzyme consists of two distinct domains, active respectively as a phosphoribosyl-AMP cyclohydrolase (PRA-CH) and phosphoribosyl-ATP pyrophosphatase (PRA-PH). The domains are analogous to single-domain enzymes encoded by bacterial hisI and hisE genes, respectively. The calculated sequence similarity networks between HISN2 analogs from prokaryotes and eukaryotes suggest that the plant enzymes are closest relatives of those in the class of Deltaproteobacteria. In this work, we obtained crystal structures of HISN2 enzyme from Medicago truncatula (MtHISN2) and described its architecture and interactions with AMP. The AMP molecule bound to the PRA-PH domain shows positioning of the N1-phosphoribosyl relevant to catalysis. AMP bound to the PRA-CH domain mimics a part of the substrate, giving insights into the reaction mechanism. The latter interaction also arises as a possible second-tier regulatory mechanism of the HBP flux, as indicated by inhibition assays and isothermal titration calorimetry.

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

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.

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

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 AtGDH1 isozyme from A. thaliana is NAD+-specific. Altogether, A. thaliana expresses three GDH isozymes (AtGDH1-3) targeted to mitochondria, of which AtGDH2 has an extra EF-hand motif and is stimulated by calcium. Our enzymatic assays of AtGDH1 established that its sensitivity to calcium is negligible. In vivo the AtGDH1-3 enzymes form homo- and heterohexamers of varied composition. We solved the crystal structure of recombinant AtGDH1 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 D 3-symmetric homohexamers. A subunit of AtGDH1 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 AtGDH1-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 AtGDH1 is insensitive to MPD in our assays, the observation of druggable sites opens a potential for non-competitive herbicide design.

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.