Molecular recognition of N-acetyltryptophan enantiomers by β-cyclodextrin

The enantioselectivity of β-cyclodextrin (β-CD) towards L- and D-N-acetyltryptophan (NAcTrp) has been studied in aqueous solution and the crystalline state. NMR studies in solution show that β-CD forms complexes of very similar but not identical geometry with both L- and D-NAcTrp and exhibits stronger binding with L-NAcTrp. In the crystalline state, only β-CD-L-NAcTrp crystallizes readily from aqueous solutions as a dimeric complex (two hosts enclosing two guest molecules). In contrast, crystals of the complex β-CD-D-NAcTrp were never obtained, although numerous conditions were tried. In aqueous solution, the orientation of the guest in both complexes is different than in the β-CD-L-NAcTrp complex in the crystal. Overall, the study shows that subtle differences observed between the β-CD-L,D-NAcTrp complexes in aqueous solution are magnified at the onset of crystallization, as a consequence of accumulation of many soft host-guest interactions and of the imposed crystallographic order, thus resulting in very dissimilar propensity of each enantiomer to produce crystals with β-CD

Crystal Structure of the Monomeric Extracellular Domain of α9 Nicotinic Receptor Subunit in Complex With α-Conotoxin RgIA: Molecular Dynamics Insights Into RgIA Binding to α9α10 Nicotinic Receptors

The α9 subunit of nicotinic acetylcholine receptors (nAChRs) exists mainly in heteropentameric assemblies with α10. Accumulating data indicate the presence of three different binding sites in α9α10 nAChRs: the α9(+)/α9(−), the α9(+)/α10(−), and the α10(+)/α9(−). The major role of the principal (+) side of the extracellular domain (ECD) of α9 subunit in binding of the antagonists methyllylcaconitine and α-bungarotoxin was shown previously by the crystal structures of the monomeric α9-ECD with these molecules. Here we present the 2.26-Å resolution crystal structure of α9-ECD in complex with α-conotoxin (α-Ctx) RgIA, a potential drug for chronic pain, the first structure reported for a complex between an nAChR domain and an α-Ctx. Superposition of this structure with those of other α-Ctxs bound to the homologous pentameric acetylcholine binding proteins revealed significant similarities in the orientation of bound conotoxins, despite the monomeric state of the α9-ECD. In addition, ligand-binding studies calculated a binding affinity of RgIA to the α9-ECD at the low micromolar range. Given the high identity between α9 and α10 ECDs, particularly at their (+) sides, the presented structure was used as template for molecular dynamics simulations of the ECDs of the human α9α10 nAChR in pentameric assemblies. Our results support a favorable binding of RgIA at α9(+)/α9(−) or α10(+)/α9(−) rather than the α9(+)/α10(−) interface, in accordance with previous mutational and functional data

Molecular structures of the bacteria ferredoxins PaFd and EcFd

Crystallization, data collection and structure analysis of the two [4Fe-4S] cluster ferredoxins (Fds) from the bacteria Pseudomonas aeruginosa (PaFd) and Escherichia coli (EcFd) are described in the present Ph.D. dissertation. Ferredoxins of the particular family that exhibit the special feature of different reduction potentials of the two iron-sulfur clusters, are referred in the literature as electron transfer proteins. Crystallization experiments of both PaFd and EcFd were carried out under inert conditions using correspondingly argon and nitrogen, as proteins of this family undergo oxidation reactions leading to their denaturation. For the preliminary crystallization tests commercially available crystallization kits were used, which were then modified combining the conditions that had been successful in other ferredoxins. The X-ray data were collected using synchrotron radiation at the European Molecular Biology Laboratory Hamburg using area detectors. PaFd was crystallized in the orthorhombic crystal system, in space group P212121, unit cell dimensions 39.1, 45.1, 51.9 A and the data were collected at the resolution 1.32 A. The structure was solved by the molecular replacement method, using the Allochromatium vinosum ferredoxin as a search model. The refinement proceeded up to R = 15% and the geometrical features of the final model were within the errors found in high resolution structures. The 1.32 A resolution, which is the highest up to now for a member of this family of Fds, allowed a detailed analysis of all intramolecular interactions, as well as of the geometry of the clusters. At the final stages of the refinement the cluster atoms were refined without any restraints, in order to determine their unbiased geometry. The PaFd structure revealed important structural information that allow the comparison of this class of ferredoxins with other high resolution ferredoxin structures, as to the impact of the structure on the reduction potentials. EcFd structure belonges in the hexagonal crystal system and was crystallized in space group P62. Its crystals were merohedral twins and thus showed higher symmetry than the real one. While the crystal belonged in the point group 6, its diffraction pattern appeared the symmetry of the higher symmetry point group 622. The initial difficulties in determining the structure overcame using detwinned data, while the real space group was proved to be P62 only after the completion of the refinement. The twin crystal was comprised of two parts in percentage 55% and 45% and the reflections h, k, l and k, h, -l were related as an effect of twinning. The refinement of the structure 116 proceeded with the least squares method, assuming for the calculated structure factors the linear combination: Fc 2 = α Fc1 2 + (1 – α) Fc2 2 where α the twin fraction and Fc1, Fc2 the calculated structure factors for the two parts of the crystal. As near perfect twinning effectively halves the number of observed reflection intensities at any given resolution, the 1.65 A resolution of the EcFd data essentially has the same information content as untwinned data at much lower resolution.Στην παρούσα διατριβή περιγράφεται η κρυστάλλωση, η συλλογή κρυσταλλογραφικών µένων και η ανάλυση της δοµής πρωτεϊνών της οικογένειας των φερρεδοξινών δύο πλειάδων 4S] από τα βακτήρια Pseudomonas aeruginosa (PaFd) και Escherichia coli (EcFd). Οι φερρεδοξίνες της συγκεκριµένης οικογένειας, οι οποίες εµφανίζουν το ιδιαίτερο χαρακτηριστικό της εγάλης διαφοράς των κανονικών δυναµικών αναγωγής µεταξύ των δύο πλειάδων σιδήρου – θείου, αναφέρονται στη βιβλιογραφία ως πρωτεΐνες µεταφοράς ηλεκτρονίων. Η κρυστάλλωση των PaFd και EcFd πραγµατοποιήθηκε υπό αδρανείς συνθήκες αργού και αζώτου αντίστοιχα, λόγω της ευαισθησίας τους κατά την έκθεση τους στο ατµοσφαιρικό οξυγόνο. Οι συνθήκες που χρησιµοποιήθηκαν αρχικά ήταν οι εµπορικά διαθέσιµες προπαρασκευασµένες συνθήκες, οι οποίες εν συνεχεία τροποποιήθηκαν συνδυάζοντας συνθήκες που χρησιµοποιήθηκαν επιτυχώς σε άλλες φερρεδοξίνες. Η συλλογή των κρυσταλλογραφικών δεδοµένων πραγµατοποιήθηκε ακτινοβολία συγχρότρου, στο Ευρωπαϊκό Εργαστήριο Μοριακής Βιολογίας Αµβούργου, σε ανιχνευτές δύο διαστάσεων. Η PaFd κρυσταλλώθηκε σε ορθοροµβικό κρυσταλλικό σύστηµα, στην οµάδα χώρου P212121, διαστάσεις µοναδιαίας κυψελίδας 39.1, 45.1, 51.9 A και σε µέγιστη διακριτική ικανότητα 1.32 A. µή επιλύθηκε µε τη µέθοδο της µοριακής αντικατάστασης χρησιµοποιώντας ως µοντέλο τη δοµή φερρεδοξίνης Allochromatium vinosum. Η δοµή βελτιστοποιήθηκε µέχρι την τιµή R = 15% και το τελικό µοντέλο δεν παρουσίαζε καµία απόκλιση από τα γεωµετρικά χαρακτηριστικά των δοµών υψηλής διακριτικής ικανότητας. Η διακριτική ικανότητα των 1.32 A είναι η υψηλότερη που έχει επιτευχθεί σε φερρεδοξίνη της οικογένειας των 2[4Fe-4S] και επέτρεψε τη λεπτοµερή ανάλυση των µοριακών αλληλεπιδράσεων και της γεωµετρίας των πλειάδων σιδήρου – θείου, οι οποίες στα τελευταία στάδια βελτιστοποιήθηκαν χωρίς κανέναν περιορισµό. Από τη δοµή της PaFd αντλήθηκαν αντικές δοµικές πληροφορίες, που επέτρεψαν τη σύγκριση της δοµής της οικογένειας αυτής µε άλλες φερρεδοξίνες υψηλής διακριτικής ικανότητας, ως προς την επίδραση της δοµής στα κανονικά δυναµικά αναγωγής. Η EcFd έδωσε κρυστάλλους εξαγωνικής συµµετρίας, οµάδας χώρου P62, οι οποίοι ήταν µεροεδρικώς διπλοί και ως εκ τούτου εµφάνιζε υψηλότερη συµµετρία από την πραγµατική. Ενώ ο κρύσταλλος ανήκε στην οµάδα σηµείου 6, η περίθλαση του έδειχνε ότι ανήκε στην οµάδα σηµείου 622, δηλαδή υψηλότερης συµµετρίας οµάδα Laue. Παρά τις δυσκολίες στην επίλυση της δοµής, επιτεύχθηκε προσδιορισµός αρχικών φάσεων µε τη µέθοδο της µοριακής αντικατάστασης, 114 χρησιµοποιώντας κρυσταλλογραφικά δεδοµένα µερικώς διορθωµένα, ενώ πιστοποιήθηκε η πραγµατική οµάδα χώρου Ρ62 µόνο όταν η βελτιστοποίηση προχώρησε σε ικανοποιητικό σηµείο. Ο διπλός κρύσταλλος βρέθηκε ότι αποτελείτο από δύο επίµέρους κρυστάλλους σε ποσοστά ~ 55% και 45%, µε αποτέλεσµα οι ανακλάσεις h, k, l και k, h, -l να σχετίζονται. Η βελτιστοποίηση έγινε µε τη µέθοδο των ελαχίστων τετραγώνων, και οι υπολογιζόµενοι παράγοντες δοµής προσδιορίστηκαν από τη σχέση: Fc 2 = α Fc1 2 + (1 – α) Fc2 2 όπου α το κλάσµα πολλαπλότητας και Fc1, Fc2 οι υπολογιζόµενοι παράγοντες δοµής των δύο επιµέρους κρυστάλλων. Τα κρυσταλλογραφικά δεδοµένα διακριτικής ικανότητας 1.65 A υποβαθµίζονται αρκετά, λόγω της ύπαρξης µεροεδρικώς διπλού κρυστάλλου, ώστε το τελικό µοντέλο να παρουσιάζει αρκετές αποκλίσεις από τα γεωµετρικά χαρακτηριστικά των δοµών υψηλής διακριτικής ικανότητας

Inhibitor-Dependent Usage of the S1′ Specificity Pocket of ER Aminopeptidase 2

Endoplasmic reticulum aminopeptidase 2 (ERAP2) is an intracellular enzyme involved in the processing of antigenic peptides intended for presentation by major histocompatibility complex class I (MHCI) molecules. Because of its role in regulating immune responses, ERAP2 is an emerging pharmacological target. Phosphinic pseudopeptides are potent transition-state analogue inhibitors of ERAP2. Previous structure–activity studies have revealed a complex but ambiguous relationship between the occupation of putative specificity pockets and the inhibitor efficacy. To address these problems, we solved crystal structures of ERAP2 in complex with two phosphinic pseudotripeptide inhibitors. Both compounds are found in the catalytic site in a canonical orientation for transition-state analogues and utilize the S1 and S2′ pockets in a similar fashion. Strikingly, their P1′ side chains exhibit different orientations and make interactions with distinct shallow pockets near the ERAP2 active site. These structures suggest that S1′ pocket usage in ERAP2 may be inhibitor-dependent and constitute useful starting templates for the further optimization of this class of compounds

Insights into the Protein Ruthenation Mechanism by Antimetastatic Metallodrugs: High-Resolution X-ray Structures of the Adduct Formed between Hen Egg-White Lysozyme and NAMI-A at Various Time Points

The pharmacological profile of medicinally relevant Ru(III) coordination compounds has been ascribed to their interactions with proteins, as several studies have provided evidence that DNA is not the primary target. In this regard, numerous spectroscopic and crystallographic studies have indicated that the Ru(III) ligands play an important role in determining the metal binding site, acting as the recognition element in the early stages of the protein–complex formation. Herein, we present a series of near-atomic-resolution X-ray crystal structures of the adducts formed between the antimetastatic metallodrug imidazolium trans-[tetrachlorido(S-dimethyl sufoxide)(1H-imidazole)ruthenate(III)] (NAMI-A) and hen egg-white lysozyme (HEWL). These structures elucidate a series of binding events starting from the noncovalent interaction of intact NAMI-A ions with HEWL (1.5 h), followed by the stepwise exchange of all Ru ligands except for 1H-imidazole (26 h) to the final “ruthenated” protein comprising one aquated Ru ion coordinated to histidine-15 of HEWL (98 h). Our structural data clearly support a two-step mechanism of protein ruthenation, illustrating the ligand-mediated recognition step of the process

Structural and Evolutionary Insights within the Polysaccharide Deacetylase Gene Family of Bacillus anthracis and Bacillus cereus

Functional and folding constraints impose interdependence between interacting sites along the protein chain that are envisaged through protein sequence evolution. Studying the influence of structure in phylogenetic models requires detailed and reliable structural models. Polysaccharide deacetylases (PDAs), members of the carbohydrate esterase family 4, perform mainly metal-dependent deacetylation of O- or N-acetylated polysaccharides such as peptidoglycan, chitin and acetylxylan through a conserved catalytic core termed the NodB homology domain. Genomes of Bacillus anthracis and its relative Bacillus cereus contain multiple genes of putative or known PDAs. A comparison of the functional domains of the recently determined PDAs from B. anthracis and B. cereus and multiple amino acid and nucleotide sequence alignments and phylogenetic analysis performed on these closely related species showed that there were distinct differences in binding site formation, despite the high conservation on the protein sequence, the folding level and the active site assembly. This may indicate that, subject to biochemical verification, the binding site-forming sequence fragments are under functionally driven evolutionary pressure to accommodate and recognize distinct polysaccharide residues according to cell location, use, or environment. Finally, we discuss the suggestion of the paralogous nature of at least two genes of B. anthracis, ba0330 and ba0331, via specific differences in gene sequence, protein structure, selection pressure and available localization patterns. This study may contribute to understanding the mechanisms under which sequences evolve in their structures and how evolutionary processes enable structural variations

High-Resolution Crystal Structure of Endoplasmic Reticulum Aminopeptidase 1 with Bound Phosphinic Transition-State Analogue Inhibitor

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that helps generate peptides presented by Major Histocompatibility Complex Class I (MHC class I) molecules and is an emerging target for immunotherapy applications. Despite almost two decades of research on ERAP1, lack of high-resolution crystal structures has hampered drug-development efforts. By optimizing the protein construct, we obtained a high-resolution (1.60 Å) crystal structure of the closed-conformation of ERAP1 with a potent phosphinic pseudopeptide inhibitor bound in its active site. The structure provides key insight on the mechanism of inhibition as well as selectivity toward homologous enzymes and allows detailed mapping of the internal cavity of the enzyme that accommodates peptide-substrates. Bis-tris propane and malic acid molecules, found bound in pockets in the internal cavity, reveal potential druggable secondary binding sites. The ability to obtain high-resolution crystal structures of ERAP1 removes a major bottleneck in the development of compounds that regulate its activity and will greatly accelerate drug-discovery efforts

Structural Insights into the Role of β3 nAChR Subunit in the Activation of Nicotinic Receptors

The β3 subunit of nicotinic acetylcholine receptors (nAChRs) participates in heteropentameric assemblies with some α and other β neuronal subunits forming a plethora of various subtypes, differing in their electrophysiological and pharmacological properties. While β3 has for several years been considered an accessory subunit without direct participation in the formation of functional binding sites, recent electrophysiology data have disputed this notion and indicated the presence of a functional (+) side on the extracellular domain (ECD) of β3. In this study, we present the 2.4 Å resolution crystal structure of the monomeric β3 ECD, which revealed rather distinctive loop C features as compared to those of α nAChR subunits, leading to intramolecular stereochemical hindrance of the binding site cavity. Vigorous molecular dynamics simulations in the context of full length pentameric β3-containing nAChRs, while not excluding the possibility of a β3 (+) binding site, demonstrate that this site cannot efficiently accommodate the agonist nicotine. From the structural perspective, our results endorse the accessory rather than functional role of the β3 nAChR subunit, in accordance with earlier functional studies on β3-containing nAChRs

High-resolution crystal structures of a “half sandwich”-type Ru(II) coordination compound bound to hen egg-white lysozyme and proteinase K

The high-resolution X-ray crystal structures of the adducts formed between the “half sandwich”-type Ru(II) coordination compound [Ru$^{II}$(1,4,7-trithiacyclononane)(ethane-1,2-diamine)Cl]$^+$ and two proteins, namely hen egg-white lysozyme and proteinase K, are presented. The structures unveil that upon reaction with both enzymes the Ru(II) compound is coordinated by solvent-exposed aspartate residues after releasing the chloride ligand (Asp101 in lysozyme, Asp200 and Asp260 in proteinase K), while retaining the two chelating ligands. The adduct with Asp101 residue at the catalytic cleft of lysozyme is accompanied by residue-specific conformational changes to accommodate the Ru(II) fragment, whereas the complexes bound at the two calcium-binding sites of proteinase K revealed minimal structural perturbation of the enzyme. To the best of our knowledge, proteinase K is used here for the first time as a model system of protein metalation and these are the first X-ray crystal structures of protein adducts of a Ru(II) coordination compound that maintains its coordination sphere almost intact upon binding. Our data demonstrate the role of ligands in stabilizing the protein adducts via hydrophobic/aromatic or hydrogen-bonding interactions, as well as their underlying role in the selection of specific sites on the electrostatic potential surface of the enzymes