Conformational study of H-NOX (heme-nitric oxide/oxygen binding) domains and their complexes with signaling molecules through NMR spectroscopy

Nitric oxide (ΝΟ) plays a key role in the the convertion of GTP to cGMP. The main receptor of NO is the sGC enzyme. sGC is a heterodimer, composed of α1 and β1 subunit, of which the latter contains the heme binding H-NOX domain, responsible for NO recognition and binding. NO binds upon the Fe of H-NOX and triggers electronic and conformational changes, leading to the signal transmission from the H-NOX domain to the catalytic and the subsequent enzyme’s activation. Impaired function of the NO/sGC/cGMP signaling pathway has been linked several diseases, such as hypertensionand heart failure. Low bioavailability of NO inhibits the pathway while heme oxidation makes sGC unresponsive to NO. As a central player in this axis, H-NOX is the focus of intense research efforts aiming to develop therapeutic molecules that enhance the pathway’s activity. Two classes of compounds have been described that directly affect the function of sGC: stimulators which activate sGC in spite of the low NO concentration and activators which replace oxidized heme, restoring the proper function of the enzyme. H-NOX domains have been identified not only in mammals but also in bacteria.In the present thesis, we present the conformational study of three bacterial H-NOX proteins, from Nostoc sp. (Ns H-NOX), Caldanaerobacter subterraneus (Cs H-NOX) and Vibrio cholerae (Vc H-NOX) and the mode of interaction with diatomic gases and chemical compounds. By applying NMR spectroscopy, we report the conformational exchange effect during the heme replacement of Ns H-NOX domain by the activators BAY 58-2667 and BAY 60-2770, leading Ns H-NOX to adopt a more rigid conformation. Same approach is applied to determine how the stimulator BAY 41-2272 interacts withNs H-NOX domain. Combination of tow different spectroscopic techniques, we study the conformational and electronic changes of the Ns H-NOX domain after the binding of NO. Site – directed mutagenesis experiments identify the role and the participation of the two tunnels of Ns H-NOX domain, in the entry and exit of NO, as well as in the general gas migration in the protein interior. Furthermore, we report the expression and purification conditions for the sample preparation of Cs H-NOX and Vc H-NOX proteins aiming their structural and functional study via NMR spectroscopy.Το μονοξείδιο του αζώτου (ΝΟ) συμμετέχει στη μετατροπή του GTP προς cGMP. Ο κύριος φυσιολογικός υποδοχέας για το ΝΟ είναι το ένζυμο sGC. Το ένζυμο sGC, είναι ένα ετεροδιμερές και αποτελείται από δύο υπομονάδες: την α1 και τη β1. Στο αμινοτελικό άκρο της β1 υπομονάδας εντοπίζεται ο Η-ΝΟΧ τομέας, ο οποίος φέρει ένα μόρι αίμης και είναι υπεύθυνος για την αναγνώριση και την πρόσδεση του ΝΟ. Η α1 υπομονάδα δεν φέρει Η-ΝΟΧ ή αντίστοιχο τομέα. Το ΝΟ προσδένεται στο Fe επάγοντας ηλεκτρονιακές και διαμορφωτικές αλλαγές, οι οποίες οδηγούν στη μετάδοση του σήματος από τον Η-ΝΟΧ στον καταλυτικό τομέα, με αποτέλεσμα την ενεργοποίηση του ενζύμου. Δυσλειτουργία του σηματοδοτικού μονοπατιού NO/sGC/cGMP έχει συσχετιστεί με παθολογικές καταστάσεις όπως υπέρταση και καρδιακή ανεπάρκεια. Χαμηλή βιοδιαθεσιμότητα του ΝΟ αναστέλλει τη λειτουργία του μονοπατιού ενώ με την οξείδωση του Fe, η sGC να χάνει τη λειτουργικότητά της. Συνεπώς, ο Η-ΝΟΧ τομέας αποτελεί φαρμακευτικό στόχο για το σχεδιασμό δραστικών ενώσεων με σκοπό τη ενίσχυση της λειτουργίας του μονοπατιού NO/sGC/cGMP. Δύο κατηγορίες ενώσεων έχουν αναπτυχθεί: οι διεγέρτες, οι οποίοι ενεργοποιούν την sGC σε συνθήκες χαμηλής βιοδιθεσιμότητας NO και οι ενεργοποιητές, οι οποίοι αντικαθιστούν την οξειδωμένη αίμη, επαναφέροντας τη λειτουργία του ενζύμου. Εκτός από τα θηλαστικά, Η-ΝΟΧεπικράτειες έχουν ταυτοποιηθεί και σε βακτήρια. Στην παρούσα διατριβή παρουσίαζεται η μελέτη της διαμόρφωσης τριώνβακτηριακών Η-ΝΟΧ επικρατειών, των Nostoc sp. (Ns H-NOX), Caldanaerobacter subterraneus (Cs H-NOX) και Vibrio cholerae (Vc H-NOX) και του τρόπου αλληλεπίδρασης με διατομικά αέρια και δραστικές ενώσεις. Με τη χρήση φασματοκοπίας NMR περιγράφεται το φαινόμενο διαμορφωτικής ανταλλαγής κατά την αντικατάσταση της αίμης της Ns H-NOX επικράτειας από τους ενεργοποιητές ΒΑΥ 58-2667 και ΒΑΥ 60-2770 με αποτέλεσμα η πρωτεΐνη να αποκτά πιο σταθερή διαμόρφωση, ενώ με τον ίδιο τρόπο προσδιορίζεται ο τρόπος αλληλεπίδρασης του διεγέρτη ΒΑΥ 41-2272 με την Ns H-NOX επικράτεια. Με το συνδυασμό δύο NMR και UV-vis μελετώνται οι διαμορφωτικές και ηλεκτρονιακές αλλαγές, τις οποίες προκαλεί η πρόσδεση του ΝΟ ενώ παράλληλα με πειράματα κατευθυνόμενης μεταλλαξιγένεσης της Ns H-NOX επικράτειας προσδιορίζεται ο τρόπος εισόδου και εξόδου του ΝΟ στο εσωτερικό της πρωτεΐνης ώστε να προσδεθεί στην αίμη. Επιπλέον, παρουσιάζεται έκφραση και η απομόνωση των Cs H-NOX και Vc H-NOX επικράτειών με σκοπό τη δομική και λειτουργική τους μελέτη μέσω φασματοσκοπίας NMR

NMR Analysis Suggests Synergy between the RRM2 and the Carboxy-Terminal Segment of Human La Protein in the Recognition and Interaction with HCV IRES

The La protein (lupus antigen) is a ubiquitous RNA-binding protein found in all human cells. It is mainly localized in the nucleus, associates with all RNA polymerase III (Pol III) transcripts, as the first factor they interact with, and modulates subsequent processing events. Export of La to the cytoplasm has been reported to stimulate the decoding of specific cellular and viral mRNAs through IRES-dependent (Internal ribosome entry site) binding and translation. Using NMR (Nuclear Magnetic Resonance) spectroscopy, we provide atomic-level-resolution structural insights on the dynamical properties of human La (hLa) protein in solution. Moreover, using a combination of NMR spectroscopy and isothermal titration calorimetry (ITC), we provide evidence about the role and ligand specificity of the C-terminal domain of the La protein (RRM2 and C-terminal region) that could mediate the recognition of HCV-IRES

NMR Analysis Suggests Synergy between the RRM2 and the Carboxy-Terminal Segment of Human La Protein in the Recognition and Interaction with HCV IRES

The La protein (lupus antigen) is a ubiquitous RNA-binding protein found in all human cells. It is mainly localized in the nucleus, associates with all RNA polymerase III (Pol III) transcripts, as the first factor they interact with, and modulates subsequent processing events. Export of La to the cytoplasm has been reported to stimulate the decoding of specific cellular and viral mRNAs through IRES-dependent (Internal ribosome entry site) binding and translation. Using NMR (Nuclear Magnetic Resonance) spectroscopy, we provide atomic-level-resolution structural insights on the dynamical properties of human La (hLa) protein in solution. Moreover, using a combination of NMR spectroscopy and isothermal titration calorimetry (ITC), we provide evidence about the role and ligand specificity of the C-terminal domain of the La protein (RRM2 and C-terminal region) that could mediate the recognition of HCV-IRES

Backbone and side chain NMR assignments of the H-NOX domain from Nostoc sp. in complex with BAY58-2667 (cinaciguat)

Soluble guanylate cyclase (sGC) enzyme is activated by the gaseous signaling agent nitric oxide (NO) and triggers the conversion of GTP (guanosine 5′-triphosphate) to cGMP (cyclic guanylyl monophosphate). It contains the heme binding H-NOX (heme- nitric oxide/oxygen binding) domain which serves as the sensor of NO and it is highly conserved across eukaryotes and bacteria as well. Many research studies focus on the synthesis of chemical compounds bearing possible therapeutic action, which mimic the heme moiety and activate the sGC enzyme. In this study, we report a preliminary solution NMR (Nuclear Magnetic Resonance) study of the H-NOX domain from Nostoc sp. cyanobacterium in complex with the chemical sGC activator cinaciguat (BAY58-2667). An almost complete sequence-specific assignment of its 1H, 15N and 13C resonances was obtained and its secondary structure predicted by TALOS+

NMR study of non-structural proteins-part II: H-1, C-13, N-15 backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV)

International audienceno abstrac

Replacement of heme by soluble guanylate cyclase (sGC) activators abolishes heme-nitric oxide/oxygen (H-NOX) domain structural plasticity

The gasotransmitter nitric oxide (NO) is a critical endogenous regulator of homeostasis, in major part via the generation of cGMP (cyclic guanosine monophosphate) from GTP (guanosine triphosphate) by NO’s main physiological receptor, the soluble guanylate cyclase (sGC). sGC is a heterodimer, composed of an alpha 1 and a beta 1 subunit, of which the latter contains the heme-nitric oxide/oxygen (H-NOX) domain, responsible for NO recognition, binding and signal initiation. The NO/sGC/cGMP axis is dysfunctional in a variety of diseases, including hypertension and heart failure, especially since oxidative stress results in heme oxidation, sGC unresponsiveness to NO and subsequent degradation. As a central player in this axis, sGC is the focus of intense research efforts aiming to develop therapeutic molecules that enhance its activity. A class of drugs named sGC “activators” aim to replace the oxidized heme of the H-NOX domain, thus stabilizing the enzyme and restoring its activity. Although numerous studies outline the pharmacology and binding behavior of these compounds, the static 3D models available so far do not allow a satisfactory understanding of the structural basis of sGC’s activation mechanism by these drugs. Herein, application NMR describes different conformational states during the replacement of the heme by a sGC activators. We show that the two sGC activators (BAY 58-2667 and BAY 60-2770) significantly decrease the conformational plasticity of the recombinant H-NOX protein domain of Nostoc sp. cyanobacterium, rendering it a lot more rigid compared to the heme-occupied H-NOX. NMR methodology also reveals, for the first time, a surprising bi-directional competition between reduced heme and these compounds, pointing to a highly dynamic regulation of the H-NOX domain. This competitive, bi-directional mode of interaction is also confirmed by monitoring cGMP generation in A7r5 vascular smooth muscle cells by these activators. We show that, surprisingly, heme’s redox state impacts differently the bioactivity of these two structurally similar compounds. In all, by NMR-based and functional approaches we contribute unique experimental insight into the dynamic interaction of sGC activators with the H-NOX domain and its dependence on the heme redox status, with the ultimate goal to permit a better design of such therapeutically important molecules

Conformational plasticity of the VEEV macro domain is important for binding of ADP-ribose

International audienceVenezuelan equine encephalitis virus (VEEV) is a new world alphavirus which can be involved in several central nervous system disorders such as encephalitis and meningitis. The VEEV genome codes for 4 non-structural proteins (nsP), of which nsP3 contains a Macro domain. Macro domains (MD) can be found as stand-alone proteins or embedded within larger proteins in viruses, bacteria and eukaryotes. Their most common feature is the binding of ADP-ribose (ADPr), while several macro domains act as ribosylation writers, erasers or readers. Alphavirus MD erase ribosylation but their precise contribution in viral replication is still under investigation. NMR-driven titration experiments of ADPr in solution with the VEEV macro domain (in apo- and complex state) show that it adopts a suitable conformation for ADPr binding. Specific experiments indicate that the flexibility of the loops β5-α3 and α3-β6 is critical for formation of the complex and assists a wrapping mechanism for ADPr binding. Furthermore, along with this sequence of events, the VEEV MD undergoes a conformational exchange process between the apo state and a low-populated "dark" conformational state