Alessandro Arcovito | Università Cattolica del Sacro Cuore (Catholic University of the Sacred Heart) (original) (raw)
Papers by Alessandro Arcovito
European Biophysics Journal With Biophysics Letters, Jan 1, 2010
Von Willebrand factor (VWF) is a large multimeric adhesive glycoprotein, with complex roles in th... more Von Willebrand factor (VWF) is a large multimeric adhesive glycoprotein, with complex roles in thrombosis and hemostasis, present in circulating blood and in secretory granules of endothelial cells and platelets. High shear stress triggers conformational changes responsible for both binding to the platelet receptor glycoprotein GpIb and its self-association, thus supporting the formation of platelet plug under flow. Ristocetin also promotes the interaction of VWF with GpIb and is able to induce platelet aggregation, and thus is largely used to mimic this effect in vitro. In this research paper, we followed the time course of VWF self-association in solution induced by ristocetin binding by light scattering and at the same time we collected atomic force microscopy images to clarify the nature of the assembly that is formed. In fact, this process evolves initially through the formation of fibrils that subsequently interact to form supramolecular structures whose dimensions would be capable of trapping platelets even in the absence of any degree of shear stress or interaction with external surfaces. This intrinsic property, that is the ability to self-aggregate, may be involved in some pathological settings that have been revealed in clinical practice.
Journal of Synchrotron Radiation, 1999
Visible light can break the Fe-CO bond in Fe (II) carbonmonoxy-myoglobin (MbCO) giving an unligat... more Visible light can break the Fe-CO bond in Fe (II) carbonmonoxy-myoglobin (MbCO) giving an unligated product (Mb*) that is almost stable at T< 30 K. Fe K-edge polarized X-ray absorption spectra (P-XAS) of the photoproduct (T= 20 K) of an oriented single crystal (0.2 ...
Infection and Immunity, 2014
We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional ... more We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of the slyA operon, ef_3001, renamed pmvE (for polyamine metabolism and virulence of E. faecalis). PmvE shares strong homologies with N 1 -spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used an E. faecalis strain carrying the recombinant plasmid pMSP3535-pmvE (V19/p3535-pmvE), which allows the induction of pmvE by addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence of E. faecalis in the Galleria mellonella infection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses an N-acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence of E. faecalis, likely through its involvement in the polyamine metabolism.
Neuroglobin and cellular prion protein (PrP C ) are expressed in the nervous system and co-locali... more Neuroglobin and cellular prion protein (PrP C ) are expressed in the nervous system and co-localized in the retinal ganglion cell layer. Both proteins do not have an unambiguously assigned function, and it was recently reported that PrP C aggregates rapidly in the presence of neuroglobin, whereas it does not aggregate in the presence of myoglobin, another globin with different tissue specificity. Electrostatic complementarity between the unstructured PrP C N-terminus and neuroglobin has been proposed to mediate this specific interaction. To verifythis hypothesis experimentally, we have used a combined approach of automated docking and molecular dynamics (MD) studies carried out on short stretches of prion protein (PrP) N-terminus to identify the minimal electrostatically interacting aminoacidic sequences with neuroglobin. Subsequently, we have performed the synthesis of these peptides by solid phase methods, and we tested their interaction with neuroglobin by surface plasmon resonance (SPR). Preliminary results confirm unequivocally the specific interaction between synthetic PrP peptides and neuroglobin suggesting a crucial role of PrP C positively charged regions in thisprotein-protein association.
Journal of Physics: Conference Series, 2009
Denitrifying bacteria control NO and NO2 cytosolic levels by regulating the expression of denitri... more Denitrifying bacteria control NO and NO2 cytosolic levels by regulating the expression of denitrification gene clusters via REDOX signalling of specific transcriptional factors that may act as NO sensors in vivo. A protein belonging to the subclass DNR (dissimilative nitrate respiration regulator) from Pseudomonas aeruginosa has been recently suggested to be a heme containing protein. Very recently the three dimensional
Fourier transform infrared (FTIR) spectroscopy in the CO stretch bands combined with temperature ... more Fourier transform infrared (FTIR) spectroscopy in the CO stretch bands combined with temperature derivative spectroscopy (TDS) was used to characterize intermediate states obtained by photolysis of two sperm whale mutant myoglobins, YQR (L29(B10)Y, H64(E7)Q, T67(E10)R) and YQRF (with an additional I107(G8)F replacement). Both mutants assume two different boundstate conformations, A 0 and A 3 , which can be distinguished by their different CO bands near 1965 and 1933 cm ؊1 . They most likely originate from different conformations of the Gln-64 side chain. Within each A substate, a number of photoproduct states have been characterized on the basis of the temperature dependence of recombination in TDS experiments. Different locations and orientations of the ligand within the protein can be distinguished by the infrared spectra of the photolyzed CO. Recombination from the primary docking site, B, near the heme dominates below 50 K. Above 60 K, ligand rebinding occurs predominantly from a secondary docking site, C, in which the CO is trapped in the Xe4 cavity on the distal side, as shown by crystallography of photolyzed YQR and L29W myoglobin CO. Another kinetic state (C) has been identified from which rebinding occurs around 130 K. Moreover, a population appearing above the solvent glass transition at ϳ180 K (D state) is assigned to rebinding from the Xe1 cavity, as suggested by the photoproduct structure of the L29W sperm whale myoglobin mutant. For both the YQR and YQRF mutants, rebinding from the B sites near the heme differs for the two A substates, supporting the view that the return of the ligand from the C, C, and D states is not governed by the recombination barrier at the heme iron but rather by migration to the active site. Comparison of YQR and YQRF shows that access to the Xe4 site (C) is severely restricted by introduction of the bulky Phe side chain at position 107.
Journal of Biological Chemistry, 2014
Background: Nucleophosmin is a nucleolar protein that interacts with G-quadruplexes. Results: Sit... more Background: Nucleophosmin is a nucleolar protein that interacts with G-quadruplexes. Results: Site-directed mutagenesis and molecular dynamics unravel the role of single residues in complex formation. Conclusion: A disordered segment of nucleophosmin contributes to G-quadruplex recognition by facilitating the formation of an encounter complex and transiently interacting with the G-quadruplex. Significance: Understanding the role played by flanking fuzziness is an important issue in protein/DNA interactions.
Physical Review Letters, 2001
We report the first quantitative analysis of the Fe K-edge polarized x-ray absorption near edge s... more We report the first quantitative analysis of the Fe K-edge polarized x-ray absorption near edge structure of the iron protein carbonmonoxy-myoglobin (MbCO) single crystal and of its cryogenic photoproduct Mb(*)CO. The CO-Fe-heme local structure has been determined using a novel fitting procedure based on the full multiple scattering approach. The extracted local structure of Mb(*)CO includes a Fe-CO distance of (3.08+/-0.07) A, with a tilting angle between the heme normal and the Fe-C vector of (37+/-7) degrees, and a bending angle between the Fe-C vector and the C-O bond of (31+/-5) degrees.
Journal of Protein Chemistry, 2002
The kinetics of the catalytic cycle of myeloperoxidase and of horseradish peroxidase reacting wit... more The kinetics of the catalytic cycle of myeloperoxidase and of horseradish peroxidase reacting with aminoglycosides have been studied by conventional and stopped-flow spectrophotometry. Aminoglycosides acted as one-electron reducing substrates converting compound I, formed when stoichiometric amounts of hydrogen peroxide were added to the enzyme, to compound II, and compound II to the resting, ferric enzyme. The latter gradually decayed into a further spectroscopic derivative ( max ϭ 540 and 403 nm) tentatively identified as a complex of ferric heme with the antibiotic oxidation product(s), and the resulting enzyme was fully inactivated. Since myeloperoxidase is the only human enzyme known to convert chloride ions into the cytotoxic hypochlorous acid, the data presented in this paper bear relevance to the pharmacological effects of aminoglycoside antibiotics, which, while inhibiting bacterial growth, also prevent oxidative cellular damage caused by hypochlorous acid aging as substrates and inhibitors of myeloperoxidase.
International Journal of Nanomedicine, 2014
The Journal of Physical Chemistry B, 2010
Polarized Fe K-edge X-ray absorption near-edge structure (XANES) spectra of murine carbonmonoxy-n... more Polarized Fe K-edge X-ray absorption near-edge structure (XANES) spectra of murine carbonmonoxy-neuroglobin (NgbCO) single crystals have been collected and compared with a number of derivatives of sperm whale myoglobin (Mb), that is, the nitrosyl (MbNO) and deoxy (Mb) derivatives, the previously reported cyanomet (MbCN) and carbonmonoxy (MbCO) derivatives, and the cryogenic photoproduct of MbCO (Mb · CO). The single crystals under study exhibit a strong XANES angular dichroism which allows the heme geometry of each sample to be analyzed with extremely high accuracy via the full multiple scattering (MS) approach. The results of two alternative methods to undergo the MS analysis have been compared with high resolution X-ray diffraction (XRD) data and with X-ray absorption spectroscopy (XAS) data in solution. As a result of the present analysis, the Fe-heme structure in solution and in the cryo-trapped NgbCO single crystal (which cracks at room temperature) are the same. Accordingly, the residual energy involved in the protein relaxation responsible of crystal cracking at room temperature after CO binding does not reside in the heme pocket. A combined approach (polarized XANES and XRD) is suggested to be applied on the same single crystals of metalloproteins at opportunely equipped synchrotron beamlines. Figure 1. Structural superimposition of murine NgbCO (blue) and sperm whale MbCO (light blue), based on the position of the backbone CR atoms.
Structure, 2006
Understanding the basis of communication within protein domains is a major challenge in structura... more Understanding the basis of communication within protein domains is a major challenge in structural biology. We present structural and dynamical evidence for allosteric effects in a PDZ domain, PDZ2 from the tyrosine phosphatase PTP-BL, upon binding to a target peptide. The NMR structures of its free and peptide-bound states differ in the orientation of helix alpha2 with respect to the remainder of the molecule, concomitant with a readjustment of the hydrophobic core. Using an ultrafast mixing instrument, we detected a deviation from simple bimolecular kinetics for the association with peptide that is consistent with a rate-limiting conformational change in the protein (k(obs) approximately 7 x 10(3) s(-1)) and an induced-fit model. Furthermore, the binding kinetics of 15 mutants revealed that binding is regulated by long-range interactions, which can be correlated with the structural rearrangements resulting from peptide binding. The homologous protein PSD-95 PDZ3 did not display a similar ligand-induced conformational change.
Proceedings of the National Academy of Sciences, 2003
Although conformational changes are essential for the function of proteins, little is known about... more Although conformational changes are essential for the function of proteins, little is known about their structural dynamics at atomic level resolution. Myoglobin (Mb) is the paradigm to investigate conformational dynamics because it is a simple globular heme protein displaying a photosensitivity of the iron-ligand bond. Upon laser photodissociation of carboxymyoglobin Mb a nonequilibrium population of protein structures is generated that relaxes over a broad time range extending from picoseconds to milliseconds. This process is associated with migration of the ligand to cavities in the matrix and with a reduction in the geminate rebinding rate by several orders of magnitude. Here we report nanosecond time-resolved Laue diffraction data to 1.55-Å resolution on a Mb mutant, which depicts the sequence of structural events associated with this extended relaxation. Motions of the distal E-helix, including the mutated residue Gln-64(E7), and of the CD-turn are found to lag significantly (100 -300 ns) behind local rearrangements around the heme such as heme tilting, iron motion out of the heme plane, and swinging of the mutated residue Tyr-29(B10), all of which occur promptly (<3 ns). Over the same delayed time range, CO is observed to migrate from a cavity distal to the heme known to bind xenon (called Xe4) to another such cavity proximal to the heme (Xe1). We propose that the extended relaxation of the globin moiety reflects reequilibration among conformational substates known to play an essential role in controlling protein function. myoglobin ͉ protein dynamics ͉ conformational states ͉ functional intermediates ͉ protein crystallography
Proceedings of the National Academy of Sciences, 2007
X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbi... more X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbing atom and therefore allows bond distances and angles of the surrounding atomic cluster to be measured with atomic resolution. By contrast, the accuracy and resolution of metalloprotein active sites obtainable from x-ray crystallography are often insufficient to analyze the electronic properties of the metals that are essential for their biological functions. Here, we demonstrate that the combination of both methods on the same metalloprotein single crystal yields a structural model of the protein with exceptional active-site resolution. To this end, we have collected an x-ray diffraction data set to 1.4-Å resolution and Fe K-edge polarized x-ray absorption near edge structure (XANES) spectra on the same cyanomet sperm whale myoglobin crystal. The XANES spectra were quantitatively analyzed by using a method based on the multiple scattering approach, which yielded Fe-heme structural parameters with ؎(0.02-0.07)-Å accuracy on the atomic distances and ؎7°on the Fe-CN angle. These XANES-derived parameters were subsequently used as restraints in the crystal structure refinement. By combining XANES and x-ray diffraction, we have obtained an cyanomet sperm whale myoglobin structural model with a higher precision of the bond lengths and angles at the active site than would have been possible with crystallographic analysis alone. myoglobin ͉ crystallographic restraints ͉ protein crystallography ͉ synchroton radiation M etalloproteins perform a wide range of biological functions essential for sustaining life; they are particularly abundant in bioenergetic pathways, e.g., photosynthesis and respiration (1, 2). Often, the metal ion is located at the active site of an enzyme, where an intricate coordination geometry finetunes the electronic properties of the metal so as to enable it to catalyze specific reactions. X-ray diffraction (XRD) on protein single crystals or high-field NMR on protein solutions are the standard techniques by which entire molecular structures of proteins including their active-site metal clusters can be analyzed. By using quantum-chemical refinement, precise structural models of metal sites have been obtained from XRD data (3). X-ray absorption spectroscopy (XAS) is an alternative technique for measuring the structure of metal sites (4, 5). Electrons are liberated from the metal ion by the photoelectric effect, and the interference effects of the electron waves are analyzed to map out the local environment of the metal. XAS can reveal structural parameters with a very high precision, comparable to that of small-molecule crystallography. Such detailed structural data are required for elucidating the function of metal centers in a metalloprotein (6), as structural changes to the metal coordination during redox or substrate-binding reactions are generally Ͻ0.1 Å and hence remain unnoticed in standard protein crys-tallography unless the resolution is exceptionally high. As yet, x-ray structures of proteins at true atomic resolution (Ͻ1.2 Å) are still scarce .
Proceedings of the National Academy of Sciences, 2006
Work carried out over the last 30 years unveiled the role of structural dynamics in controlling p... more Work carried out over the last 30 years unveiled the role of structural dynamics in controlling protein function. Cavity networks modulate structural dynamics trajectories and are functionally relevant; in globins they have been assigned a role in ligand migration and docking. These findings raised renewed interest for time-resolved structural investigations of myoglobin (Mb), a simple heme protein displaying a photosensitive iron-ligand bond. Photodissociation of MbCO generates a nonequilibrium population of protein structures relaxing over a time range extending from picoseconds to milliseconds. This process triggers ligand migration to matrix cavities with clear-cut effects on the rate and yield of geminate rebinding. Here, we report subnanosecond time-resolved Laue diffraction data on the triple mutant YQR-Mb [Leu-29(B10)Tyr, His-64(E7)Gln, Thr-67(E10)Arg] that depict the sequence of structural events associated with heme and protein relaxation from 100 ps to 316 ns and above. The photodissociated ligand rapidly (<0.1 ns) populates the Xe-binding cavity distal to the heme. Moreover, the heme relaxation toward the deoxy configuration is heterogeneous, with a slower phase (Ϸns) evident in these experiments. Damping of the heme response appears to result from a strain exerted by the E-helix via the CD-turn; Phe-43(CD1), in close contact with heme, opposes tilt until the strain is relieved. A comparison with crystallographic data on wild-type Mb and mutants Leu(29)Phe or Leu(29)Trp suggests that the internal structure controls the rate and amplitude of the relaxation events. A correlation between structural dynamics as unveiled by Laue crystallography and functional properties of Mb is presented.
Nucleic Acids Research, 2013
Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and expor... more Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex-binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplexbinding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.
Journal of Thrombosis and Haemostasis, 2013
Background: This work was aimed at characterizing the interaction of b 2 -glycoprotein I (b 2 GPI... more Background: This work was aimed at characterizing the interaction of b 2 -glycoprotein I (b 2 GPI), an abundant plasma protein of unknown function, with human thrombin, the final effector protease in the coagulation cascade. Methods: The b 2 GPI-thrombin interaction was studied by surface plasmon resonance (SPR), fluorescence, and molecular modeling. The effect of b 2 GPI on the procoagulant (fibrin generation and platelet aggregation) and anticoagulant (protein C activation) functions of thrombin were investigated with turbidimetric, immunocytofluorimetric and enzymatic assays. Results: SPR and fluorescence data indicated that b 2 GPI tightly bound thrombin (K d = 34 nM) by interacting with both protease exosites, while leaving the active site accessible. This picture is fully consistent with the theoretical model of the b 2 GPI-thrombin complex. In particular, blockage of thrombin exosites with binders specific for exosite-1 (hirugen and HD1 aptamer) or exosite-2 (fibrinogen c′-peptide and HD22 aptamer) impaired the b 2 GPI-thrombin interaction. Identical results were obtained with thrombin mutants having one of the two exosites selectively compromised by mutation (Arg73Ala and Arg101Ala). Fluorescence measurements indicated that b 2 GPI did not affect the affinity of the enzyme for active site inhibitors, such as p-aminobenzamidine and the hirudin(1-47) domain, in agreement with the structural model. b 2 GPI dose-dependently prolonged the thrombin clotting time and ecarin clotting time in b 2 GPI-deficient plasma. b 2 GPI inhibited thrombin-induced platelet aggregation (IC 50 = 0.36 lM) by impairing thrombin cleavage of protease-activated receptor 1 (PAR1) (IC 50 = 0.32 lM), both on gel-filtered platelets and in whole blood. Strickingly, b 2 GPI did not affect thrombin-mediated generation of the anticoagulant protein C. Conclusions: b 2 GPI functions as a physiologic anticoagulant by inhibiting the key procoagulant activities of thrombin without affecting its unique anticoagulant function.
Journal of the American Chemical Society, 2010
The effect of structural disorder on the X-ray absorption near-edge structure (XANES) spectrum of... more The effect of structural disorder on the X-ray absorption near-edge structure (XANES) spectrum of a heme protein has been investigated using the dynamical description of the system derived from molecular dynamics (MD) simulations. The XANES spectra of neuroglobin (Ngb) and carbonmonoxy-neuroglobin (NgbCO) have been quantitatively reproduced, starting from the MD geometrical configurations, without carrying out any optimization in the structural parameter space. These results provide an important experimental validation of the reliability of the potentials used in the MD simulations and accordingly corroborate the consistency of the structural dynamic information on the metal center, related to its biological function. This analysis allowed us to demonstrate that the configurational disorder associated with the distortion of the heme plane and with the different orientations of the axial ligands can affect the XANES features at very low energy. Neglecting configurational disorder in the XANES quantitative analysis of heme proteins is a source of systematic errors in the determination of Fe coordination geometry. The combined use of XANES and MD is a novel strategy to enhance the resolution and reliability of the structural information obtained on metalloproteins, making the combination of these techniques powerful for metalloprotein investigations.
Journal of Synchrotron Radiation, 1999
Visible light can break the Fe-CO bond in Fe (II) carbonmonoxy-myoglobin (MbCO) giving an unligat... more Visible light can break the Fe-CO bond in Fe (II) carbonmonoxy-myoglobin (MbCO) giving an unligated product (Mb*) that is almost stable at T&lt; 30 K. Fe K-edge polarized X-ray absorption spectra (P-XAS) of the photoproduct (T= 20 K) of an oriented single crystal (0.2 ...
European Biophysics Journal With Biophysics Letters, Jan 1, 2010
Von Willebrand factor (VWF) is a large multimeric adhesive glycoprotein, with complex roles in th... more Von Willebrand factor (VWF) is a large multimeric adhesive glycoprotein, with complex roles in thrombosis and hemostasis, present in circulating blood and in secretory granules of endothelial cells and platelets. High shear stress triggers conformational changes responsible for both binding to the platelet receptor glycoprotein GpIb and its self-association, thus supporting the formation of platelet plug under flow. Ristocetin also promotes the interaction of VWF with GpIb and is able to induce platelet aggregation, and thus is largely used to mimic this effect in vitro. In this research paper, we followed the time course of VWF self-association in solution induced by ristocetin binding by light scattering and at the same time we collected atomic force microscopy images to clarify the nature of the assembly that is formed. In fact, this process evolves initially through the formation of fibrils that subsequently interact to form supramolecular structures whose dimensions would be capable of trapping platelets even in the absence of any degree of shear stress or interaction with external surfaces. This intrinsic property, that is the ability to self-aggregate, may be involved in some pathological settings that have been revealed in clinical practice.
Journal of Synchrotron Radiation, 1999
Visible light can break the Fe-CO bond in Fe (II) carbonmonoxy-myoglobin (MbCO) giving an unligat... more Visible light can break the Fe-CO bond in Fe (II) carbonmonoxy-myoglobin (MbCO) giving an unligated product (Mb*) that is almost stable at T&lt; 30 K. Fe K-edge polarized X-ray absorption spectra (P-XAS) of the photoproduct (T= 20 K) of an oriented single crystal (0.2 ...
Infection and Immunity, 2014
We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional ... more We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of the slyA operon, ef_3001, renamed pmvE (for polyamine metabolism and virulence of E. faecalis). PmvE shares strong homologies with N 1 -spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used an E. faecalis strain carrying the recombinant plasmid pMSP3535-pmvE (V19/p3535-pmvE), which allows the induction of pmvE by addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence of E. faecalis in the Galleria mellonella infection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses an N-acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence of E. faecalis, likely through its involvement in the polyamine metabolism.
Neuroglobin and cellular prion protein (PrP C ) are expressed in the nervous system and co-locali... more Neuroglobin and cellular prion protein (PrP C ) are expressed in the nervous system and co-localized in the retinal ganglion cell layer. Both proteins do not have an unambiguously assigned function, and it was recently reported that PrP C aggregates rapidly in the presence of neuroglobin, whereas it does not aggregate in the presence of myoglobin, another globin with different tissue specificity. Electrostatic complementarity between the unstructured PrP C N-terminus and neuroglobin has been proposed to mediate this specific interaction. To verifythis hypothesis experimentally, we have used a combined approach of automated docking and molecular dynamics (MD) studies carried out on short stretches of prion protein (PrP) N-terminus to identify the minimal electrostatically interacting aminoacidic sequences with neuroglobin. Subsequently, we have performed the synthesis of these peptides by solid phase methods, and we tested their interaction with neuroglobin by surface plasmon resonance (SPR). Preliminary results confirm unequivocally the specific interaction between synthetic PrP peptides and neuroglobin suggesting a crucial role of PrP C positively charged regions in thisprotein-protein association.
Journal of Physics: Conference Series, 2009
Denitrifying bacteria control NO and NO2 cytosolic levels by regulating the expression of denitri... more Denitrifying bacteria control NO and NO2 cytosolic levels by regulating the expression of denitrification gene clusters via REDOX signalling of specific transcriptional factors that may act as NO sensors in vivo. A protein belonging to the subclass DNR (dissimilative nitrate respiration regulator) from Pseudomonas aeruginosa has been recently suggested to be a heme containing protein. Very recently the three dimensional
Fourier transform infrared (FTIR) spectroscopy in the CO stretch bands combined with temperature ... more Fourier transform infrared (FTIR) spectroscopy in the CO stretch bands combined with temperature derivative spectroscopy (TDS) was used to characterize intermediate states obtained by photolysis of two sperm whale mutant myoglobins, YQR (L29(B10)Y, H64(E7)Q, T67(E10)R) and YQRF (with an additional I107(G8)F replacement). Both mutants assume two different boundstate conformations, A 0 and A 3 , which can be distinguished by their different CO bands near 1965 and 1933 cm ؊1 . They most likely originate from different conformations of the Gln-64 side chain. Within each A substate, a number of photoproduct states have been characterized on the basis of the temperature dependence of recombination in TDS experiments. Different locations and orientations of the ligand within the protein can be distinguished by the infrared spectra of the photolyzed CO. Recombination from the primary docking site, B, near the heme dominates below 50 K. Above 60 K, ligand rebinding occurs predominantly from a secondary docking site, C, in which the CO is trapped in the Xe4 cavity on the distal side, as shown by crystallography of photolyzed YQR and L29W myoglobin CO. Another kinetic state (C) has been identified from which rebinding occurs around 130 K. Moreover, a population appearing above the solvent glass transition at ϳ180 K (D state) is assigned to rebinding from the Xe1 cavity, as suggested by the photoproduct structure of the L29W sperm whale myoglobin mutant. For both the YQR and YQRF mutants, rebinding from the B sites near the heme differs for the two A substates, supporting the view that the return of the ligand from the C, C, and D states is not governed by the recombination barrier at the heme iron but rather by migration to the active site. Comparison of YQR and YQRF shows that access to the Xe4 site (C) is severely restricted by introduction of the bulky Phe side chain at position 107.
Journal of Biological Chemistry, 2014
Background: Nucleophosmin is a nucleolar protein that interacts with G-quadruplexes. Results: Sit... more Background: Nucleophosmin is a nucleolar protein that interacts with G-quadruplexes. Results: Site-directed mutagenesis and molecular dynamics unravel the role of single residues in complex formation. Conclusion: A disordered segment of nucleophosmin contributes to G-quadruplex recognition by facilitating the formation of an encounter complex and transiently interacting with the G-quadruplex. Significance: Understanding the role played by flanking fuzziness is an important issue in protein/DNA interactions.
Physical Review Letters, 2001
We report the first quantitative analysis of the Fe K-edge polarized x-ray absorption near edge s... more We report the first quantitative analysis of the Fe K-edge polarized x-ray absorption near edge structure of the iron protein carbonmonoxy-myoglobin (MbCO) single crystal and of its cryogenic photoproduct Mb(*)CO. The CO-Fe-heme local structure has been determined using a novel fitting procedure based on the full multiple scattering approach. The extracted local structure of Mb(*)CO includes a Fe-CO distance of (3.08+/-0.07) A, with a tilting angle between the heme normal and the Fe-C vector of (37+/-7) degrees, and a bending angle between the Fe-C vector and the C-O bond of (31+/-5) degrees.
Journal of Protein Chemistry, 2002
The kinetics of the catalytic cycle of myeloperoxidase and of horseradish peroxidase reacting wit... more The kinetics of the catalytic cycle of myeloperoxidase and of horseradish peroxidase reacting with aminoglycosides have been studied by conventional and stopped-flow spectrophotometry. Aminoglycosides acted as one-electron reducing substrates converting compound I, formed when stoichiometric amounts of hydrogen peroxide were added to the enzyme, to compound II, and compound II to the resting, ferric enzyme. The latter gradually decayed into a further spectroscopic derivative ( max ϭ 540 and 403 nm) tentatively identified as a complex of ferric heme with the antibiotic oxidation product(s), and the resulting enzyme was fully inactivated. Since myeloperoxidase is the only human enzyme known to convert chloride ions into the cytotoxic hypochlorous acid, the data presented in this paper bear relevance to the pharmacological effects of aminoglycoside antibiotics, which, while inhibiting bacterial growth, also prevent oxidative cellular damage caused by hypochlorous acid aging as substrates and inhibitors of myeloperoxidase.
International Journal of Nanomedicine, 2014
The Journal of Physical Chemistry B, 2010
Polarized Fe K-edge X-ray absorption near-edge structure (XANES) spectra of murine carbonmonoxy-n... more Polarized Fe K-edge X-ray absorption near-edge structure (XANES) spectra of murine carbonmonoxy-neuroglobin (NgbCO) single crystals have been collected and compared with a number of derivatives of sperm whale myoglobin (Mb), that is, the nitrosyl (MbNO) and deoxy (Mb) derivatives, the previously reported cyanomet (MbCN) and carbonmonoxy (MbCO) derivatives, and the cryogenic photoproduct of MbCO (Mb · CO). The single crystals under study exhibit a strong XANES angular dichroism which allows the heme geometry of each sample to be analyzed with extremely high accuracy via the full multiple scattering (MS) approach. The results of two alternative methods to undergo the MS analysis have been compared with high resolution X-ray diffraction (XRD) data and with X-ray absorption spectroscopy (XAS) data in solution. As a result of the present analysis, the Fe-heme structure in solution and in the cryo-trapped NgbCO single crystal (which cracks at room temperature) are the same. Accordingly, the residual energy involved in the protein relaxation responsible of crystal cracking at room temperature after CO binding does not reside in the heme pocket. A combined approach (polarized XANES and XRD) is suggested to be applied on the same single crystals of metalloproteins at opportunely equipped synchrotron beamlines. Figure 1. Structural superimposition of murine NgbCO (blue) and sperm whale MbCO (light blue), based on the position of the backbone CR atoms.
Structure, 2006
Understanding the basis of communication within protein domains is a major challenge in structura... more Understanding the basis of communication within protein domains is a major challenge in structural biology. We present structural and dynamical evidence for allosteric effects in a PDZ domain, PDZ2 from the tyrosine phosphatase PTP-BL, upon binding to a target peptide. The NMR structures of its free and peptide-bound states differ in the orientation of helix alpha2 with respect to the remainder of the molecule, concomitant with a readjustment of the hydrophobic core. Using an ultrafast mixing instrument, we detected a deviation from simple bimolecular kinetics for the association with peptide that is consistent with a rate-limiting conformational change in the protein (k(obs) approximately 7 x 10(3) s(-1)) and an induced-fit model. Furthermore, the binding kinetics of 15 mutants revealed that binding is regulated by long-range interactions, which can be correlated with the structural rearrangements resulting from peptide binding. The homologous protein PSD-95 PDZ3 did not display a similar ligand-induced conformational change.
Proceedings of the National Academy of Sciences, 2003
Although conformational changes are essential for the function of proteins, little is known about... more Although conformational changes are essential for the function of proteins, little is known about their structural dynamics at atomic level resolution. Myoglobin (Mb) is the paradigm to investigate conformational dynamics because it is a simple globular heme protein displaying a photosensitivity of the iron-ligand bond. Upon laser photodissociation of carboxymyoglobin Mb a nonequilibrium population of protein structures is generated that relaxes over a broad time range extending from picoseconds to milliseconds. This process is associated with migration of the ligand to cavities in the matrix and with a reduction in the geminate rebinding rate by several orders of magnitude. Here we report nanosecond time-resolved Laue diffraction data to 1.55-Å resolution on a Mb mutant, which depicts the sequence of structural events associated with this extended relaxation. Motions of the distal E-helix, including the mutated residue Gln-64(E7), and of the CD-turn are found to lag significantly (100 -300 ns) behind local rearrangements around the heme such as heme tilting, iron motion out of the heme plane, and swinging of the mutated residue Tyr-29(B10), all of which occur promptly (<3 ns). Over the same delayed time range, CO is observed to migrate from a cavity distal to the heme known to bind xenon (called Xe4) to another such cavity proximal to the heme (Xe1). We propose that the extended relaxation of the globin moiety reflects reequilibration among conformational substates known to play an essential role in controlling protein function. myoglobin ͉ protein dynamics ͉ conformational states ͉ functional intermediates ͉ protein crystallography
Proceedings of the National Academy of Sciences, 2007
X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbi... more X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbing atom and therefore allows bond distances and angles of the surrounding atomic cluster to be measured with atomic resolution. By contrast, the accuracy and resolution of metalloprotein active sites obtainable from x-ray crystallography are often insufficient to analyze the electronic properties of the metals that are essential for their biological functions. Here, we demonstrate that the combination of both methods on the same metalloprotein single crystal yields a structural model of the protein with exceptional active-site resolution. To this end, we have collected an x-ray diffraction data set to 1.4-Å resolution and Fe K-edge polarized x-ray absorption near edge structure (XANES) spectra on the same cyanomet sperm whale myoglobin crystal. The XANES spectra were quantitatively analyzed by using a method based on the multiple scattering approach, which yielded Fe-heme structural parameters with ؎(0.02-0.07)-Å accuracy on the atomic distances and ؎7°on the Fe-CN angle. These XANES-derived parameters were subsequently used as restraints in the crystal structure refinement. By combining XANES and x-ray diffraction, we have obtained an cyanomet sperm whale myoglobin structural model with a higher precision of the bond lengths and angles at the active site than would have been possible with crystallographic analysis alone. myoglobin ͉ crystallographic restraints ͉ protein crystallography ͉ synchroton radiation M etalloproteins perform a wide range of biological functions essential for sustaining life; they are particularly abundant in bioenergetic pathways, e.g., photosynthesis and respiration (1, 2). Often, the metal ion is located at the active site of an enzyme, where an intricate coordination geometry finetunes the electronic properties of the metal so as to enable it to catalyze specific reactions. X-ray diffraction (XRD) on protein single crystals or high-field NMR on protein solutions are the standard techniques by which entire molecular structures of proteins including their active-site metal clusters can be analyzed. By using quantum-chemical refinement, precise structural models of metal sites have been obtained from XRD data (3). X-ray absorption spectroscopy (XAS) is an alternative technique for measuring the structure of metal sites (4, 5). Electrons are liberated from the metal ion by the photoelectric effect, and the interference effects of the electron waves are analyzed to map out the local environment of the metal. XAS can reveal structural parameters with a very high precision, comparable to that of small-molecule crystallography. Such detailed structural data are required for elucidating the function of metal centers in a metalloprotein (6), as structural changes to the metal coordination during redox or substrate-binding reactions are generally Ͻ0.1 Å and hence remain unnoticed in standard protein crys-tallography unless the resolution is exceptionally high. As yet, x-ray structures of proteins at true atomic resolution (Ͻ1.2 Å) are still scarce .
Proceedings of the National Academy of Sciences, 2006
Work carried out over the last 30 years unveiled the role of structural dynamics in controlling p... more Work carried out over the last 30 years unveiled the role of structural dynamics in controlling protein function. Cavity networks modulate structural dynamics trajectories and are functionally relevant; in globins they have been assigned a role in ligand migration and docking. These findings raised renewed interest for time-resolved structural investigations of myoglobin (Mb), a simple heme protein displaying a photosensitive iron-ligand bond. Photodissociation of MbCO generates a nonequilibrium population of protein structures relaxing over a time range extending from picoseconds to milliseconds. This process triggers ligand migration to matrix cavities with clear-cut effects on the rate and yield of geminate rebinding. Here, we report subnanosecond time-resolved Laue diffraction data on the triple mutant YQR-Mb [Leu-29(B10)Tyr, His-64(E7)Gln, Thr-67(E10)Arg] that depict the sequence of structural events associated with heme and protein relaxation from 100 ps to 316 ns and above. The photodissociated ligand rapidly (<0.1 ns) populates the Xe-binding cavity distal to the heme. Moreover, the heme relaxation toward the deoxy configuration is heterogeneous, with a slower phase (Ϸns) evident in these experiments. Damping of the heme response appears to result from a strain exerted by the E-helix via the CD-turn; Phe-43(CD1), in close contact with heme, opposes tilt until the strain is relieved. A comparison with crystallographic data on wild-type Mb and mutants Leu(29)Phe or Leu(29)Trp suggests that the internal structure controls the rate and amplitude of the relaxation events. A correlation between structural dynamics as unveiled by Laue crystallography and functional properties of Mb is presented.
Nucleic Acids Research, 2013
Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and expor... more Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex-binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplexbinding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.
Journal of Thrombosis and Haemostasis, 2013
Background: This work was aimed at characterizing the interaction of b 2 -glycoprotein I (b 2 GPI... more Background: This work was aimed at characterizing the interaction of b 2 -glycoprotein I (b 2 GPI), an abundant plasma protein of unknown function, with human thrombin, the final effector protease in the coagulation cascade. Methods: The b 2 GPI-thrombin interaction was studied by surface plasmon resonance (SPR), fluorescence, and molecular modeling. The effect of b 2 GPI on the procoagulant (fibrin generation and platelet aggregation) and anticoagulant (protein C activation) functions of thrombin were investigated with turbidimetric, immunocytofluorimetric and enzymatic assays. Results: SPR and fluorescence data indicated that b 2 GPI tightly bound thrombin (K d = 34 nM) by interacting with both protease exosites, while leaving the active site accessible. This picture is fully consistent with the theoretical model of the b 2 GPI-thrombin complex. In particular, blockage of thrombin exosites with binders specific for exosite-1 (hirugen and HD1 aptamer) or exosite-2 (fibrinogen c′-peptide and HD22 aptamer) impaired the b 2 GPI-thrombin interaction. Identical results were obtained with thrombin mutants having one of the two exosites selectively compromised by mutation (Arg73Ala and Arg101Ala). Fluorescence measurements indicated that b 2 GPI did not affect the affinity of the enzyme for active site inhibitors, such as p-aminobenzamidine and the hirudin(1-47) domain, in agreement with the structural model. b 2 GPI dose-dependently prolonged the thrombin clotting time and ecarin clotting time in b 2 GPI-deficient plasma. b 2 GPI inhibited thrombin-induced platelet aggregation (IC 50 = 0.36 lM) by impairing thrombin cleavage of protease-activated receptor 1 (PAR1) (IC 50 = 0.32 lM), both on gel-filtered platelets and in whole blood. Strickingly, b 2 GPI did not affect thrombin-mediated generation of the anticoagulant protein C. Conclusions: b 2 GPI functions as a physiologic anticoagulant by inhibiting the key procoagulant activities of thrombin without affecting its unique anticoagulant function.
Journal of the American Chemical Society, 2010
The effect of structural disorder on the X-ray absorption near-edge structure (XANES) spectrum of... more The effect of structural disorder on the X-ray absorption near-edge structure (XANES) spectrum of a heme protein has been investigated using the dynamical description of the system derived from molecular dynamics (MD) simulations. The XANES spectra of neuroglobin (Ngb) and carbonmonoxy-neuroglobin (NgbCO) have been quantitatively reproduced, starting from the MD geometrical configurations, without carrying out any optimization in the structural parameter space. These results provide an important experimental validation of the reliability of the potentials used in the MD simulations and accordingly corroborate the consistency of the structural dynamic information on the metal center, related to its biological function. This analysis allowed us to demonstrate that the configurational disorder associated with the distortion of the heme plane and with the different orientations of the axial ligands can affect the XANES features at very low energy. Neglecting configurational disorder in the XANES quantitative analysis of heme proteins is a source of systematic errors in the determination of Fe coordination geometry. The combined use of XANES and MD is a novel strategy to enhance the resolution and reliability of the structural information obtained on metalloproteins, making the combination of these techniques powerful for metalloprotein investigations.
Journal of Synchrotron Radiation, 1999
Visible light can break the Fe-CO bond in Fe (II) carbonmonoxy-myoglobin (MbCO) giving an unligat... more Visible light can break the Fe-CO bond in Fe (II) carbonmonoxy-myoglobin (MbCO) giving an unligated product (Mb*) that is almost stable at T&lt; 30 K. Fe K-edge polarized X-ray absorption spectra (P-XAS) of the photoproduct (T= 20 K) of an oriented single crystal (0.2 ...