Sofia R. Pauleta
Universidade Nova de Lisboa
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Featured researches published by Sofia R. Pauleta.
Proceedings of the National Academy of Sciences of the United States of America | 2014
Andrea Saponaro; Sofia R. Pauleta; Francesca Cantini; Manolis Matzapetakis; Christian Hammann; Chiara Donadoni; Lei Hu; Gerhard Thiel; Lucia Banci; Bina Santoro; Anna Moroni
Significance cAMP regulation of ion channels controls higher brain functions, such as sleep, memory, and cognition. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by the direct binding of cAMP to their cytoplasmic tail and inhibited by the neuronal β-subunit tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), which prevents cAMP binding. Understanding the molecular mechanisms of regulation of this family of ion channels is critical because it pertains to the physiological processes and diseases associated with dysfunctions in the HCN current. Here, we explain the dual regulatory system of HCN2 channels in atomic detail. cAMP and TRIP8b do not compete for the same binding site on the HCN2 cytoplasmic tail; rather, they exert their mutual competition by promoting and stabilizing two different conformational states of the protein. cAMP signaling in the brain mediates several higher order neural processes. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels directly bind cAMP through their cytoplasmic cyclic nucleotide binding domain (CNBD), thus playing a unique role in brain function. Neuronal HCN channels are also regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit that antagonizes the effects of cAMP by interacting with the channel CNBD. To unravel the molecular mechanisms underlying the dual regulation of HCN channel activity by cAMP/TRIP8b, we determined the NMR solution structure of the HCN2 channel CNBD in the cAMP-free form and mapped on it the TRIP8b interaction site. We reconstruct here the full conformational changes induced by cAMP binding to the HCN channel CNBD. Our results show that TRIP8b does not compete with cAMP for the same binding region; rather, it exerts its inhibitory action through an allosteric mechanism, preventing the cAMP-induced conformational changes in the HCN channel CNBD.
Biochemistry | 2008
Simone Dell’Acqua; Sofia R. Pauleta; Enrico Monzani; Alice S. Pereira; Luigi Casella; José J. G. Moura; Isabel Moura
The multicopper enzyme nitrous oxide reductase (N 2OR) catalyzes the final step of denitrification, the two-electron reduction of N 2O to N 2. This enzyme is a functional homodimer containing two different multicopper sites: CuA and CuZ. CuA is a binuclear copper site that transfers electrons to the tetranuclear copper sulfide CuZ, the catalytic site. In this study, Pseudomonas nautica cytochrome c 552 was identified as the physiological electron donor. The kinetic data show differences when physiological and artificial electron donors are compared [cytochrome vs methylviologen (MV)]. In the presence of cytochrome c 552, the reaction rate is dependent on the ET reaction and independent of the N 2O concentration. With MV, electron donation is faster than substrate reduction. From the study of cytochrome c 552 concentration dependence, we estimate the following kinetic parameters: K m c 552 = 50.2 +/- 9.0 muM and V max c 552 = 1.8 +/- 0.6 units/mg. The N 2O concentration dependence indicates a K mN 2 O of 14.0 +/- 2.9 muM using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c 552 is used as the electron donor (p K a = 6.6 or 8.3, respectively). The kinetic study also revealed the hydrophobic nature of the interaction, and direct electron transfer studies showed that CuA is the center that receives electrons from the physiological electron donor. The formation of the electron transfer complex was observed by (1)H NMR protein-protein titrations and was modeled with a molecular docking program (BiGGER). The proposed docked complexes corroborated the ET studies giving a large number of solutions in which cytochrome c 552 is placed near a hydrophobic patch located around the CuA center.
Journal of Biological Inorganic Chemistry | 2011
Simone Dell’Acqua; Sofia R. Pauleta; Isabel Moura; José J. G. Moura
This review focuses on the novel CuZ center of nitrous oxide reductase, an important enzyme owing to the environmental significance of the reaction it catalyzes, reduction of nitrous oxide, and the unusual nature of its catalytic center, named CuZ. The structure of the CuZ center, the unique tetranuclear copper center found in this enzyme, opened a novel area of research in metallobiochemistry. In the last decade, there has been progress in defining the structure of the CuZ center, characterizing the mechanism of nitrous oxide reduction, and identifying intermediates of this reaction. In addition, the determination of the structure of the CuZ center allowed a structural interpretation of the spectroscopic data, which was supported by theoretical calculations. The current knowledge of the structure, function, and spectroscopic characterization of the CuZ center is described here. We would like to stress that although many questions have been answered, the CuZ center remains a scientific challenge, with many hypotheses still being formed.
Journal of Biological Inorganic Chemistry | 2008
Isabel Moura; Sofia R. Pauleta; José J. G. Moura
Metalloenzymes control enzymatic activity by changing the characteristics of the metal centers where catalysis takes place. The conversion between inactive and active states can be tuned by altering the coordination number of the metal site, and in some cases by an associated conformational change. These processes will be illustrated using heme proteins (cytochrome c nitrite reductase, cytochrome c peroxidase and cytochrome cd1 nitrite reductase), non-heme proteins (superoxide reductase and [NiFe]-hydrogenase), and copper proteins (nitrite and nitrous oxide reductases) as examples. These examples catalyze electron transfer reactions that include atom transfer, abstraction and insertion.
Journal of Bacteriology | 2011
Anouchka Fiévet; Laetitia My; Eric Cascales; Mireille Ansaldi; Sofia R. Pauleta; Isabel Moura; Zorah Dermoun; Christophe S. Bernard; Alain Dolla; Corinne Aubert
Analysis of sequenced bacterial genomes revealed that the genomes encode more than 30% hypothetical and conserved hypothetical proteins of unknown function. Among proteins of unknown function that are conserved in anaerobes, some might be determinants of the anaerobic way of life. This study focuses on two divergent clusters specifically found in anaerobic microorganisms and mainly composed of genes encoding conserved hypothetical proteins. We show that the two gene clusters DVU2103-DVU2104-DVU2105 (orp2) and DVU2107-DVU2108-DVU2109 (orp1) form two divergent operons transcribed by the σ(54)-RNA polymerase. We further demonstrate that the σ(54)-dependent transcriptional regulator DVU2106, located between orp1 and orp2, collaborates with σ(54)-RNA polymerase to orchestrate the simultaneous expression of the divergent orp operons. DVU2106, whose structural gene is transcribed by the σ(70)-RNA polymerase, negatively retrocontrols its own expression. By using an endogenous pulldown strategy, we identify a physiological complex composed of DVU2103, DVU2104, DVU2105, DVU2108, and DVU2109. Interestingly, inactivation of DVU2106, which is required for orp operon transcription, induces morphological defects that are likely linked to the absence of the ORP complex. A putative role of the ORP proteins in positioning the septum during cell division is discussed.
Journal of Inorganic Biochemistry | 2009
Rui M. Almeida; Sofia R. Pauleta; Isabel Moura; José J. G. Moura
The paramagnetic effect due to the presence of a metal center with unpaired electrons is no longer considered a hindrance in protein NMR spectroscopy. In the present work, the paramagnetic effect due to the presence of a metal center with unpaired electrons was used to map the interface of an electron transfer complex. Desulfovibrio gigas cytochrome c(3) was chosen as target to study the effect of the paramagnetic probe, Fe-rubredoxin, which produced specific line broadening in the heme IV methyl resonances M2(1) and M18(1). The rubredoxin binding surface in the complex with cytochrome c(3) was identified in a heteronuclear 2D NMR titration. The identified heme methyls on cytochrome c(3) are involved in the binding interface of the complex, a result that is in agreement with the predicted complexes obtained by restrained molecular docking, which shows a cluster of possible solutions near heme IV. The use of a paramagnetic probe in (1)HNMR titration and the mapping of the complex interface, in combination with a molecular simulation algorithm proved to be a valuable strategy to study electron transfer complexes involving non-heme iron proteins and cytochromes.
Philosophical Transactions of the Royal Society B | 2012
Simone Dell'Acqua; Sofia R. Pauleta; José J. G. Moura; Isabel Moura
Nitrous oxide reductase (N2OR) catalyses the final step of the denitrification pathway—the reduction of nitrous oxide to nitrogen. The catalytic centre (CuZ) is a unique tetranuclear copper centre bridged by inorganic sulphur in a tetrahedron arrangement that can have different oxidation states. Previously, Marinobacter hydrocarbonoclasticus N2OR was isolated with the CuZ centre as CuZ*, in the [1Cu2+ : 3Cu+] redox state, which is redox inert and requires prolonged incubation under reductive conditions to be activated. In this work, we report, for the first time, the isolation of N2OR from M. hydrocarbonoclasticus in the ‘purple’ form, in which the CuZ centre is in the oxidized [2Cu2+ : 2Cu+] redox state and is redox active. This form of the enzyme was isolated in the presence of oxygen from a microaerobic culture in the presence of nitrate and also from a strictly anaerobic culture. The purple form of the enzyme was biochemically characterized and was shown to be a redox active species, although it is still catalytically non-competent, as its specific activity is lower than that of the activated fully reduced enzyme and comparable with that of the enzyme with the CuZ centre in either the [1Cu2+ : 3Cu+] redox state or in the redox inactive CuZ* state.
Journal of Inorganic Biochemistry | 2009
Xiangjin Xie; Ryan G. Hadt; Sofia R. Pauleta; Pablo J. González; Sun Un; Isabel Moura; Edward I. Solomon
The blue or Type 1 (T1) copper site of Paracoccuspantotrophus pseudoazurin exhibits significant absorption intensity in both the 450 and 600 nm regions. These are sigma and pi S(Cys) to Cu(2+) charge transfer (CT) transitions. The temperature dependent absorption, EPR, and resonance Raman (rR) vibrations enhanced by these bands indicate that a single species is present at all temperatures. This contrasts the temperature dependent behavior of the T1 center in nitrite reductase [S. Ghosh, X. Xie, A. Dey, Y. Sun, C. Scholes, E. Solomon, Proc. Natl. Acad. Sci. 106 (2009) 4969-4974] which has a thioether ligand that is unconstrained by the protein. The lack of temperature dependence in the T1 site in pseudoazurin indicates the presence of a protein constraint similar to the blue Cu site in plastocyanin where the thioether ligand is constrained at 2.8 A. However, plastocyanin exhibits only pi CT. This spectral difference between pseudoazurin and plastocyanin reflects a coupled distortion of the site where the axial thioether in pseudoazurin is also constrained, but at a shorter Cu-S(Met) bond length. This leads to an increase in the Cu(2+)-S(Cys) bond length, and the site undergoes a partial tetragonal distortion in pseudoazurin. Thus, its ground state wavefunction has both sigma and pi character in the Cu(2+)-S(Cys) bond.
Biomolecular Nmr Assignments | 2007
Sofia R. Pauleta; Américo G. Duarte; Marta S. P. Carepo; Alice S. Pereira; Pedro Tavares; Isabel Moura; José J. G. Moura
We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo–Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using 13C-detection experiments.
Journal of Inorganic Biochemistry | 2012
Ryan G. Hadt; Xiangjin Xie; Sofia R. Pauleta; Isabel Moura; Edward I. Solomon
The short Cu(2+)-S(Met) bond in pseudoazurin (PAz) results in the presence of two relatively intense S(p)(π) and S(p)(σ) charge transfer (CT) transitions. This has enabled resonance Raman (rR) data to be obtained for each excited state. The rR data show very different intensity distribution patterns for the vibrations in the 300-500 cm(-1) region. Time-dependent density functional theory (TDDFT) calculations have been used to determine that the change in intensity distribution between the S(p)(π) and S(p)(σ) excited states reflects the differential enhancement of S(Cys) backbone modes with Cu-S(Cys)-C(β) out-of-plane (oop) and in-plane (ip) bend character in their respective potential energy distributions (PEDs). The rR excited state distortions have been related to ground state reorganization energies (λ s) and predict that, in addition to M-L stretches, the Cu-S(Cys)-C(β) oop bend needs to be considered. DFT calculations predict a large distortion in the Cu-S(Cys)-C(β) oop bending coordinate upon reduction of a blue copper (BC) site; however, this distortion is not present in the X-ray crystal structures of reduced BC sites. The lack of Cu-S(Cys)-C(β) oop distortion upon reduction corresponds to a previously unconsidered constraint on the thiolate ligand orientation in the reduced state of BC proteins and can be considered as a contribution to the entatic/rack nature of BC sites.