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De Schutter, A, Correia HD, Freire DM, Rivas MG, Rizzi A, Santos-Silva T, González PJ, Van Doorslaer S.  2015.  Ligand Binding to Chlorite Dismutase from Magnetospirillum sp, October. The journal of physical chemistry. B. 119:13859—13869., Number 43 AbstractWebsite
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dos Santos, R, Romão MJ, Roque ACA, Carvalho AL.  2021.  Magnetic particles used in a new approach for designed protein crystallization. CrystEngComm. 23:1083-1090.: The Royal Society of Chemistry AbstractWebsite

After more than one hundred and thirty thousand protein structures determined by X-ray crystallography{,} the challenge of protein crystallization for 3D structure determination remains. In the quest for additives for efficient protein crystallization{,} inorganic materials emerge as an alternative. Magnetic particles (MPs) are versatile inorganic materials{,} easy to use{,} modify and manipulate in a wide range of biological assays. The potential of using functionalised MPs as crystallization chaperones for protein crystallization was shown in this work. MPs with distinct coatings were rationally designed to promote protein crystallization by affinity-triggered heterogeneous nucleation. Hen egg white lysozyme (HEWL) and trypsin{,} were crystallized in the presence of MPs either bare or coated with a polysaccharide (chitin) or a protein (casein){,} respectively. The addition of MPs was characterized in terms of bound protein to the MPs{,} crystal morphology{,} time-lapse of crystal emergence{,} crystallization yield fold change and crystal diffraction quality for structure determination. The MPs additives have shown to bind to the respective target protein{,} and to promote nucleation and crystal growth without compromising crystal morphology. On the other hand{,} MPs addition led to faster detectable crystal emergence and up to 13 times higher crystallization yield{,} addressing some the challenges in protein crystallization{,} the main bottleneck of macromolecular crystallography. Structure determination of the protein crystallized in the presence of MPs revealed that the structural characteristics of the protein remained unchanged{,} as shown by the superposition with PDB annotated proteins. Moreover{,} and unlike most reported cases{,} it was possible to exclude the inhibitor benzamidine during trypsin crystallisation{,} which is a remarkable result opening new prospects in enzyme engineering and drug design. Our results show that MPs coated with affinity ligands to target proteins can be used as controlled and tailor-made crystallization inducers.

dos Santos, R, Iria I, Manuel AM, Leandro AP, Madeira CAC, Goncalves J, Carvalho AL, Roque AC.  2020.  Magnetic Precipitation: A New Platform for Protein Purification, 2020. Biotechnology JournalBiotechnology Journal. n/a(n/a):2000151.: John Wiley & Sons, Ltd AbstractWebsite

One of the trends in downstream processing comprises the use of ?anything-but-chromatography? methods to overcome the current downfalls of standard packed-bed chromatography. Precipitation and magnetic separation are two techniques already proven to accomplish protein purification from complex media, yet never used in synergy. With the aim to capture antibodies directly from crude extracts, a new approach combining precipitation and magnetic separation was developed and named as affinity magnetic precipitation. A precipitation screening, based on the Hofmeister series, and a commercial precipitation kit were tested with affinity magnetic particles to assess the best condition for antibody capture from human serum plasma and clarified cell supernatant. The best conditions were obtained when using PEG3350 as precipitant at 4°C for 1h, reaching 80% purity and 50% recovery of polyclonal antibodies from plasma, and 99% purity with 97% recovery yield of anti-TNFα mAb from cell supernatants. These results show that the synergetic use of precipitation and magnetic separation can represent an alternative for the efficient capture of antibodies. This article is protected by copyright. All rights reserved

Garattini, E, Mendel R, Romao MJ, Wright R, Terao M.  2003.  Mammalian molybdo-flavoenzymes, an expanding family of proteins: structure, genetics, regulation, function and pathophysiology. Biochemical Journal. 372:15-32. AbstractWebsite
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Correia, VG, Trovão F, Pinheiro BA, Brás JLA, Silva LM, Nunes C, Coimbra MA, Liu Y, Feizi T, Fontes CMGA, Mulloy B, Chai W, Carvalho AL, Palma AS.  2021.  Mapping Molecular Recognition of β1,3-1,4-Glucans by a Surface Glycan-Binding Protein from the Human Gut Symbiont Bacteroides ovatus, December. Microbiology spectrum. 9:e0182621., Number 3 AbstractWebsite

A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of <i>Bacteroidetes</i> in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage β1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBP<sub>MLG</sub>-A protein encoded by the <i>BACOVA_2743</i> gene is a SusD-like protein that plays a key role in the PUL's specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBP<sub>MLG</sub>-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBP<sub>MLG</sub>-A with a β1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward β1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of β1,3 linkages. The interaction is defined by a 41-Å-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial β1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-π interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the β-glucan backbone imposed by the β1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBP<sub>MLG</sub>-A to import long β1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows <i>Bacteroidetes</i> to outcompete bacteria that lack this PUL for utilization of β1,3-1,4-glucans. <b>IMPORTANCE</b> With the knowledge of bacterial gene systems encoding proteins that target dietary carbohydrates as a source of nutrients and their importance for human health, major efforts are being made to understand carbohydrate recognition by various commensal bacteria. Here, we describe an integrative strategy that combines carbohydrate microarray technology with structural studies to further elucidate the molecular determinants of carbohydrate recognition by BoSGBP<sub>MLG</sub>-A, a key protein expressed at the surface of Bacteroides ovatus for utilization of mixed-linkage β1,3-1,4-glucans. We have mapped at high resolution interactions that occur at the binding site of BoSGBP<sub>MLG</sub>-A and provide evidence for the role of key water-mediated interactions for fine specificity and affinity. Understanding at the molecular level how commensal bacteria, such as prominent members of <i>Bacteroidetes</i>, can differentially utilize dietary carbohydrates with potential prebiotic activities will shed light on possible ways to modulate the microbiome to promote human health.

Mota, C, Webster M, Saidi M, Kapp U, Zubieta C, Giachin G, Manso JA, de Sanctis D.  2024.  Metal ion activation and DNA recognition by the Deinococcus radiodurans manganese sensor DR2539. bioRxiv. : Cold Spring Harbor Laboratory AbstractWebsite

The accumulation of manganese ions is crucial for scavenging reactive oxygen species (ROS) and protecting the proteome of Deinococcus radiodurans (Dr). However, metal homeostasis still needs to be tightly regulated to avoid toxicity. DR2539, a dimeric transcription regulator, plays a key role in Dr manganese homeostasis. Despite comprising three well-conserved domains: a DNA binding domain, a dimerization domain, and an ancillary domain, both the metal ion activation mechanism and the DNA recognition mechanism remain elusive. In this study, we present biophysical analyses and the structure of the dimerization and DNA binding domains of DR2539 in its holo form and in complex with the 21 bp pseudo-palindromic repeat of the dr1709 promotor region. These findings shed light into the activation and recognition mechanisms. The dimer presents eight manganese binding sites that induce structural conformations essential for DNA binding. The analysis of the protein-DNA interfaces elucidates the significance of Tyr59 and helix H3 sequence in the interaction with the DNA. Finally, the structure in solution as determined by small angle X-ray scattering experiments and supported by AlphaFold modelling provides a model illustrating the conformational changes induced upon metal binding.Competing Interest StatementThe authors have declared no competing interest.

Moura, JJG, Brondino CD, Trincao J, Romao MJ.  2004.  Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases. Journal of Biological Inorganic Chemistry. 9:791-799., Number 7 AbstractWebsite
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Garcia-Alvarez, B, Melero R, Dias FMV, Prates JAM, Fontes CMGA, Smith SP, Romao MJ, Carvalho AL, Llorca O.  2011.  Molecular Architecture and Structural Transitions of a Clostridium thermocellum Mini-Cellulosome. Journal of Molecular Biology. 407:571-580., Number 4 AbstractWebsite
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Moura, I, Cabrito I, Almeida G, Cunha C, Romao MJ, Moura JJG.  2003.  Molecular aspects of denitrification/nitrate dissimilation. Journal of Inorganic Biochemistry. 96:195-195., Number 1 AbstractWebsite
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Ribeiro, DO, Viegas A, Pires VMR, Medeiros-Silva J, Bule P, Chai W, Marcelo F, Fontes CMGA, Cabrita EJ, Palma AS, Carvalho AL.  2020.  Molecular basis for the preferential recognition of β1,3-1,4-glucans by the family 11 carbohydrate-binding module from Clostridium thermocellum. The FEBS Journal. 287:2723-2743., Number 13 AbstractWebsite

Understanding the specific molecular interactions between proteins and β1,3-1,4-mixed-linked d-glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for β1,3-1,4-mixed-linked over β1,4-linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to β1,3-1,4-linked glucose oligosaccharides showed that both the chain length and the position of the β1,3-linkage are important for recognition, and identified the tetrasaccharide Glcβ1,4Glcβ1,4Glcβ1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site-directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of β1,3-1,4-mixed-linked glucans. This is mediated by a conformation–selection mechanism of the ligand in the binding cleft through CH-π stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a β1,3-linkage at the reducing end and at specific positions along the β1,4-linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed-linked β-glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture. Database Structural data are available in the Protein Data Bank under the accession codes 6R3M and 6R31.

Viegas, A, Bras NF, Cerqueira NMFSA, Fernandes PA, Prates JAM, Fontes CMGA, Bruix M, Romao MJ, Carvalho AL, Ramos MJ, Macedo AL, Cabrita EJ.  2008.  Molecular determinants of ligand specificity in family 11 carbohydrate binding modules - an NMR, X-ray crystallography and computational chemistry approach. Febs Journal. 275:2524-2535., Number 10 AbstractWebsite
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Ali, MS, Muthukumaran J, Jain M, Santos-Silva T, Al-Lohedan HA, Al-Shuail NS.  2021.  Molecular interactions of cefoperazone with bovine serum albumin: Extensive experimental and computational investigations, 2021. 337:116354. AbstractWebsite

We investigated the binding of the cephalosporin-class drug cefoperazone (CFP) with bovine serum albumin (BSA) using spectroscopic techniques and in silico methods. The aim of this study was to (i) emphasize the importance of correcting for the inner filter effect in this type of study and (ii) understand the binding mechanism of CFP with BSA by addressing protein conformation and plausible binding sites. Formation of the complex was confirmed by UV–visible spectroscopy. Quenching of BSA fluorescence in the presence of CFP was also observed. Because of the high absorption of CFP in the fluorescence emission range of BSA, the fluorescence emission spectra were corrected for the inner filter effect. Fluorescence emission was studied at excitation wavelengths of 280 and 295 nm. The uncorrected data showed a significant contribution of tyrosine at the excitation wavelength of 280 nm; however, after correction, this contribution became negligible. The static-type mechanism was found to be involved in quenching, with almost 1:1 binding between BSA and CFP. Hydrogen bonding and hydrophobic forces were found to dominate the protein–ligand interactions with a slight decrease in the α-helical contents. Synchronous fluorescence spectral data (at Δλ = 15 and 60 nm) were also corrected for the inner filter effect, with the results being similar to those of excitation at 280 and 295 nm. Molecular docking and molecular dynamics (MD) simulation results suggest that, apart from the two known drug binding sites (drug site I and II), one putative binding site (binding site III) located between BSA domains 1 and 3 was also possible for CFP. MD simulations of the previously reported drug binding sites (drug site I and II) and putative binding site III revealed that binding site III showed excellent binding profiles and could be a target for future research related to BSA-drug binding.

Santarsia, S, Grosso AS, Trovão F, Jiménez-Barbero J, Carvalho AL, Nativi C, Marcelo F.  2018.  Molecular recognition of a Thomsen-Friedenreich antigen mimetic targeting human galectin-3, 2018. ChemMedChem. Aug 9. doi: 10.1002/cmdc.201800525. [Epub ahead of print](ja): Wiley-Blackwell AbstractWebsite

Overexpression of the Thomsen-Friedenreich (TF) antigen in cell membrane proteins occurs in 90% of adenocarcinomas. Additionally, the binding of the TF-antigen to human galectin-3 (Gal-3), also frequently overexpressed in malignancy, promotes cancer progression and metastasis. In this context, structures that interfere with this specific interaction display the potential to prevent cancer metastasis. Herein, a multidisciplinary approach, combining the optimized synthesis of a TF-antigen mimetic with NMR, X-ray crystallography methods and isothermal titration calorimetry assays has been employed to unravel the molecular structural details that govern the Gal-3/TF-mimetic interaction. The TF-mimetic presents a binding affinity for Gal-3 similar to the TF-natural antigen and retains the binding epitope and the bioactive conformation observed for the native antigen. Furthermore, from a thermodynamic perspective a decrease in the enthalpic contribution was observed for the Gal-3/TF-mimetic complex, however this behaviour is compensated by a favourable entropy gain. From a structural perspective, these results establish our TF-mimetic as a scaffold to design multivalent solutions to potentially interfere with Gal-3 aberrant interactions and likely be used to hamper Gal-3-mediated cancer cells adhesion and metastasis.

Thoenes, U, Flores OL, Neves A, Devreese B, Van Beeumen JJ, Huber R, Romao MJ, Legall J, Moura JJG, Rodriguespousada C.  1994.  MOLECULAR-CLONING AND SEQUENCE-ANALYSIS OF THE GENE OF THE MOLYBDENUM-CONTAINING ALDEHYDE OXIDOREDUCTASE OF DESULFOVIBRIO-GIGAS - THE DEDUCED AMINO-ACID-SEQUENCE SHOWS SIMILARITY TO XANTHINE DEHYDROGENASE. European Journal of Biochemistry. 220:901-910., Number 3 AbstractWebsite
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Romao, MJ.  2009.  Molybdenum and tungsten enzymes: a crystallographic and mechanistic overview. Dalton Transactions. :4053-4068., Number 21 AbstractWebsite
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Brondino, CD, Romao MJ, Moura I, Moura JJG.  2006.  Molybdenum and tungsten enzymes: the xanthine oxidase family. Current Opinion in Chemical Biology. 10:109-114., Number 2 AbstractWebsite
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Romao, MJ, Cunha CA, Brondino CD, Moura JJG.  2002.  Molybdenum enzymes in reactions involving aldehydes and acids. Molybdenum and Tungsten: Their Roles in Biological Processes. 39:539-570. AbstractWebsite
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Leisico, F, Godinho LM, Gonçalves IC, Silva SP, Carneiro B, Romão MJ, Santos-Silva T, de Sá-Nogueira I.  2020.  Multitask ATPases (NBDs) of bacterial ABC importers type I and their interspecies exchangeability, 2020. 10(1):19564. AbstractWebsite

ATP-binding cassette (ABC) type I importers are widespread in bacteria and play a crucial role in its survival and pathogenesis. They share the same modular architecture comprising two intracellular nucleotide-binding domains (NBDs), two transmembrane domains (TMDs) and a substrate-binding protein. The NBDs bind and hydrolyze ATP, thereby generating conformational changes that are coupled to the TMDs and lead to substrate translocation. A group of multitask NBDs that are able to serve as the cellular motor for multiple sugar importers was recently discovered. To understand why some ABC importers share energy-coupling components, we used the MsmX ATPase from Bacillus subtilis as a model for biological and structural studies. Here we report the first examples of functional hybrid interspecies ABC type I importers in which the NBDs could be exchanged. Furthermore, the first crystal structure of an assigned multitask NBD provides a framework to understand the molecular basis of the broader specificity of interaction with the TMDs.

Hettmann, T, Siddiqui RA, Frey C, Santos-Silva T, Romao MJ, Diekmann S.  2004.  Mutagenesis study on amino acids around the molybdenum centre of the periplasmic nitrate reductase from Ralstonia eutropha. Biochemical and Biophysical Research Communications. 320:1211-1219., Number 4 AbstractWebsite
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Hettmann, T, Siddiqui RA, van Langen J, Frey C, Romao MJ, Diekmann S.  2003.  Mutagenesis study on the role of a lysine residue highly conserved in formate dehydrogenases and periplasmic nitrate reductases. Biochemical and Biophysical Research Communications. 310:40-47., Number 1 AbstractWebsite
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Peixoto, D, Malta G, Cruz H, Barroso S, Carvalho AL, Ferreira LM, Branco PS.  2019.  N-Heterocyclic olefin catalysis for the ring opening of cyclic amidine compounds: a pathway to the synthesis of ε-caprolactam and γ-lactam-derived amines, 2019. The Journal of Organic Chemistry. : American Chemical Society AbstractWebsite

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Ferreira, P, Cerqueira NMFSA, Coelho C, Fernandes PA, Romão MJ, Ramos MJ.  2019.  New insights about the monomer and homodimer structures of the human AOX1, 2019. Physical Chemistry Chemical Physics. 21(25):13545-13554.: The Royal Society of Chemistry AbstractWebsite

Human aldehyde oxidase (hAOX1) is a molybdenum dependent enzyme that plays an important role in the metabolism of various compounds either endogenous or xenobiotics. Due to its promiscuity, hAOX1 plays a major role in the pharmacokinetics of many drugs and therefore has gathered a lot of attention from the scientific community and, particularly, from the pharmaceutical industry. In this work, homology modelling, molecular docking and molecular dynamics simulations were used to study the structure of the monomer and dimer of human AOX. The results with the monomer of hAOX1 allowed to shed some light on the role played by thioridazine and two malonate ions that are co-crystalized in the recent X-ray structure of hAOX1. The results show that these molecules endorse several conformational rearrangements in the binding pocket of the enzyme and these changes have an impact in the active site topology as well as in the stability of the substrate (phthalazine). The results show that the presence of both molecules open two gates located at the entrance of the binding pocket, from which results the flooding of the active site. They also endorse several modifications in the shape of the binding pocket (namely the position of Lys893) that, together with the presence of the solvent molecules, favour the release of the substrate to the solvent. Further insights were also obtained with the assembled homodimer of hAOX1. The allosteric inhibitor (THI) binds closely to the region where the dimerization of both monomers occur. These findings suggest that THI can interfere with protein dimerization.

Santos, MFA, Seixas JD, Coelho AC, Mukhopadhyay A, Reis PM, Romao MJ, Romao CC, Santos-Silva T.  2012.  New insights into the chemistry of fac- Ru(CO)(3) (2+) fragments in biologically relevant conditions: The CO releasing activity of Ru(CO)(3)Cl-2(1,3-thiazole) , and the X-ray crystal structure of its adduct with lysozyme. Journal of Inorganic Biochemistry. 117:285-291. AbstractWebsite
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Pessoa, JC, Gonçalves G, Roy S, Correia I, Mehtab S, Santos MFA, Santos-Silva T.  2014.  New insights on vanadium binding to human serum transferrin. Inorganica Chimica Acta. 420:60-68. AbstractWebsite

Abstract The knowledge on the binding of vanadium ions and complexes to serum proteins and how vanadium might be transported in blood and up-taken by cells has received much attention during the last decade, particularly as far as the transport of VIVO2+ is concerned. In this work we revise and discuss some relevant aspects of previous research, namely the two main types of binding proposed for transport of VIVO(carrier)2 complexes. New results, obtained by circular dichroism (CD), \{EPR\} and gel electrophoresis, regarding the binding of vanadium to hTF in the oxidation states +5 and +3 are also presented. Namely, evidences for the binding of VV-species to diferric-transferrin, designated by (FeIII)2hTF, as well as to (AlIII)2hTF, are presented and discussed, the possibility of up-take of vanadate by cells through (FeIII)2hTF endocytosis being suggested. It is also confirmed that \{VIII\} binds strongly to hTF, forming di-vanadium(III)-transferrin, designated by (VIII)2hTF, and gel electrophoresis experiments indicate that (VIII)2hTF corresponds to a ‘closed conformation’ similar to (FeIII)2hTF.