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Cerqueira, NMFSA, Coelho C, Bras NF, Fernandes PA, Garattini E, Terao M, Romao MJ, Ramos MJ.  2015.  Insights into the structural determinants of substrate specificity and activity in mouse aldehyde oxidases. Journal of Biological Inorganic Chemistry. 20:209-217., Number 2 AbstractWebsite

In this work, a combination of homology modeling and molecular dynamics (MD) simulations was used to investigate the factors that modulate substrate specificity and activity of the mouse AOX isoforms: mAOX1, mAOX2 (previously mAOX3l1), mAOX3 and mAOX4. The results indicate that the AOX isoform structures are highly preserved and even more conserved than the corresponding amino acid sequences. The only differences are at the protein surface and substrate-binding site region. The substrate-binding site of all isoforms consists of two regions: the active site, which is highly conserved among all isoforms, and a isoform-specific region located above. We predict that mAOX1 accepts a broader range of substrates of different shape, size and nature relative to the other isoforms. In contrast, mAOX4 appears to accept a more restricted range of substrates. Its narrow and hydrophobic binding site indicates that it only accepts small hydrophobic substrates. Although mAOX2 and mAOX3 are very similar to each other, we propose the following pairs of overlapping substrate specificities: mAOX2/mAOX4 and mAOX3/mAXO1. Based on these considerations, we propose that the catalytic activity between all isoforms should be similar but the differences observed in the binding site might influence the substrate specificity of each enzyme. These results also suggest that the presence of several AOX isoforms in mouse allows them to oxidize more efficiently a wider range of substrates. This contrasts with the same or other organisms that only express one isoform and are less efficient or incapable of oxidizing the same type of substrates.

Kowacz, M, Marchel M, Juknaite L, Esperanca J, Romao MJ, Carvalho AL, Rebelo LPN.  2015.  Ionic-Liquid-Functionalized Mineral Particles for Protein Crystallization. Crystal Growth & Design. 15:2994-3003., Number 6 AbstractWebsite

Nucleation is a critical step determining the outcome of the entire crystallization process. Finding an effective nucleant for protein crystallization is of utmost importance for structural biology. The latter relies on good-quality crystals to solve the three-dimensional structures of macromolecules. In this study we show that crystalline barium sulfate (BaSO4) with an etched and/or ionic liquid (IL)-functionalized surface (1) can induce protein nucleation at concentrations well below the concentration needed to promote crystal growth under control conditions, (2) can shorten the nucleation time, (3) can increase the growth rate, and finally (4) may help to improve the protein crystal morphology. These effects were shown for lysozyme, RNase A, trypsin, proteinase K, myoglobin, and hemoglobin. Therefore, the use of BaSO4 particles enables us to reduce the amount of protein in crystallization trials and increases the chance of obtaining protein crystals of the desired quality. In the context of the underlying mechanism, it is shown that the protein-solid contact formation is governed by the interaction of the polar compartments of the biomacromolecule with the support. The tendency of a protein to concentrate near the solid surface is enhanced by both the hydrophobicity of the protein and that of the surface (tuned by the functionalizing IL). These mechanisms of interaction of biomacromolecules with inorganic hydrophilic solids correspond to the principles of amphiphilic IL-mineral interactions.

Palma, AS, Liu Y, Zhang H, Zhang Y, McCleary BV, Yu G, Huang Q, Guidolin LS, Ciocchini AE, Torosantucci A, Wang D, Carvalho AL, Fontes CM, Mulloy B, Childs RA, Feizi T, Chai W.  2015.  Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry. Mol Cell Proteomics. AbstractWebsite

Glucans are polymers of D-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes including immunomodulation, anti-cancer activities, pathogen virulence and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure-function studies and their exploitation. We describe construction of a glucome microarray, the first sequence-defined glycome-scale microarray, using a designer approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. The negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear homo and hetero and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signalling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.

Otrelo-Cardoso, AR, Nair RR, Correia MAS, Rivas MG, Santos-Silva T.  2014.  TupA: A Tungstate Binding Protein in the Periplasm of Desulfovibrio alaskensis G20, 2014/05/29/accep. International Journal of Molecular Sciences. 15(7):11783-11798.: MDPI AbstractWebsite

The TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis as the energy source (the reaction catalyzed by the ModC component). We report the heterologous expression, purification, determination of affinity binding constants and crystallization of the Desulfovibrio alaskensis G20 TupA. The tupA gene (locus tag Dde_0234) was cloned in the pET46 Enterokinase/Ligation-Independent Cloning (LIC) expression vector, and the construct was used to transform BL21 (DE3) cells. TupA expression and purification were optimized to a final yield of 10 mg of soluble pure protein per liter of culture medium. Native polyacrylamide gel electrophoresis was carried out showing that TupA binds both tungstate and molybdate ions and has no significant interaction with sulfate, phosphate or perchlorate. Quantitative analysis of metal binding by isothermal titration calorimetry was in agreement with these results, but in addition, shows that TupA has higher affinity to tungstate than molybdate. The protein crystallizes in the presence of 30% (w/v) polyethylene glycol 3350 using the hanging-drop vapor diffusion method. The crystals diffract X-rays beyond 1.4 Å resolution and belong to the P2(1) space group, with cell parameters a = 52.25 Å, b = 42.50 Å, c = 54.71 Å, β = 95.43°. A molecular replacement solution was found, and the structure is currently under refinement.

Otrelo-Cardoso, AR, Schwuchow V, Rodrigues D, Cabrita EJ, Leimkuehler S, Romao MJ, Santos-Silva T.  2014.  Biochemical, Stabilization and Crystallization Studies on a Molecular Chaperone (PaoD) Involved in the Maturation of Molybdoenzymes. Plos One. 9, Number 1 AbstractWebsite
Gao, C, Liu Y, Zhang H, Zhang Y, Fukuda MN, Palma AS, Kozak RP, Childs RA, Nonaka M, Li Z, Siegel DL, Hanfland P, Peehl DM, Chai W, Greene MI, Feizi T.  2014.  Carbohydrate sequence of the prostate cancer-associated antigen F77 assigned by a mucin O-glycome designer array. J Biol Chem. 289:16462-77., Number 23 AbstractWebsite

Monoclonal antibody F77 was previously raised against human prostate cancer cells and has been shown to recognize a carbohydrate antigen, but the carbohydrate sequence of the antigen was elusive. Here, we make multifaceted approaches to characterize F77 antigen, including binding analyses with the glycolipid extract of the prostate cancer cell line PC3, microarrays with sequence-defined glycan probes, and designer arrays from the O-glycome of an antigen-positive mucin, in conjunction with mass spectrometry. Our results reveal F77 antigen to be expressed on blood group H on a 6-linked branch of a poly-N-acetyllactosamine backbone. We show that mAb F77 can also bind to blood group A and B analogs but with lower intensities. We propose that the close association of F77 antigen with prostate cancers is a consequence of increased blood group H expression together with up-regulated branching enzymes. This is in contrast to other epithelial cancers that have up-regulated branching enzymes but diminished expression of H antigen. With knowledge of the structure and prevalence of F77 antigen in prostate cancer, the way is open to explore rationally its application as a biomarker to detect F77-positive circulating prostate cancer-derived glycoproteins and tumor cells.

Suits, MD, Pluvinage B, Law A, Liu Y, Palma AS, Chai W, Feizi T, Boraston AB.  2014.  Conformational analysis of the Streptococcus pneumoniae hyaluronate lyase and characterization of its hyaluronan-specific carbohydrate-binding module. J Biol Chem. 289:27264-77., Number 39 AbstractWebsite

For a subset of pathogenic microorganisms, including Streptococcus pneumoniae, the recognition and degradation of host hyaluronan contributes to bacterial spreading through the extracellular matrix and enhancing access to host cell surfaces. The hyaluronate lyase (Hyl) presented on the surface of S. pneumoniae performs this role. Using glycan microarray screening, affinity electrophoresis, and isothermal titration calorimetry we show that the N-terminal module of Hyl is a hyaluronan-specific carbohydrate-binding module (CBM) and the founding member of CBM family 70. The 1.2 Å resolution x-ray crystal structure of CBM70 revealed it to have a β-sandwich fold, similar to other CBMs. The electrostatic properties of the binding site, which was identified by site-directed mutagenesis, are distinct from other CBMs and complementary to its acidic ligand, hyaluronan. Dynamic light scattering and solution small angle x-ray scattering revealed the full-length Hyl protein to exist as a monomer/dimer mixture in solution. Through a detailed analysis of the small angle x-ray scattering data, we report the pseudoatomic solution structures of the monomer and dimer forms of the full-length multimodular Hyl.

Palma, AS, Feizi T, Childs RA, Chai W, Liu Y.  2014.  The neoglycolipid (NGL)-based oligosaccharide microarray system poised to decipher the meta-glycome. Curr Opin Chem Biol. 18:87-94. AbstractWebsite

The neoglycolipid (NGL) technology is the basis of a state-of-the-art oligosaccharide microarray system. The NGL-based microarray system in the Glycosciences Laboratory Imperial College London ( is one of the two leading platforms for glycan microarrays, being offered for screening analyses to the broad biomedical community. Highlighted in this review are the sensitivity of the analysis system and, coupled with mass spectrometry, the provision for generating 'designer' microarrays from glycomes to identify novel ligands of biological relevance. Among recent applications are assignments of ligands for apicomplexan parasites, pandemic 2009 influenza virus, polyoma and reoviruses, an innate immune receptor against fungal pathogens, Dectin-1, and a novel protein of the endoplasmic reticulum, malectin; also the characterization of an elusive cancer-associated antigen. Some other contemporary advances in glycolipid-containing arrays and microarrays are also discussed.

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.

Otrelo-Cardoso, AR, da Silva Correia MA, Schwuchow V, Svergun DI, Romao MJ, Leimkuehler S, Santos-Silva T.  2014.  Structural Data on the Periplasmic Aldehyde Oxidoreductase PaoABC from Escherichia coli: SAXS and Preliminary X-ray Crystallography Analysis. International Journal of Molecular Sciences. 15:2223-2236., Number 2 AbstractWebsite
Ribeiro, D, Kulakova A, Quaresma P, Pereira E, Bonifacio C, Romao MJ, Franco R, Carvalho AL.  2014.  Use of Gold Nanoparticles as Additives in Protein Crystallization. Crystal Growth & Design. 14:222-227., Number 1 AbstractWebsite

Gold nanoparticles (AuNPs) exhibit unique properties that have made them a very attractive material for application in biological assays. Given the potentially interesting interactions between AuNPs and biological macromolecules, we investigated AuNPs-induced protein crystal growth. Differently functionalized AuNPs were tested as additives in cocrystallization studies with model proteins (hen egg white lysozyme (HEWL), ribonuclease A (RNase A), and proteinase K) as well as with case studies where there were problems in obtaining well-diffracting crystals. Trials were performed considering different crystallization drawbacks, from total absence of crystals to improvement of crystal morphology, size, twinning, and number of crystals per drop. Improvement of some of these factors was observed in the cases of HEWL, RNase A, phenylalanine hydroxylase (PAR), myoglobin, native aldehyde oxidase (AOH), and human albumin. In these proteins, the presence of the AuNPs promoted an increase in the size and/or better crystal morphology. From the systematic trials and subsequent observations, it can be concluded that the introduction of AuNPs should definitely be considered in crystal optimization trials to improve previously determined crystallization conditions.

Santos, MFA, Correia I, Oliveira AR, Garribba E, Pessoa JC, Santos-Silva T.  2014.  Vanadium Complexes as Prospective Therapeutics: Structural Characterization of a VIV Lysozyme Adduct. European Journal of Inorganic Chemistry. :n/a–n/a.: WILEY-VCH Verlag AbstractWebsite

The biological activity of vanadium complexes, namely, as insulin enhancers, is well known. We report a combined X-ray crystallography, electron paramagnetic resonance, and density functional theory study of the interaction of vanadium picolinate complexes with hen egg white lysozyme (HEWL). We show that the VIVO(pic)2 complex covalently binds to the COO– group of the side chain of Asp52 of HEWL. The long VIV=O bond obtained in the X-ray study is explained to be due to reduction of VIV to VIII during exposure of the crystals to the intense X-ray beam.

Crusat, M, Liu J, Palma AS, Childs RA, Liu Y, Wharton SA, Lin YP, Coombs PJ, Martin SR, Matrosovich M, Chen Z, Stevens DJ, Hien VM, Thanh TT, le Nhu NT, Nguyet LA, do Ha Q, van Doorn HR, Hien TT, Conradt HS, Kiso M, Gamblin SJ, Chai W, Skehel JJ, Hay AJ, Farrar J, de Jong MD, Feizi T.  2013.  Changes in the hemagglutinin of H5N1 viruses during human infection–influence on receptor binding. Virology. 447:326-37., Number 1-2 AbstractWebsite

As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process.

Seixas, JD, Mukhopadhyay A, Santos-Silva T, Otterbein LE, Gallo DJ, Rodrigues SS, Guerreiro BH, Goncalves AML, Penacho N, Marques AR, Coelho AC, Reis PM, Romao MJ, Romao CC.  2013.  Characterization of a versatile organometallic pro-drug (CORM) for experimental CO based therapeutics. Dalton Transactions. 42:5985-5998., Number 17 AbstractWebsite
Mukhopadhyay, A, Bursakov SA, Ramos JL, Wittich RM, Kladova AV, Romao MJ, van Dillewijn P, Carvalho AL.  2013.  Determinants of selective group reduction in the TNT-bound xenobiotic reductase B from P. putida. European Biophysics Journal with Biophysics Letters. 42:S179-S179. AbstractWebsite
Mahro, M, Bras NF, Cerqueira NMFSA, Teutloff C, Coelho C, Romao MJ, Leimkuehler S.  2013.  Identification of Crucial Amino Acids in Mouse Aldehyde Oxidase 3 That Determine Substrate Specificity. Plos One. 8, Number 12 AbstractWebsite
Coelho, C, Marangon J, Rodrigues D, Moura JJG, Romao MJ, Paes de Sousa PM, Correia dos Santos MM.  2013.  Induced peroxidase activity of haem containing nitrate reductases revealed by protein film electrochemistry. Journal of Electroanalytical Chemistry. 693:105-113. AbstractWebsite
Mehtab, S, Goncalves G, Roy S, Tomaz AI, Santos-Silva T, Santos MFA, Romao MJ, Jakusch T, Kiss T, Pessoa JC.  2013.  Interaction of vanadium(IV) with human serum apo-transferrin. Journal of Inorganic Biochemistry. 121:187-195. AbstractWebsite
Marangon, J, Correia HD, Brondino CD, Moura JJG, Romao MJ, Gonzalez PJ, Santos-Silva T.  2013.  Kinetic and Structural Studies of Aldehyde Oxidoreductase from Desulfovibrio gigas Reveal a Dithiolene-Based Chemistry for Enzyme Activation and Inhibition by H2O2. Plos One. 8, Number 12 AbstractWebsite
Verma, AK, Goyal A, Freire F, Bule P, Venditto I, Bras JLA, Santos H, Cardoso V, Bonifacio C, Thompson A, Romao MJ, Prates JAM, Ferreira LMA, Fontes CMGA, Najmudin S.  2013.  Overexpression, crystallization and preliminary X-ray crystallographic analysis of glucuronoxylan xylanohydrolase (Xyn30A) from Clostridium thermocellum. Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 69:1440-1442. AbstractWebsite
Viegas, A, Sardinha J, Freire F, Duarte DF, Carvalho AL, Fontes CMGA, Romao MJ, Macedo AL, Cabrita EJ.  2013.  Solution structure, dynamics and binding studies of a family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11). Biochemical Journal. 451:289-300. AbstractWebsite
Palma, AS, Pinheiro B, Liu Y, Takeda Y, Chai W, Ito Y, Romao MJ, Carvalho AL, Feizi T.  2013.  The Structural Basis of the Recognition of Di-glucosylated N-glycans by the ER Lectin Malectin. Glycobiology. 23:1368-1369., Number 11 AbstractWebsite
Neu, U, Allen SA, Blaum BS, Liu Y, Frank M, Palma AS, Ströh LJ, Feizi T, Peters T, Atwood WJ, Stehle T.  2013.  A structure-guided mutation in the major capsid protein retargets BK polyomavirus. PLoS Pathog. 9:e1003688., Number 10 AbstractWebsite

Viruses within a family often vary in their cellular tropism and pathogenicity. In many cases, these variations are due to viruses switching their specificity from one cell surface receptor to another. The structural requirements that underlie such receptor switching are not well understood especially for carbohydrate-binding viruses, as methods capable of structure-specificity studies are only relatively recently being developed for carbohydrates. We have characterized the receptor specificity, structure and infectivity of the human polyomavirus BKPyV, the causative agent of polyomavirus-associated nephropathy, and uncover a molecular switch for binding different carbohydrate receptors. We show that the b-series gangliosides GD3, GD2, GD1b and GT1b all can serve as receptors for BKPyV. The crystal structure of the BKPyV capsid protein VP1 in complex with GD3 reveals contacts with two sialic acid moieties in the receptor, providing a basis for the observed specificity. Comparison with the structure of simian virus 40 (SV40) VP1 bound to ganglioside GM1 identifies the amino acid at position 68 as a determinant of specificity. Mutation of this residue from lysine in BKPyV to serine in SV40 switches the receptor specificity of BKPyV from GD3 to GM1 both in vitro and in cell culture. Our findings highlight the plasticity of viral receptor binding sites and form a template to retarget viruses to different receptors and cell types.

Neu, U, Khan ZM, Schuch B, Palma AS, Liu Y, Pawlita M, Feizi T, Stehle T.  2013.  Structures of B-lymphotropic polyomavirus VP1 in complex with oligosaccharide ligands. PLoS Pathog. 9:e1003714., Number 10 AbstractWebsite

B-Lymphotropic Polyomavirus (LPyV) serves as a paradigm of virus receptor binding and tropism, and is the closest relative of the recently discovered Human Polyomavirus 9 (HPyV9). LPyV infection depends on sialic acid on host cells, but the molecular interactions underlying LPyV-receptor binding were unknown. We find by glycan array screening that LPyV specifically recognizes a linear carbohydrate motif that contains α2,3-linked sialic acid. High-resolution crystal structures of the LPyV capsid protein VP1 alone and in complex with the trisaccharide ligands 3'-sialyllactose and 3'-sialyl-N-acetyl-lactosamine (3SL and 3SLN, respectively) show essentially identical interactions. Most contacts are contributed by the sialic acid moiety, which is almost entirely buried in a narrow, preformed cleft at the outer surface of the capsid. The recessed nature of the binding site on VP1 and the nature of the observed glycan interactions differ from those of related polyomaviruses and most other sialic acid-binding viruses, which bind sialic acid in shallow, more exposed grooves. Despite their different modes for recognition, the sialic acid binding sites of LPyV and SV40 are half-conserved, hinting at an evolutionary strategy for diversification of binding sites. Our analysis provides a structural basis for the observed specificity of LPyV for linear glycan motifs terminating in α2,3-linked sialic acid, and links the different tropisms of known LPyV strains to the receptor binding site. It also serves as a useful template for understanding the ligand-binding properties and serological crossreactivity of HPyV9.

Pinheiro, BA, Carvalho AL, Romao MJ, Fontes CM.  2013.  Study of the cohesin-dockerin interaction and its role in the C. thermocellum cellulosome assembly. European Biophysics Journal with Biophysics Letters. 42:S180-S180. AbstractWebsite