Publications of Moreno Galleni
Bookmark and Share    
Full Text
See detailDetection and Characterization of VIM-52, a New Variant of VIM-1 from a Klebsiella pneumoniae Clinical Isolate
de Barsy, Marie; Mercuri, Paola ULiege; Oueslati, Saoussen et al

in Antimicrobial Agents and Chemotherapy (2021), 65(Issue 11), 02660-20

ABSTRACT Over the last two decades, antimicrobial resistance has become a global health problem. In Gram-negative bacteria, metallo-b-lactamases (MBLs), which inactivate virtually all b-lactams ... [more ▼]

ABSTRACT Over the last two decades, antimicrobial resistance has become a global health problem. In Gram-negative bacteria, metallo-b-lactamases (MBLs), which inactivate virtually all b-lactams, increasingly contribute to this phenomenon. The aim of this study is to characterize VIM-52, a His224Arg variant of VIM-1, identified in a Klebsiella pneumoniae clinical isolate. VIM-52 conferred lower MICs to cefepime and ceftazidime compared to VIM-1. These results were confirmed by steady-state kinetic measurements, where VIM-52 yielded a lower activity toward ceftazidime and cefepime but not against carbapenems. Residue 224 is part of the L10 loop (residues 221 to 241), which borders the active site. As Arg 224 and Ser 228 both play an important and interrelated role in enzymatic activity, stability, and substrate specificity for the MBLs, targeted mutagenesis at both positions was performed and further confirmed their crucial role for substrate specificity. [less ▲]

Detailed reference viewed: 29 (6 ULiège)
Full Text
See detailConversion of electrospun chitosan into chitin: a robust strategy to tune the properties of 2D biomimetic nanofiber scaffolds
Toncheva-Moncheva, Natalia; Aqil, Abdelhafid ULiege; Galleni, Moreno ULiege et al

in Polysaccharides (2021), 2(2), 271-286

New biomimetic micro- and nano-CsU-based fibrous scaffolds electrospun from solution containing high purity-medical grade chitosan (CsU) of fungus origin (CsU1, Mv ~174,000 and CsU2, 205,000, degree of ... [more ▼]

New biomimetic micro- and nano-CsU-based fibrous scaffolds electrospun from solution containing high purity-medical grade chitosan (CsU) of fungus origin (CsU1, Mv ~174,000 and CsU2, 205,000, degree of deacetylation (DDA) ~65%) and polyethylene oxide (PEO, Mv ~ 900,000), in the presence of given amounts of Triton X-100 (from 0.01 to 0.5 wt%) as surfactant were fabricated. We demonstrate that by carefully selecting compositions and surfactant levels, porous mats with CsU content up to 90% (at this molecular weight and DDA) were achieved. Remarkable long-term stability in water or phosphate buffer solution storage were obtained by developing post-electrospinning treatment allowing the complete elimination of the PEO from the CsU-fibers as demonstrated by TGA, DSC and ESEM analysis. Subsequent reacetylation procedure was applied to convert 2D biomimetic chitosan mats to chitin (CsE)-based ones while preserving the nanofiber structure. This innovative procedure allows tuning and modifying the thermal, mechanical properties and more importantly the biodegradation abilities (fast enzymatic biodegradation in some cases and slower on the others) of the prepared nanofibrous mats. The established reproducible method offers the unique advantage to modulate the membrane properties leading to stable 2D biomimetic CsU and/or chitin (CsE) scaffolds tailor-made for specific purposes in the field of tissue engineering. [less ▲]

Detailed reference viewed: 29 (3 ULiège)
Full Text
See detailLaboratory Variants GESG170L, GESG170K, and GESG170H Increase Carbapenem Hydrolysis and Confer Resistance to Clavulanic Acid
Piccirilli, Alessandra; Mercuri, Paola ULiege; Segatore, Bernardetta et al

in Antimicrobial Agents and Chemotherapy (2021), 65(6), 01931-20

Detailed reference viewed: 27 (8 ULiège)
Full Text
See detailGlycoside hydrolase family 5: structural snapshots highlighting the involvement of two conserved residues in catalysis
Collet, Laetitia ULiege; Vander Wauven, Corinne; Dutoit, Raphaël et al

in Acta Crystallographica. Section D, Structural Biology (2021)

The ability of retaining glycoside hydrolases (GHs) to transglycosylate is inherent to the double‐displacement mechanism. Studying reaction intermediates, such as the glycosyl‐enzyme intermediate (GEI ... [more ▼]

The ability of retaining glycoside hydrolases (GHs) to transglycosylate is inherent to the double‐displacement mechanism. Studying reaction intermediates, such as the glycosyl‐enzyme intermediate (GEI) and the Michaelis complex, could provide valuable information to better understand the molecular factors governing the catalytic mechanism. Here, the GEI structure of RBcel1, an endo‐1,4‐β‐glucanase of the GH5 family endowed with transglycosylase activity, is reported. It is the first structure of a GH5 enzyme covalently bound to a natural oligosaccharide with the two catalytic glutamate residues present. The structure of the variant RBcel1_E135A in complex with cellotriose is also reported, allowing a description of the entire binding cleft of RBcel1. Taken together, the structures deliver different snapshots of the double‐displacement mechanism. The structural analysis revealed a significant movement of the nucleophilic glutamate residue during the reaction. Enzymatic assays indicated that, as expected, the acid/base glutamate residue is crucial for the glycosylation step and partly contributes to deglycosylation. Moreover, a conserved tyrosine residue in the −1 subsite, Tyr201, plays a determinant role in both the glycosylation and deglycosylation steps, since the GEI was trapped in the RBcel1_Y201F variant. The approach used to obtain the GEI presented here could easily be transposed to other retaining GHs in clan GH‐A. [less ▲]

Detailed reference viewed: 29 (8 ULiège)
Full Text
See detailIdentification of b-Lactamase-Encoding (bla) Genes in Phenotypically b-Lactam-Resistant Escherichia coli Isolated from Young Calves in Belgium
Guérin, Virginie ULiege; Thiry, Damien ULiege; Lucas, Perrick et al

in Microbial Drug Resistance: Mechanism, Epidemiology, and Disease (2021)

The bla genes identification present in 94 phenotypically resistant Escherichia coli isolated from feces or intestinal contents of young calves with diarrhea or enteritis in Belgium was performed by ... [more ▼]

The bla genes identification present in 94 phenotypically resistant Escherichia coli isolated from feces or intestinal contents of young calves with diarrhea or enteritis in Belgium was performed by microarrays (MA) and whole genome sequencing (WGS). According to their resistance phenotypes to 8 b-lactams at the disk diffusion assay these 94 E. coli produced a narrow-spectrum-b-lactamase (NSBL), an extended-spectrum-b-lactamase (ESBL) or a cephalosporinase (AmpC). All ESBL-encoding genes identified by MA and WGS belonged to the blaCTX-M family, with a majority to the blaCTX-M-1 subfamily. Two different genes encoding an AmpC, blaCMY-2, and blaDHA-1 were detected in isolates with an AmpC phenotype. The blaTEM-1 and the blaOXA-1 were detected alone in isolates with a NSBL phenotype or in combination with ESBL-/AmpC-encoding bla genes. Furthermore, the WGS identified mutations in the ampC gene promoter at nucleotides -42 (C>T) and/or -18 (G>A) that could not be identified by MA, in several isolates with an AmpC-like resistance phenotype. No carbapenemaseencoding gene was detected. To our knowledge this is the first survey on the identification of bla genes in E. coli isolated from young diarrheic or septicemic calves in Belgium. [less ▲]

Detailed reference viewed: 45 (10 ULiège)
Full Text
See detailExploring the Role of L10 Loop in New Delhi Metallo- beta-lactamase (NDM-1): Kinetic and Dynamic Studies
Piccirilli, Alessandra; Criscuolo, Emanuele; Brisdelli, Fabrizia et al

in Molecules (2021), 26

Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid ... [more ▼]

Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some -lactams. Significant reduction of kcat values of L218T and L221T for carbapenems, cefazolin, cefoxitin and cefepime was observed. The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of TmB and TmD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some -lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2- OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues. [less ▲]

Detailed reference viewed: 24 (4 ULiège)
Full Text
See detail4-Alkyl-1,2,4-triazole-3-thione analogues as metallo-β-lactamase inhibitors
Gavara, L.; Legru, A.; Verdirosa, F. et al

in Bioorganic Chemistry (2021), 113

In Gram-negative bacteria, the major mechanism of resistance to β-lactam antibiotics is the production of one or several β-lactamases (BLs), including the highly worrying carbapenemases. Whereas ... [more ▼]

In Gram-negative bacteria, the major mechanism of resistance to β-lactam antibiotics is the production of one or several β-lactamases (BLs), including the highly worrying carbapenemases. Whereas inhibitors of these enzymes were recently marketed, they only target serine-carbapenemases (e.g. KPC-type), and no clinically useful inhibitor is available yet to neutralize the class of metallo-β-lactamases (MBLs). We are developing compounds based on the 1,2,4-triazole-3-thione scaffold, which binds to the di-zinc catalytic site of MBLs in an original fashion, and we previously reported its promising potential to yield broad-spectrum inhibitors. However, up to now only moderate antibiotic potentiation could be observed in microbiological assays and further exploration was needed to improve outer membrane penetration. Here, we synthesized and characterized a series of compounds possessing a diversely functionalized alkyl chain at the 4-position of the heterocycle. We found that the presence of a carboxylic group at the extremity of an alkyl chain yielded potent inhibitors of VIM-type enzymes with Ki values in the μM to sub-μM range, and that this alkyl chain had to be longer or equal to a propyl chain. This result confirmed the importance of a carboxylic function on the 4-substituent of 1,2,4-triazole-3-thione heterocycle. As observed in previous series, active compounds also preferentially contained phenyl, 2-hydroxy-5-methoxyphenyl, naphth-2-yl or m-biphenyl at position 5. However, none efficiently inhibited NDM-1 or IMP-1. Microbiological study on VIM-2-producing E. coli strains and on VIM-1/VIM-4-producing multidrug-resistant K. pneumoniae clinical isolates gave promising results, suggesting that the 1,2,4-triazole-3-thione scaffold worth continuing exploration to further improve penetration. Finally, docking experiments were performed to study the binding mode of alkanoic analogues in the active site of VIM-2. © 2021 Elsevier Inc. [less ▲]

Detailed reference viewed: 21 (4 ULiège)
Full Text
See detailMutational Effects on Carbapenem Hydrolysis of YEM-1, a New Subclass B2 Metallo-β-Lactamase from Yersinia mollaretii
Mercuri, Paola ULiege; Esposito, Roberto; Blétard, Sylvie et al

in Antimicrobial Agents and Chemotherapy (2020)

Detailed reference viewed: 64 (18 ULiège)
Full Text
See detaildi-Cysteine Residues of the Arabidopsis thaliana HMA4 C-Terminus Are Only Partially Required for Cadmium Transport
Ceasar, S. A.; Lekeux, Gilles ULiege; Motte, Patrick ULiege et al

in Frontiers in Plant Science (2020), 11

Cadmium (Cd) is highly toxic to the environment and humans. Plants are capable of absorbing Cd from the soil and of transporting part of this Cd to their shoot tissues. In Arabidopsis, the plasma membrane ... [more ▼]

Cadmium (Cd) is highly toxic to the environment and humans. Plants are capable of absorbing Cd from the soil and of transporting part of this Cd to their shoot tissues. In Arabidopsis, the plasma membrane Heavy Metal ATPase 4 (HMA4) transporter mediates Cd xylem loading for export to shoots, in addition to zinc (Zn). A recent study showed that di-Cys motifs present in the HMA4 C-terminal extension (AtHMA4c) are essential for high-affinity Zn binding and transport in planta. In this study, we have characterized the role of the AtHMA4c di-Cys motifs in Cd transport in planta and in Cd-binding in vitro. In contrast to the case for Zn, the di-Cys motifs seem to be partly dispensable for Cd transport as evidenced by limited variation in Cd accumulation in shoot tissues of hma2hma4 double mutant plants expressing native or di-Cys mutated variants of AtHMA4. Expression analysis of metal homeostasis marker genes, such as AtIRT1, excluded that maintained Cd accumulation in shoot tissues was the result of increased Cd uptake by roots. In vitro Cd-binding assays further revealed that mutating di-Cys motifs in AtHMA4c had a more limited impact on Cd-binding than it has on Zn-binding. The contributions of the AtHMA4 C-terminal domain to metal transport and binding therefore differ for Zn and Cd. Our data suggest that it is possible to identify HMA4 variants that discriminate Zn and Cd for transport. © Copyright © 2020 Ceasar, Lekeux, Motte, Xiao, Galleni and Hanikenne. [less ▲]

Detailed reference viewed: 42 (4 ULiège)
Full Text
See detail4-(N-Alkyl- and -Acyl-amino)-1,2,4-triazole-3-thione Analogs as Metallo-Beta-Lactamase Inhibitors: Impact of 4-Linker on Potency and Spectrum of Inhibition
Gavara, L.; Verdirosa, F.; Legru, A. et al

in Biomolecules (2020), 10(8), 1094

To fight the increasingly worrying bacterial resistance to antibiotics, the discovery and development of new therapeutics is urgently needed. Here, we report on a new series of 1,2,4-triazole-3-thione ... [more ▼]

To fight the increasingly worrying bacterial resistance to antibiotics, the discovery and development of new therapeutics is urgently needed. Here, we report on a new series of 1,2,4-triazole-3-thione compounds as inhibitors of metallo-β-lactamases (MBLs), which represent major resistance determinants to β-lactams, and especially carbapenems, in Gram-negative bacteria. These molecules are stable analogs of 4-amino-1,2,4-triazole-derived Schiff bases, where the hydrazone-like bond has been reduced (hydrazine series) or the 4-amino group has been acylated (hydrazide series); the synthesis and physicochemical properties thereof are described. The inhibitory potency was determined on the most clinically relevant acquired MBLs (IMP-, VIM-, and NDM-types subclass B1 MBLs). When compared with the previously reported hydrazone series, hydrazine but not hydrazide analogs showed similarly potent inhibitory activity on VIM-type enzymes, especially VIM-2 and VIM-4, with Ki values in the micromolar to submicromolar range. One of these showed broad-spectrum inhibition as it also significantly inhibited VIM-1 and NDM-1. Restoration of β-lactam activity in microbiological assays was observed for one selected compound. Finally, the binding to the VIM-2 active site was evaluated by isothermal titration calorimetry and a modeling study explored the effect of the linker structure on the mode of binding with this MBL. [less ▲]

Detailed reference viewed: 54 (15 ULiège)
Full Text
See detailA standard numbering scheme for class C β-lactamases
Mack, A. R.; Barnes, M. D.; Taracila, M. A. et al

in Antimicrobial Agents and Chemotherapy (2020), 64(3),

Unlike for classes A and B, a standardized amino acid numbering scheme has not been proposed for the class C (AmpC) β-lactamases, which complicates communication in the field. Here, we propose a scheme ... [more ▼]

Unlike for classes A and B, a standardized amino acid numbering scheme has not been proposed for the class C (AmpC) β-lactamases, which complicates communication in the field. Here, we propose a scheme developed through a collaborative approach that considers both sequence and structure, preserves traditional numbering of catalytically important residues (Ser64, Lys67, Tyr150, and Lys315), is adaptable to new variants or enzymes yet to be discovered and includes a variation for genetic and epidemiological applications. Copyright © 2020 American Society for Microbiology. All Rights Reserved. [less ▲]

Detailed reference viewed: 33 (4 ULiège)
Full Text
See detail4-Amino-1,2,4-triazole-3-thione-derived Schiff bases as metallo-beta-lactamase inhibitors
Gavara, L.; Sevaille, L.; De Luca, F. et al

in European Journal of Medicinal Chemistry (2020), 208

Resistance to β-lactam antibiotics in Gram-negatives producing metallo-β-lactamases (MBLs) represents a major medical threat and there is an extremely urgent need to develop clinically useful inhibitors ... [more ▼]

Resistance to β-lactam antibiotics in Gram-negatives producing metallo-β-lactamases (MBLs) represents a major medical threat and there is an extremely urgent need to develop clinically useful inhibitors. We previously reported the original binding mode of 5-substituted-4-amino/H-1,2,4-triazole-3-thione compounds in the catalytic site of an MBL. Moreover, we showed that, although moderately potent, they represented a promising basis for the development of broad-spectrum MBL inhibitors. Here, we synthesized and characterized a large number of 4-amino-1,2,4-triazole-3-thione-derived Schiff bases. Compared to the previous series, the presence of an aryl moiety at position 4 afforded an average 10-fold increase in potency. Among 90 synthetic compounds, more than half inhibited at least one of the six tested MBLs (L1, VIM-4, VIM-2, NDM-1, IMP-1, CphA) with Ki values in the microM to sub-microM range. Several were broad-spectrum inhibitors, also inhibiting the most clinically relevant VIM-2 and NDM-1. Active compounds generally contained halogenated, bicyclic aryl or phenolic moieties at position 5, and one substituent among o-benzoic, 2,4-dihydroxyphenyl, p-benzyloxyphenyl or 3-(m-benzoyl)-phenyl at position 4. The crystallographic structure of VIM-2 in complex with an inhibitor showed the expected binding between the triazole-thione moiety and the dinuclear centre and also revealed a network of interactions involving Phe61, Tyr67, Trp87 and the conserved Asn233. Microbiological analysis suggested that the compound antibacterial activity was limited by poor outer membrane penetration. This was supported by the ability of one compound to restore the susceptibility of an NDM-1-producing E. coli clinical strain toward several beta-lactams in the presence only of a sub-inhibitory concentration of colistin, a permeabilizing agent. Finally, some compounds were tested against the structurally similar di-zinc human glyoxalase II and found weaker inhibitors of the latter enzyme, thus showing a promising selectivity towards MBLs. [less ▲]

Detailed reference viewed: 47 (10 ULiège)
Full Text
See detailMutational Effects in Carbapenem Hydrolysis of YEM-1, a New Sub-Class B2 Metallo-beta-Lactamase from Yersinia mollaretii
Mercuri, Paola ULiege; Esposito, Roberto; Blétard, Sylvie et al

Poster (2019, June 22)

Background The genome of Yersinia mollaretii ATCC43969 was totally sequenced (NCBI NZ_AALD0200 0006.1). Its analysis indicated the presence of two β-lactamase genes, namely blaB and yem-1 coding for an ... [more ▼]

Background The genome of Yersinia mollaretii ATCC43969 was totally sequenced (NCBI NZ_AALD0200 0006.1). Its analysis indicated the presence of two β-lactamase genes, namely blaB and yem-1 coding for an AmpC-like and a metallo β-lactamase respectively. Up to date, many studies on BlaB were performed (1,2). Our work aimed to characterize the kinetic profile of YEM-1 and the residues essential for its activity. Methods YEM-1 was produced in E. coli Rosetta and purified to homogeneity. A survey of its kinetic properties was performed against different β-lactam antibiotics. A model of the three-dimensional structure of YEM-1 was build using the known structure of Aeromonas hydrophila CphA as template. Its analysis highlighted the major residues substitution between CphA and YEM-1. These residues were mutated by site-directed mutagenesis. The kinetic properties of the different mutants were analyzed. Results We noted that, at 37°C, YEM-1 is produced as inclusion bodies. We obtained a soluble form of YEM-1 when the culture was grown at 18°C in presence of IPTG 0.1 mM. The MBL was purified in three chromatographic steps that include a HiTRAP SP, an IMAC and a molecular sieve. The kinetic analysis of the YEM-1 showed that only imipenem was hydrolyzed efficiently. We confirmed that it belongs to the subclass B2 MBL family. As expected, the structure of YEM-1 has a similar fold than CphA. We found that the main differences between CphA and YEM-1 were the residues in position 67, 156 and 236 (BBL numbering) respectively. By site-directed mutagenesis, we made the YEM-1 single mutants (Y67V, T156F, S236F), the double mutant T156F-S236F and the triple mutant (Y67T156FS236F) in order to design the active site of CphA in YEM-1. The substitution Y67V yielded a global increase of the catalytic efficiency to carbapenems, with the exception of the imipenem. The mutations of T156 and/or S236 in phenylalanine globally decrease the enzymatic activity of the enzyme. Finally the double mutant and triple mutants did not increase the catalytic efficiencies of YEM-1 against carbapenems. Conclusions Despite a high sequence's similarity between CphA and YEM-1 (57%), we showed that the Yersinia MBL was less effective than CphA against carbapemens. We also highlighted the substitution Y67V in YEM-1 that increased its efficiency against all the carbapemen tested and in particular ertapenem and meropenem. [less ▲]

Detailed reference viewed: 78 (6 ULiège)
Full Text
See detailExploring the suitability of RanBP2-type Zinc Fingers for RNA-binding protein design
De Franco, Simona ULiege; Vandenameele, Julie ULiege; Brans, Alain ULiege et al

in Scientific Reports (2019), 9(1), 2484

Detailed reference viewed: 103 (24 ULiège)
See detailEnzyme Immobilization on Ordered Mesoporous Materials
Belet, Artium ULiege; Dondelinger, Mathieu; Gommes, Cédric ULiege et al

Conference (2019)

In the last decades, concerns about the hazards of carcinogenic compounds found in indoor air have risen. Among those compounds, formaldehyde, which is well-known to be found in paints, tobacco smoke ... [more ▼]

In the last decades, concerns about the hazards of carcinogenic compounds found in indoor air have risen. Among those compounds, formaldehyde, which is well-known to be found in paints, tobacco smoke, open fireplaces, insulating foams, lacquers, glues, varnishes, paper and household products, is of grave concern. With the aim of removing formaldehyde from indoor air, our work focused on the immobilization of an enzyme (FDH) catalyzing the following reaction in the presence of NAD+ as an enzyme cofactor: H2CO + H2O + NAD+ + FDH ⇌ HCOOH + H+ + NADH + FDH One of the main challenges in this work was to preserve the activity of an enzyme immobilized in a harsh environment and to maintain its activity for a long time. Ordered mesoporous silica and periodic mesoporous organosilicates materials have shown to be good candidates. They offer a large specific surface area and their physico-chemical properties as well as their structural properties can be tailored during (or after) their sol-gel syntheses [1,2]. FDH was produced and protein-engineered to have a His-Tag sequence, allowing purification on a Ni2+ chromatography column. Pure FDH was successfully immobilized on different supports and its activity was assessed by UV-Vis spectrometry of NADH. The immobilization was characterized by solid state 29Si NMR and by IR spectroscopy while enzyme loading was characterized by TG-DSC. The geometrical properties of supports were characterized by Hg porosimetry, nitrogen adsorption/desorption, TEM (cf. Figure 2) and by small-angle X-ray scattering. Altogether, these results allowed us to investigate relationships between the activity of immobilized enzymes and the environment provided by their supports. [less ▲]

Detailed reference viewed: 20 (2 ULiège)
Full Text
See detailHomology modeling and in vivo functional characterization of the zinc permeation pathway in a heavy metal P-type ATPase
Lekeux, Gilles ULiege; Crowet, Jean-Marc ULiege; Nouet, Cécile ULiege et al

in Journal of Experimental Botany (2019), 70

The P1B ATPase Heavy Metal ATPase 4 (HMA4) is responsible for zinc and cadmium translocation from roots to shoots in the plant Arabidopsis thaliana. It couples ATP hydrolysis to cytosolic domain movements ... [more ▼]

The P1B ATPase Heavy Metal ATPase 4 (HMA4) is responsible for zinc and cadmium translocation from roots to shoots in the plant Arabidopsis thaliana. It couples ATP hydrolysis to cytosolic domain movements enabling metal transport across the membrane. Thanks to high conservation level within the P-type ATPase family, the role of the HMA4 cytoplasmic catalytic domains can be inferred from well characterized pumps. In contrast, the function of its terminal cytosolic extensions as well as the metal permeation mechanism through the membrane remains elusive. Here, homology modeling of the HMA4 transmembrane region was conducted based on the crystal structure of a ZntA bacterial homolog. The analysis highlighted amino acids forming a metal permeation pathway, whose importance was subsequently investigated functionally through mutagenesis and complementation experiments in plants. Although the zinc pathway displayed overall conservation among the two proteins, significant differences were observed, especially in the entrance area with altered electronegativity and the presence of a salt bridge/H-bond network. The analysis also newly identified amino acids whose mutation results in total or partial loss of the protein function. In addition, comparison of zinc and cadmium accumulation in shoots of A. thaliana complemented lines revealed a number of HMA4 mutants exhibiting different abilities in zinc and cadmium translocation. These observations could be instrumental to design low cadmium accumulating crops, hence decreasing human cadmium exposure . [less ▲]

Detailed reference viewed: 117 (31 ULiège)
Full Text
See detailUnderstanding the significance and implications of antibody numbering and antigen-binding surface/residue definition
Dondelinger, Mathieu ULiege; Filée, Patrice; Sauvage, Eric ULiege et al

in Frontiers in Immunology (2018), 9(OCT),

Detailed reference viewed: 71 (7 ULiège)
Full Text
See detailCombinatorial Design of a Nanobody that Specifically Targets Structured RNAs
Cawez, Frédéric ULiege; Duray, Elodie ULiege; Hu, Y et al

in Journal of Molecular Biology (2018), 430(11), 1652-1670

Detailed reference viewed: 194 (18 ULiège)