Two previously reported holoprotein crystal forms of the flavodoxin-like
E. coli protein WrbA, diffracting to 2.6 and 2.0
Å resolution, and new crystals of WrbA apoprotein diffracting to 1.85
Å, are ...refined and analysed comparatively through the lens of flavodoxin structures. The results indicate that differences between apo- and holoWrbA crystal structures are manifested on many levels of protein organization as well as in the FMN-binding sites. Evaluation of the influence of crystal contacts by comparison of lattice packing reveals the protein's global response to FMN binding. Structural changes upon cofactor binding are compared with the monomeric flavodoxins. Topologically non-equivalent residues undergo remarkably similar local structural changes upon FMN binding to WrbA or to flavodoxin, despite differences in multimeric organization and residue types at the binding sites. Analysis of the three crystal structures described here, together with flavodoxin structures, rationalizes functional similarities and differences of the WrbAs relative to flavodoxins, leading to a new understanding of the defining features of WrbAs. The results suggest that WrbAs are not a remote and unusual branch of the flavodoxin family as previously thought but rather a central member with unifying structural features.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The Escherichia coli protein WrbA, an FMN‐dependent NAD(P)H:quinone oxidoreductase, was crystallized under new conditions in the presence of FAD or the native cofactor FMN. Slow‐growing deep yellow ...crystals formed with FAD display the tetragonal bipyramidal shape typical for WrbA and diffract to 1.2 Å resolution, the highest yet reported. Faster‐growing deep yellow crystals formed with FMN display an atypical shape, but diffract to only ∼1.6 Å resolution and are not analysed further here. The 1.2 Å resolution structure detailed here revealed only FMN in the active site and no electron density that can accommodate the missing parts of FAD. The very high resolution supports the modelling of the FMN isoalloxazine with a small but distinct propeller twist, apparently the first experimental observation of this predicted conformation, which appears to be enforced by the protein through a network of hydrogen bonds. Comparison of the electron density of the twisted isoalloxazine ring with the results of QM/MM simulations is compatible with the oxidized redox state. The very high resolution also supports the unique refinement of Met10 as the sulfoxide, confirmed by mass spectrometry. Bond lengths, intramolecular distances, and the pattern of hydrogen‐bond donors and acceptors suggest the cofactor may interact with Met10. Slow incorporation of FMN, which is present as a trace contaminant in stocks of FAD, into growing crystals may be responsible for the near‐atomic resolution, but a direct effect of the conformation of FMN and/or Met10 sulfoxide cannot be ruled out.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The Escherichia coli protein WrbA, an FMN-dependent NAD(P)H:quinone oxidoreductase, was crystallized under new conditions in the presence of FAD or the native cofactor FMN. Slow-growing deep yellow ...crystals formed with FAD display the tetragonal bipyramidal shape typical for WrbA and diffract to 1.2Å resolution, the highest yet reported. Faster-growing deep yellow crystals formed with FMN display an atypical shape, but diffract to only 1.6Å resolution and are not analysed further here. The 1.2Å resolution structure detailed here revealed only FMN in the active site and no electron density that can accommodate the missing parts of FAD. The very high resolution supports the modelling of the FMN isoalloxazine with a small but distinct propeller twist, apparently the first experimental observation of this predicted conformation, which appears to be enforced by the protein through a network of hydrogen bonds. Comparison of the electron density of the twisted isoalloxazine ring with the results of QM/MM simulations is compatible with the oxidized redox state. The very high resolution also supports the unique refinement of Met10 as the sulfoxide, confirmed by mass spectrometry. Bond lengths, intramolecular distances, and the pattern of hydrogen-bond donors and acceptors suggest the cofactor may interact with Met10. Slow incorporation of FMN, which is present as a trace contaminant in stocks of FAD, into growing crystals may be responsible for the near-atomic resolution, but a direct effect of the conformation of FMN and/or Met10 sulfoxide cannot be ruled out. PUBLICATION ABSTRACT
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The Escherichia coli protein WrbA, an FMN-dependent NAD(P)H:quinone oxidoreductase, was crystallized under new conditions in the presence of FAD or the native cofactor FMN. Slow-growing deep yellow ...crystals formed with FAD display the tetragonal bipyramidal shape typical for WrbA and diffract to 1.2Aa resolution, the highest yet reported. Faster-growing deep yellow crystals formed with FMN display an atypical shape, but diffract to only similar to 1.6Aa resolution and are not analysed further here. The 1.2Aa resolution structure detailed here revealed only FMN in the active site and no electron density that can accommodate the missing parts of FAD. The very high resolution supports the modelling of the FMN isoalloxazine with a small but distinct propeller twist, apparently the first experimental observation of this predicted conformation, which appears to be enforced by the protein through a network of hydrogen bonds. Comparison of the electron density of the twisted isoalloxazine ring with the results of QM/MM simulations is compatible with the oxidized redox state. The very high resolution also supports the unique refinement of Met10 as the sulfoxide, confirmed by mass spectrometry. Bond lengths, intramolecular distances, and the pattern of hydrogen-bond donors and acceptors suggest the cofactor may interact with Met10. Slow incorporation of FMN, which is present as a trace contaminant in stocks of FAD, into growing crystals may be responsible for the near-atomic resolution, but a direct effect of the conformation of FMN and/or Met10 sulfoxide cannot be ruled out.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The flavoprotein WrbA from Escherichia coli is considered to be the prototype of a new family of multimeric flavodoxin‐like proteins that are implicated in cell protection against oxidative stress. ...The present study is aimed at structural characterization of the E. coli protein with respect to its recently revealed oxidoreductase activity. Crystals of WrbA holoprotein in complex with the oxidized flavin cofactor (FMN) were obtained using standard vapour‐diffusion techniques. Deep yellow tetragonal crystals obtained from differing crystallization conditions display different space groups and unit‐cell parameters. X‐ray crystal structures of the WrbA holoprotein have been determined to resolutions of 2.0 and 2.6 Å.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Deux gènes paralogues de la bactérie Lactobacillus johnsonii NCC533 (La1), issue de la collection de culture Nestlé, montrent une forte similitude avec le gène de Lactobacillus gasseri 4B2 codant une ...protéine appelée " aggregation promoting factor " (APF). Ces deux gènes ont été clonés et caractérisés. L'analyse transcriptionnelle a montré que le taux d'expression des deux gènes est maximal durant la phase exponentielle de croissance, alors que ce taux est très faible durant la phase stationnaire. Les deux gènes sont fortement induits lorsque L. johnsonii NCC533 se développe sur un milieu solide en comparaison à la croissance en milieu liquide. La surexpression des gènes apf de la souche L. gasseri 4B2 et de la souche L. johnsonii NCC533, dans la souche L. johnsonii NCC533, ne conduit pas à un phénotype visible. Cependant, après introduction de ces mêmes constructions de surexpression chez L. gasseri 4B2, nous pouvons observer une augmentation significative du phénotype d'agrégation de cette bactérie ainsi qu'un changement de sa forme cellulaire, montrant ainsi que le phénotype résultant de la protéine APF est dépendante de la souche utilisée. Ces données indiquent que la protéine APF n'est pas seulement impliquée dans le phénomène d'agrégation, mais influence aussi, de manière directe ou indirecte, la forme cellulaire finale de L. gasseri 4B2
PDF
