Biomedical subjects

Olivier Walker

Publications and source records attributed to Olivier Walker.

6 recordsLinked to original sources

PubMed2006

Protein-protein interactions within peroxiredoxin systems.

Peroxiredoxin systems in plants were demonstrated involved in crucial roles related to reactive oxygenated species (ROS) metabolism and the linked cell signalling to ROS. Peroxiredoxins function as peroxidasic systems that combine at least a reactivating reductant agent like thioredoxins, and sometimes glutaredoxins and glutathion. In the past three years a number of peroxiredoxin structures were solved by crystallography in different experimental crystallisation conditions. The structures have revealed a significant propensity of peroxiredoxins for oligomerism that was confirmed by biophysical studies in solution using NMR and other methods as analytical ultra-centrifugation. These studies showed that quaternary structures of peroxiredoxins involve specific protein-protein interaction interfaces that rely upon the peroxiredoxin types and/or their redox conditions. The protein-protein interactions with the reactivating redoxins essentially lead to transient unstable complexes. We review herein the different protein-protein interactions characterized or deduced from those reports.

Models, Molecular↗

PubMed2006

Glutathionylation induces the dissociation of 1-Cys D-peroxiredoxin non-covalent homodimer.

1-Cys peroxiredoxins (1-Cys Prxs) are antioxidant enzymes that catalyze the reduction of hydroperoxides into alcohols using a strictly conserved cysteine. 1-Cys B-Prxs, homologous to human PrxVI, were recently shown to be reactivated by glutathione S-transferase (GST) pi via the formation of a GST-Prx heterodimer and Prx glutathionylation. In contrast, 1-Cys D-Prxs, homologous to human PrxV, are reactivated by the glutaredoxin-glutathione system through an unknown mechanism. To investigate the mechanistic events that mediate the 1-Cys D-Prx regeneration, interaction of the Prx with glutathione was studied by mass spectrometry and NMR. This work reveals that the Prx can be glutathionylated on its active site cysteine. Evidences are reported that the glutathionylation of 1-Cys D-Prx induces the dissociation of the Prx non-covalent homodimer, which can be recovered by reduction with dithiothreitol. This work demonstrates for the first time the existence of a redox-dependent dimer-monomer switch in the Prx family, similar to the decamer-dimer switch for the 2-Cys Prxs.

Binding Sites↗

PubMed2005

NMR reveals a novel glutaredoxin-glutaredoxin interaction interface.

Glutaredoxins (Grx) represent a large family of glutathione (GSH)-dependent oxidoreductases that catalyse the reduction of disulfides or glutathione mixed disulfide. Grx domains from pathogenic bacteria and plant Grxs have been recently reported to target specific peroxiredoxins (Prxs). The specificity that triggers the interaction between Grx and Prx is poorly understood and is only based on the structure of Haemophilus influenzae Prx-Grx hybrid (hyPrx5). We report here an NMR study of the Populus tremula Grx C4 that targets a P.tremula D-type II Prx. We show that Grx C4 specifically self-associates in a monomer-dimer equilibrium with an apparent K(d) of ca 2.6 mM. Grx C4 homodimer was docked under experimental restraints. The results reveal a novel Grx-Grx interface that is unrelated to the hyPrx5 Grx-Grx dimer interface. Chemical-shift perturbations and 15N spin-relaxation measurements show that the auto-association surface comprises both the active site and the GSH binding site. Reduced GSH is demonstrated to bind reduced Grx with a K(d) of ca 8.6 mM. The potential biological significance of the new Grx-Grx interaction interface is discussed.

Amino Acid Sequence↗

PubMed2005

Crystal structure and solution NMR dynamics of a D (type II) peroxiredoxin glutaredoxin and thioredoxin dependent: a new insight into the peroxiredoxin oligomerism.

Peroxiredoxins (Prxs) constitute a family of thiol peroxidases that reduce hydrogen peroxide, peroxinitrite, and hydroperoxides using a strictly conserved cysteine. Very abundant in all organisms, Prxs are produced as diverse isoforms characterized by different catalytic mechanisms and various thiol-containing reducing agents. The oligomeric state of Prxs and the link with their functionality is a subject of intensive research. We present here a combined X-ray and nuclear magnetic resonance (NMR) study of a plant Prx that belongs to the D-Prx (type II) subfamily. The Populus trichocarpa Prx is the first Prx shown to be regenerated in vitro by both the glutaredoxin and thioredoxin systems. The crystal structure and solution NMR provide evidence that the reduced protein is a specific noncovalent homodimer both in the crystal and in solution. The dimer interface is roughly perpendicular to the plane of the central beta sheet and differs from the interface of A- and B-Prx dimers, where proteins associate in the plane parallel to the beta sheet. The homodimer interface involves residues strongly conserved in the D (type II) Prxs, suggesting that all Prxs of this family can homodimerize. The study provides a new insight into the Prx oligomerism and the basis for protein-protein and enzyme-substrate interaction studies by NMR.

Amino Acid Sequence↗

PubMed2004

Efficient and accurate determination of the overall rotational diffusion tensor of a molecule from (15)N relaxation data using computer program ROTDIF.

We present a computer program ROTDIF for efficient determination of a complete rotational diffusion tensor of a molecule from NMR relaxation data. The derivation of the rotational diffusion tensor in the case of a fully anisotropic model is based on a six-dimensional search, which could be very time consuming, particularly if a grid search in the Euler angle space is involved. Here, we use an efficient Levenberg-Marquardt algorithm combined with Monte Carlo generation of initial guesses. The result is a dramatic, up to 50-fold improvement in the computational efficiency over the previous approaches. This method is demonstrated on a computer-generated and real protein systems. We also address the issue of sensitivity of the diffusion tensor determination from (15)N relaxation measurements to experimental errors in the relaxation rates and discuss possible artifacts from applying higher-symmetry tensor model and how to recognize them.

Algorithms↗

PubMed2002

Structural properties of polyubiquitin chains in solution.

Because polyubiquitin chain structure modulates Ub-mediated signaling, knowledge of the physiological conformations of chain signals should provide insights into specific recognition. Here, we characterized the solution conformations of K48-linked Ub(2) and Ub(4) using a combination of NMR techniques, including chemical shift mapping of the interdomain interface, domain orientation measurements on the basis of 15N relaxation and residual dipolar couplings, and the solvent accessibility studies. Our data indicate a switch in the conformation of Ub(2), from open to closed, with increasing pH. The closed conformation features a well-defined interface that is related to, but distinguishable from, that observed in the Ub(2) crystal structure. This interface is dynamic in solution, such that important hydrophobic residues (L8, I44, V70) that are sequestered at the interface in the closed conformation may be accessible for direct interactions with recognition factors. Our results suggest that the distal two units of Ub(4), which is the minimum signal for efficient proteasomal degradation, may adopt the closed Ub(2) conformation.

Amides↗