FHA Domain pThr Binding Specificity: It's All about Me

The FHA domain is a phospho-peptide binding module involved in a wide range of cellular pathways, with a striking specificity for phospho-threonine over phospho-serine binding partners. Biochemical, structural, and dynamic simulations analysis allowed Pennell and colleagues to unravel the molecular basis of FHA domain phospho-threonine specificity

Sampling the conformational energy landscape of a hyperthermophilic protein by engineering key substitutions

Proteins exist as a dynamic ensemble of interconverting substates, which defines their conformational energy landscapes. Recent work has indicated that mutations that shift the balance between conformational substates (CSs) are one of the main mechanisms by which proteins evolve new functions. In the present study, we probe this assertion by examining phenotypic protein adaptation to extreme conditions, using the allosteric tetrameric lactate dehydrogenase (LDH) from the hyperthermophilic bacterium Thermus thermophilus (Tt) as a model enzyme. In the presence of fructose 1, 6 bis-phosphate (FBP), allosteric LDHs catalyze the conversion of pyruvate to lactate with concomitant oxidation of nicotinamide adenine dinucleotide, reduced form (NADH). The catalysis involves a structural transition between a low-affinity inactive 'T-state' and a high-affinity active 'R-state' with bound FBP. During this structural transition, two important residues undergo changes in their side chain conformations. These are R171 and H188, which are involved in substrate and FBP binding, respectively. We designed two mutants of Tt-LDH with one ('1-Mut') and five ('5-Mut') mutations distant from the active site and characterized their catalytic, dynamical, and structural properties. In 1-Mut Tt-LDH, without FBP, the KmPyr is reduced compared with that of the wild type, which is consistent with a complete shifting of the CS equilibrium of H188 to that observed in the R-state. By contrast, the CS populations of R171, kcat and protein stability are little changed. In 5-Mut Tt-LDH, without FBP, KmPyr approaches the values it has with FBP and becomes almost temperature independent, kcat increases substantially, and the CS populations of R171 shift toward those of the R-state. These changes are accompanied by a decrease in protein stability at higher temperature, which is consistent with an increased flexibility at lower temperature. Together, these results show that the thermal properties of an enzyme can be strongly modified by only a few or even a single mutation, which serve to alter the equilibrium and, hence, the relative populations of functionally important native-state CSs, without changing the nature of the CSs themselves. They also provide insights into the types of mutational pathways by which protein adaptation to temperature is achieved.</p

Opportunities and challenges in neutron crystallography

Neutron and X-ray crystallography are complementary to each other. While X-ray scattering is directly proportional to the number of electrons of an atom, neutrons interact with the atomic nuclei themselves. Neutron crystallography therefore provides an excellent alternative in determining the positions of hydrogens in a biological molecule. In particular, since highly polarized hydrogen atoms (H+) do not have electrons, they cannot be observed by X-rays. Neutron crystallography has its own limitations, mainly due to inherent low flux of neutrons sources, and as a consequence, the need for much larger crystals and for different data collection and analysis strategies. These technical challenges can however be overcome to yield crucial structural insights about protonation states in enzyme catalysis, ligand recognition, as well as the presence of unusual hydrogen bonds in proteins

Opportunities and challenges in neutron crystallography

Neutron and X-ray crystallography are complementary to each other. While X-ray scattering is directly proportional to the number of electrons of an atom, neutrons interact with the atomic nuclei themselves. Neutron crystallography therefore provides an excellent alternative in determining the positions of hydrogens in a biological molecule. In particular, since highly polarized hydrogen atoms (H+) do not have electrons, they cannot be observed by X-rays. Neutron crystallography has its own limitations, mainly due to inherent low flux of neutrons sources, and as a consequence, the need for much larger crystals and for different data collection and analysis strategies. These technical challenges can however be overcome to yield crucial structural insights about protonation states in enzyme catalysis, ligand recognition, as well as the presence of unusual hydrogen bonds in proteins

Mécanismes moléculaires d'adaptation aux conditions physico-chimiques extrêmes dans la famille des lactate-malate déshydrogénases

La vie est présente partout, ou presque, sur Terre, et notamment dans des environnements considérés comme extrêmes. Ces organismes extrêmophiles, non contents de subsister sous ces contraintes physico-chimiques extrêmes, s'y complaisent. Plusieurs mécanismes adaptatifs, à divers niveaux de l'organisation cellulaire, ont été mis en place. Les études présentées ici s'intéressent aux mécanismes d'adaptation moléculaire des protéines en utilisant la famille des lactate-malate déshydrogénases comme modèle. Il semblerait qu'une réorganisation des interactions au sein de la structure assure la stabilité, la solubilité et l'activité de la protéine sous cette contrainte physico-chimique extrême.Dans une première partie, les propriétés biochimiques et structurales des lactates déshydrogénases de la bactérie thermophile Thermus thermophilus (TtLDH1 de la bactérie mésophile Deinococcus radiodurans (DrLDH) et du poisson psychrophile de Champsocepha/us gunnari (CgLDH) ont été déterminées. Les deux premières ont été comparées pour étudier la transition thermophile/mésophile. La dernière a été comparée à la LDH de Squa/us acanthias (SaLDH) pour comprendre la transition mésophile/psychrophile. Peu de substitutions semblent être à l'origine des différences de propriétés thermiques entre ces enzymes. Cette hypothèse a pu être vérifiée par la caractérisation d'un mutant de TtLDH. Cinq substitutions judicieusement choisies ont permis d'altérer ses propriétés thermiques.L'adaptation moléculaire aux fortes concentrations en sel a été, pendant longtemps, étudiée en utilisant la malate désydrogénase de Ha/oarcu/a marismortui (HmMalDH). Nous présentons ici les caractéristiques biochimiques et structurales de la malate désydrogénase issue de Sa/inibacter ruber (SrMaIDH), la seule bactérie halophile extrême connue à ce jour. Les propriétés de cette enzyme semblent intermédiaires entre une enzyme non halophile et une enzyme totalement efficace aux fortes concentrations en sel. Cela a permis, pour la première fois, de proposer un découplage des différents effets affectant la stabilité conformationnelle,l'activité enzymatique et la solubilité. Enfin, l'irradiation des cristaux de protéine provoque des dommages au sein de la structure de la macromolécule, et touche notamment les groupements carboxyle des chaînes latérales des résidus acides. La forme apo (enzyme seule) et le complexe ternaire (enzyme :NADH :analogue de substrat) de TtLDH ont été irradiés et, à dose absorbée équivalente, l'amplitude des dommages entre les deux formes a été comparée. La fixation du cofacteur et du substrat ne protège pas des radiations le résidu acide présent dans le site actif.Life is found everywhere, or almost, on earth and particularly in environments considered as extreme. These extremophilic organisms do not only subsist, but thrive in these conditions. Several adaptative mechanisms, at different cellular levels, have beendevelopped. Our studies focus on molecular mechanisms of proteins adaptation, using the lactate-malate dehydrogenases family as model. An intramolecular reorganization of interactions seems to be sufficient to en sure conformational stability, enzymatic activity and solubility of the protein in the se eXtreme conditions.First, biochemical and structural properties of lactate dehydrogenases from the thermophilic bacterium Thermus thermophilus (TtLDH), the mesophilic bacterium Deinococcus radiodurans (DrLDH) and the psychrophilic fish Champsocepha/us gunnari (CgLDH) have been determined. The first two have been compared to understand the thermophilic/mesophilic transition. The latter has been compared to the LDH from Squa/us acanthias (SaLDH) to study the mesophilic/psychrophilc transition. Few substitutions seems to be responsible for the differences in thermal properties between these enzymes. This hypothesis has been validated using a molecular variant of TtLDH. Five amino acid substitutions have modified its thermal properties.Molecular adaptation to high salt concentrations has been studied for a long time using the Ha/oarcu/a marismortui malatedehydrogenase (HmMaIDH). We present here the biochemical and structural properties of the malate dehydrogenase tTom Sa/inibacter ruber (SrLDH), the only extreme halophilic bacterium known to date. Properties of this enzyme appear to be 'intermediate' between a non halophilic enzyme and a fully active enzyme in high salt. For the first time, different effects that modiry conformational stability, enzymatic activity or solubility have been uncoupled.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

An experimental point of view on hydration/solvation in halophilic proteins.

International audienceProtein-solvent interactions govern the behaviors of proteins isolated from extreme halophiles. In this work, we compared the solvent envelopes of two orthologous tetrameric malate dehydrogenases (MalDHs) from halophilic and non-halophilic bacteria. The crystal structure of the MalDH from the non-halophilic bacterium Chloroflexus aurantiacus (Ca MalDH) solved, de novo, at 1.7 Å resolution exhibits numerous water molecules in its solvation shell. We observed that a large number of these water molecules are arranged in pentagonal polygons in the first hydration shell of Ca MalDH. Some of them are clustered in large networks, which cover non-polar amino acid surface. The crystal structure of MalDH from the extreme halophilic bacterium Salinibacter ruber (Sr) solved at 1.55 Å resolution shows that its surface is strongly enriched in acidic amino acids. The structural comparison of these two models is the first direct observation of the relative impact of acidic surface enrichment on the water structure organization between a halophilic protein and its non-adapted counterpart. The data show that surface acidic amino acids disrupt pentagonal water networks in the hydration shell. These crystallographic observations are discussed with respect to halophilic protein behaviors in solution

Xtrapol8 enables automatic elucidation of low-occupancy intermediate-states in crystallographic studies

International audienceAbstract Unstable states studied in kinetic, time-resolved and ligand-based crystallography are often characterized by a low occupancy, which hinders structure determination by conventional methods. To automatically extract structural information pertaining to these states, we developed Xtrapol8, a program which (i) applies various flavors of Bayesian-statistics weighting to generate the most informative Fourier difference maps; (ii) determines the occupancy of the intermediate states by use of methods hitherto not available; (iii) calculates extrapolated structure factors using the various proposed formalisms while handling the issue of negative structure factor amplitudes, and (iv) refines the corresponding structures in real and reciprocal-space. The use of Xtrapol8 could accelerate data processing in kinetic and time-resolved crystallographic studies, and as well foster the identification of drug-targetable states in ligand-based crystallography

Impact of BRCA1 BRCT Domain Missense Substitutions on Phosphopeptide Recognition

The BRCA1 BRCT domain binds pSer-x-x-Phe motifs in partner proteins to regulate the cellular response to DNA damage. Approximately 120 distinct missense variants have been identified in the BRCA1 BRCT through breast cancer screening, and several of these have been linked to an increased cancer risk. Here we probe the structures and peptide-binding activities of variants that affect the BRCA1 BRCT phosphopeptide-binding groove. The results obtained from the G1656D and T1700A variants illustrate the role of Ser1655 in pSer recognition. Mutations at Arg1699 (R1699W and R1699Q) significantly reduce peptide binding through loss of contacts to the main chain of the Phe(+3) residue and, in the case of R1699W, to a destabilization of the BRCT fold. The R1835P and E1836K variants do not dramatically reduce peptide binding, in spite of the fact that these mutations significantly alter the structure of the walls of the Phe(+3) pocket

Development of potent reversible selective inhibitors of butyrylcholinesterase as fluorescent probes

Brain butyrylcholinesterase (BChE) is an attractive target for drugs designed for the treatment of Alzheimer\u27s disease (AD) in its advanced stages. It also potentially represents a biomarker for progression of this disease. Based on the crystal structure of previously described highly potent, reversible, and selective BChE inhibitors, we have developed the fluorescent probes that are selective towards human BChE. The most promising probes also maintain their inhibition of BChE in the low nanomolar range with high selectivity over acetylcholinesterase. Kinetic studies of probes reveal a reversible mixed inhibition mechanism, with binding of these fluorescent probes to both the free and acylated enzyme. Probes show environment-sensitive emission, and additionally, one of them also shows significant enhancement of fluorescence intensity upon binding to the active site of BChE. Finally, the crystal structures of probes in complex with human BChE are reported, which offer an excellent base for further development of this library of compounds

Biochemical, structural and dynamical studies reveal strong differences in the thermal-dependent allosteric behavior of two extremophilic lactate dehydrogenases

International audienceIn this work, we combined biochemical and structural investigations with molecular dynamics (MD) simulations to analyze the very different thermal-dependent allosteric behavior of two lactate dehydrogenases (LDH) from thermophilic bacteria. We found that the enzyme from Petrotoga mobilis (P. mob) necessitates an absolute requirement of the allosteric effector (fructose 1, 6-bisphosphate) to ensure functionality. In contrast, even without allosteric effector, the LDH from Thermus thermophilus (T. the) is functional when the temperature is raised. We report the crystal structure of P. mob LDH in the Apo state solved at 1.9 Å resolution. We used this structure and the one from T. the, obtained previously, as a starting point for MD simulations at various temperatures. We found clear differences between the thermal dynamics, which accounts for the behavior of the two enzymes. Our work demonstrates that, within an allosteric enzyme, some areas act as local gatekeepers of signal transmission, allowing the enzyme to populate either the T-inactive or the R-active states with different degrees of stringency