Divide, conquer and reconstruct: How to solve the 3D structure of recalcitrant Micro-Exon Gene (MEG) protein from Schistosoma mansoni.

Micro-Exon Genes are a widespread class of genes known for their high variability, widespread in the genome of parasitic trematodes such as Schistosoma mansoni. In this study, we present a strategy that allowed us to solve the structures of three alternatively spliced isoforms from the Schistoma mansoni MEG 2.1 family for the first time. All isoforms are hydrophobic, intrinsically disordered, and recalcitrant to be expressed in high yield in heterologous hosts. We resorted to the chemical synthesis of shorter pieces, before reconstructing the entire sequence. Here, we show that isoform 1 partially folds in a-helix in the presence of trifluoroethanol while isoform 2 features two rigid elbows, that maintain the peptide as disordered, preventing any structuring. Finally, isoform 3 is dominated by the signal peptide, which folds into a-helix. We demonstrated that combining biophysical techniques, like circular dichroism and nuclear magnetic resonance at natural abundance, with in silico molecular dynamics simulation for isoform 1 only, was the key to solve the structure of MEG 2.1. Our results provide a crucial piece to the puzzle of this elusive and highly variable class of proteins.

Early detection of ureteropelvic junction obstruction in neonates with prenatal diagnosis of renal pelvis dilatation using 1H NMR urinary metabolomics.

Renal pelvis dilatation (RPD) is diagnosed in utero on prenatal ultrasonography (US) and can resolve spontaneously. However, isolated RPD can also reflect ureteropelvic junction obstruction (UPJO), which requires surgical treatment to prevent progressive renal deterioration. The diagnosis of UPJO can only be confirmed after birth with repeat US and renal isotope studies. 1H Nuclear Magnetic Resonance spectroscopy (NMR) was performed on urine of newborns with prenatally diagnosed unilateral RPD and healthy controls to identify specific urinary biomarkers for UPJO. The original combination of EigenMS normalization and sparse partial-least-squares discriminant analysis improved selectivity and sensitivity. In total, 140 urine samples from newborns were processed and 100 metabolites were identified. Correlation network identified discriminant metabolites in lower concentrations in UPJO patients. Two main metabolic pathways appeared to be impaired in patients with UPJO i.e. amino acid and betaine metabolism. In this prospective study, metabolic profiling of urine samples by NMR clearly distinguishes patients who required surgery for UPJO from patients with transient dilatations and controls. This study will pave the way for the use of metabolomics for the diagnosis of prenatal hydronephrosis in clinical routine.

The Candida glabrata glycogen branching enzyme structure reveals unique features of branching enzymes of the Saccharomycetaceae phylum.

Branching enzymes (BE) are responsible for the formation of branching points at the 1,6 position in glycogen and starch, by catalyzing the cleavage of α-1,4-linkages and the subsequent transfer by introducing α-1,6-linked glucose branched points. BEs are found in the large GH13 family, eukaryotic BEs being mainly classified in the GH13_8 subfamily, GH13_9 grouping almost exclusively prokaryotic enzymes. With the aim of contributing to the understanding of the mode of recognition and action of the enzymes belonging to GH13_8, and to the understanding of features distinguishing these enzymes from those belonging to subfamily 13_9, we solved the crystal structure of the glycogen branching enzyme (GBE) from the yeast Candida glabrata, CgGBE, in ligand-free forms and in complex with a maltotriose. The structures revealed the presence of a domain already observed in Homo sapiens and Oryza sativa BEs that we named α-helical N-terminal domain, in addition to the three conserved domains found in BE. We confirmed by phylogenetic analysis that this α-helical N-terminal domain is always present in the GH13_8 enzymes suggesting that it could actually present a signature for this subfamily. We identified two binding sites in the α-helical N-terminal domain and in the carbohydrate binding module 48 (CBM48), respectively, which show a unique structural organization only present in the Saccharomycotina phylum. Our structural and phylogenetic investigation provides new insight into the structural characterization of GH13_8 GBE revealing that unique structural features only present in the Saccharomycotina phylum thereby conferring original properties to this group of enzymes.

NMR Reveals the Interplay among the AMSH SH3 Binding Motif, STAM2, and Lys63-Linked Diubiquitin.

AMSH [associated molecule with a Src homology 3 domain of signal transducing adaptor molecule (STAM)] is one of the deubiquitinating enzymes associated in the regulation of endocytic cargo trafficking. It shows an exquisite selectivity for Lys63-linked polyubiquitin chains that are the main chains involved in cargo sorting. The first step requires the ESCRT-0 complex that comprises the STAM and hepatocyte growth factor-regulated substrate (Hrs) proteins. Previous studies have shown that the presence of the STAM protein increases the efficiency of Lys63-linked polyubiquitin chain cleavage by AMSH, one of the deubiquitinating enzyme involved in lysosomal degradation. In the present study, we are seeking to understand if a particular structural organization among these three key players is responsible for the stimulation of the catalytic activity of AMSH. To address this question, we first monitored the interaction between the ubiquitin interacting motif (UIM)-SH3 construct of STAM2 and the Lys63-linked diubiquitin (Lys63-Ub2) chains by means of NMR. We show that Lys63-Ub2 is able to bind either the UIM or the SH3 domain without any selectivity. We further demonstrate that the SH3 binding motif (SBM) of AMSH (AMSH-SBM) outcompetes Lys63-Ub2 for binding SH3. Additionally, we show how different AMSH-SBM variants, modified by their sequence and length, exhibit similar equilibrium dissociation constants when binding SH3 but significantly differ in their dissociation rate constants. Finally, we report the solution NMR structure of the AMSH-SBM/SH3 complex and propose a structural organization where the AMSH-SBM interacts with the STAM2-SH3 domain and contributes to the correct positioning of AMSH prior to polyubiquitin chains' cleavage.

Pectin gelation with chlorhexidine: Physico-chemical studies in dilute solutions.

Low methoxyl pectin is known to gel with divalent cations (e.g. Ca(2+), Zn(2+)). In this study, a new way of pectin gelation in the presence of an active pharmaceutical ingredient, chlorhexidine (CX), was highlighted. Thus chlorhexidine interactions with pectin were investigated and compared with the well-known pectin/Ca(2+) binding model. Gelation mechanisms were studied by several physico-chemical methods such as zeta potential, viscosity, size measurements and binding isotherm was determined by Proton Nuclear Magnetic Resonance Spectroscopy ((1)H NMR). The binding process exhibited similar first two steps for both divalent ions: a stoichiometric monocomplexation of the polymer followed by a dimerization step. However, stronger interactions were observed between pectin and chlorhexidine. Moreover, the dimerization step occurred under stoichiometric conditions with chlorhexidine whereas non-stoichiometric conditions were involved with calcium ions. In the case of chlorhexidine, an additional intermolecular binding occurred in a third step.

Predicting and Understanding the Enzymatic Inhibition of Human Peroxiredoxin 5 by 4-Substituted Pyrocatechols by Combining Funnel Metadynamics, Solution NMR, and Steady-State Kinetics.

Funnel metadynamics is a kind of computational simulation used to enhance the sampling of protein-ligand binding events in solution. By characterization of the binding interaction events, an estimated absolute binding free energy can be calculated. Nuclear magnetic resonance and funnel metadynamics were used to evaluate the binding of pyrocatechol derivatives (catechol, 4-methylcatechol, and 4-tert-butylcatechol) to human peroxiredoxin 5. Human peroxiredoxins are peroxidases involved in cellular peroxide homeostasis. Recently, overexpressed or suppressed peroxiredoxin levels have been linked to various diseases. Here, the catechol derivatives were found to be inhibitors against human peroxiredoxin 5 through a partial mixed type noncompetitive mechanism. Funnel metadynamics provided a microscopic model for interpreting the inhibition mechanism. Correlations were observed between the inhibition constants and the absolute binding free energy. Overall, this study showcases the fact that funnel metadynamics simulations can be employed as a preliminary approach to gain an in-depth understanding of potential enzyme inhibitors.

Funnel-metadynamics and solution NMR to estimate protein-ligand affinities.

One of the intrinsic properties of proteins is their capacity to interact selectively with other molecules in their environment, inducing many chemical equilibria each differentiated by the mutual affinities of the components. A comprehensive understanding of these molecular binding processes at atomistic resolution requires formally the complete description of the system dynamics and statistics at the relevant time scales. While solution NMR observables are averaged over different time scales, from picosecond to second, recent new molecular dynamics protocols accelerated considerably the simulation time of realistic model systems. Based on known ligands recently discovered either by crystallography or NMR for the human peroxiredoxin 5, their affinities were for the first time accurately evaluated at atomistic resolution comparing absolute binding free-energy estimated by funnel-metadynamics simulations and solution NMR experiments. In particular, free-energy calculations are demonstrated to discriminate two closely related ligands as pyrocatechol and 4-methylpyrocathecol separated just by 1 kcal/mol in aqueous solution. The results provide a new experimental and theoretical basis for the estimation of ligand-protein affinities.

NMR-based analysis of protein-ligand interactions.

Physiological processes are mainly controlled by intermolecular recognition mechanisms involving protein-protein and protein-ligand (low molecular weight molecules) interactions. One of the most important tools for probing these interactions is high-field solution nuclear magnetic resonance (NMR) through protein-observed and ligand-observed experiments, where the protein receptor or the organic compounds are selectively detected. NMR binding experiments rely on comparison of NMR parameters of the free and bound states of the molecules. Ligand-observed methods are not limited by the protein molecular size and therefore have great applicability for analysing protein-ligand interactions. The use of these NMR techniques has considerably expanded in recent years, both in chemical biology and in drug discovery. We review here three major ligand-observed NMR methods that depend on the nuclear Overhauser effect-transferred nuclear Overhauser effect spectroscopy, saturation transfer difference spectroscopy and water-ligand interactions observed via gradient spectroscopy experiments-with the aim of reporting recent developments and applications for the characterization of protein-ligand complexes, including affinity measurements and structural determination.

BcL-xL conformational changes upon fragment binding revealed by NMR.

Protein-protein interactions represent difficult but increasingly important targets for the design of therapeutic compounds able to interfere with biological processes. Recently, fragment-based strategies have been proposed as attractive approaches for the elaboration of protein-protein surface inhibitors from fragment-like molecules. One major challenge in targeting protein-protein interactions is related to the structural adaptation of the protein surface upon molecular recognition. Methods capable of identifying subtle conformational changes of proteins upon fragment binding are therefore required at the early steps of the drug design process. In this report we present a fast NMR method able to probe subtle conformational changes upon fragment binding. The approach relies on the comparison of experimental fragment-induced Chemical Shift Perturbation (CSP) of amine protons to CSP simulated for a set of docked fragment poses, considering the ring-current effect from fragment binding. We illustrate the method by the retrospective analysis of the complex between the anti-apoptotic Bcl-xL protein and the fragment 4'-fluoro-[1,1'-biphenyl]-4-carboxylic acid that was previously shown to bind one of the Bcl-xL hot spots. The CSP-based approach shows that the protein undergoes a subtle conformational rearrangement upon interaction, for residues located in helices [Formula: see text]2, [Formula: see text]3 and the very beginning of [Formula: see text]5. Our observations are corroborated by residual dipolar coupling measurements performed on the free and fragment-bound forms of the Bcl-xL protein. These NMR-based results are in total agreement with previous molecular dynamic calculations that evidenced a high flexibility of Bcl-xL around the binding site. Here we show that CSP of protein amine protons are useful and reliable structural probes. Therefore, we propose to use CSP simulation to assess protein conformational changes upon ligand binding in the fragment-based drug design approach.

Solution structure of an archaeal DNA binding protein with an eukaryotic zinc finger fold.

While the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1. The sequence of the protein consists of the classical eukaryotic C2H2 motif with the fourth histidine coordinating zinc missing, as well as of N- and C-terminal extensions. We showed that the protein AFV1p06 binds zinc and solved its solution structure by NMR. AFV1p06 displays a zinc finger fold with a novel structure extension and disordered N- and C-termini. Structure calculations show that a glutamic acid residue that coordinates zinc replaces the fourth histidine of the C2H2 motif. Electromobility gel shift assays indicate that the protein binds to DNA with different affinities depending on the DNA sequence. AFV1p06 is the first experimentally characterised archaeal zinc finger protein with a DNA binding activity. The AFV1p06 protein family has homologues in diverse viruses of hyperthermophilic archaea. A phylogenetic analysis points out a common origin of archaeal and eukaryotic C2H2 zinc fingers.

Competitive binding of UBPY and ubiquitin to the STAM2 SH3 domain revealed by NMR.

To date, the signal transducing adaptor molecule 2 (STAM2) was shown to harbour two ubiquitin binding domains (UBDs) known as the VHS and UIM domains, while the SH3 domain of STAM2 was reported to interact with deubiquitinating enzymes (DUBs) like UBPY and AMSH. In the present study, NMR evidences the interaction of the STAM2 SH3 domain with ubiquitin, demonstrating that SH3 constitutes the third UBD of STAM2. Furthermore, we show that a UBPY-derived peptide can outcompete ubiquitin for SH3 binding and vice versa. These results suggest that the SH3 domain of STAM2 plays versatile roles in the context of ubiquitin mediated receptor sorting.

Structure, function, and targets of the transcriptional regulator SvtR from the hyperthermophilic archaeal virus SIRV1.

We have characterized the structure and the function of the 6.6-kDa protein SvtR (formerly called gp08) from the rod-shaped virus SIRV1, which infects the hyperthermophilic archaeon Sulfolobus islandicus that thrives at 85 degrees C in hot acidic springs. The protein forms a dimer in solution. The NMR solution structure of the protein consists of a ribbon-helix-helix (RHH) fold between residues 13 and 56 and a disordered N-terminal region (residues 1-12). The structure is very similar to that of bacterial RHH proteins despite the low sequence similarity. We demonstrated that the protein binds DNA and uses its beta-sheet face for the interaction like bacterial RHH proteins. To detect all the binding sites on the 32.3-kb SIRV1 linear genome, we designed and performed a global genome-wide search of targets based on a simplified electrophoretic mobility shift assay. Four targets were recognized by the protein. The strongest binding was observed with the promoter of the gene coding for a virion structural protein. When assayed in a host reconstituted in vitro transcription system, the protein SvtR (Sulfolobus virus transcription regulator) repressed transcription from the latter promoter, as well as from the promoter of its own gene.

NMR-based identification of peptides that specifically recognize the d-arm of tRNA.

Human tRNA3(Lys) is used by HIV virus as a primer for the reverse transcription of its genome. The 18 nucleotides at the 3'-end of the tRNA3(Lys) are hybridized to a complementary sequence of the viral RNA called the primer-binding site. A screen against the human tRNA3(Lys) over a peptide library designed to target RNA has been performed. Of the 175 hexapeptides tested, three were found to bind to the d-stem of tRNA3(Lys). Alanine-scanning was used to define the determinants of the interaction between the peptides and tRNA3(Lys). They also bind to two other tested tRNAs, also at the level of the d-stem and loop, although the nucleotide sequence of the stem differs in one of them. These short peptides thus recognize specific structural features within the d-stem and loop of tRNAs. Associated with other pharmacophores, they could be useful to design optimized ligands targeting specific tRNAs such as retroviral replication primers.