Mariana Gallo

2´-C-Methyluridine phosphoramidite: a new building block for the preparation of RNA analogues carrying the 2´-hydroxyl group

A new stereoselective synthesis of 2′-C-methyluridine was carried out. The preparation of the corresponding protected phosphoramidite suitable for the automated synthesis of oligonucleotides, including the regioselective protection of the 2′-hydroxyl group, is described here. This new modified nucleotide is expected to generate RNA analogues potentially useful in applications where proper RNA folding is required since the 2′-hydroxyl is conserved.

Solution structure of ApaG from Xanthomonas axonopodis pv. citri reveals a fibronectin-3 fold.

ApaG proteins are found in a wide variety of bacterial genomes but their function is as yet unknown. Some eukaryotic proteins involved in protein‐protein interactions, such as the human polymerase δ‐interacting protein (PDIP38) and the F Box A (FBA) proteins, contain ApaG homology domains. We have used NMR to determine the solution structure of ApaG protein from the plant pathogen Xanthomonas axonopodis pv. citri (ApaGXac) with the aim to shed some light on its molecular function. ApaGXac is characterized by seven antiparallel β strands forming two β sheets, one containing three strands (ABE) and the other four strands (GFCC′). Relaxation measurements indicate that the protein has a quite rigid structure. In spite of the presence of a putative GXGXXG pyrophosphate binding motif ApaGXac does not bind ATP or GTP, in vitro. On the other hand, ApaGXac adopts a fibronectin type III (Fn3) fold, which is consistent with the hypothesis that it is involved in mediating protein‐protein interactions. The fact that the proteins of ApaG family do not display significant sequence similarity with the Fn3 domains found in other eukaryotic or bacterial proteins suggests that Fn3 domain may have arisen earlier in evolution than previously estimated. Proteins 2007

A strained DNA binding helix is conserved for site recognition, folding nucleation, and conformational modulation†

Nucleic acid recognition is often mediated by α‐helices or disordered regions that fold into α‐helix on binding. A peptide bearing the DNA recognition helix of HPV16 E2 displays type II polyproline (PII) structure as judged by pH, temperature, and solvent effects on the CD spectra. NMR experiments indicate that the canonical α‐helix is stabilized at the N‐terminus, while the PII forms at the C‐terminus half of the peptide. Re‐examination of the dihedral angles of the DNA binding helix in the crystal structure and analysis of the NMR chemical shift indexes confirm that the N‐terminus half is a canonical α‐helix, while the C‐terminal half adopts a 310 helix structure. These regions precisely match two locally driven folding nucleii, which partake in the native hydrophobic core and modulate a conformational switch in the DNA binding helix. The peptide shows only weak and unspecific residual DNA binding, 104‐fold lower affinity, and 500‐fold lower discrimination capacity compared with the domain. Thus, the precise side chain conformation required for modulated and tight physiological binding by HPV E2 is largely determined by the noncanonical strained α‐helix conformation, “presented” by this unique architecture.

Synthesis and properties of (2′S)- and (2′R)-2′-deoxy-2′-C-methyl oligonucleotides

The synthesis of (2′S)- and (2′R)-2′-deoxy-2′-C-methyl-N3-(4-t-butylbenzoyl) uridine, (2′S)-2′-deoxy-2′-C-methyluridine and (2′S)-2′-deoxy-2′-C-methyl-N4-isobutyryl cytidine building blocks are here described. The preparation of oligonucleotides carrying these monomers in all positions but 3′-end is presented and the binding affinity between these new fragments and the complementary DNA and RNA sequences is also assessed. (2′R) substituted oligonucleotides did not hybridize with either the complementary DNA or RNA sequences. However, the first derivative of melting curves of hybrids containing (2′S) modified oligonucleotides indicated melting transitions.

Structural characterization of the Hepatitis C Virus NS3 protease from genotype 3a: The basis of the genotype 1b vs. 3a inhibitor potency shift

The first structural characterization of the genotype 3a Hepatitis C Virus NS3 protease is reported, providing insight into the differential susceptibility of 1b and 3a proteases to certain inhibitors. Interaction of the 3a NS3 protease with a P2-P4 macrocyclic and a linear phenethylamide inhibitor was investigated. In addition, the effect of the NS4A cofactor binding on the conformation of the protease was analyzed. Complexation of NS3 with the phenethylamide inhibitor significantly stabilizes the protease but binding does not involve residues 168 and 123, two key amino acids underlying the different inhibition of genotype 1b vs. 3a proteases by P2-P4 macrocycles. Therefore, we studied the dynamic behavior of these two residues in the phenethylamide complex, serving as a model of the situation in the apo 3a protein, in order to explore the structural basis of the inhibition potency shift between the proteases of the genotypes 1b and 3a.

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) drug discovery: Biochemical toolbox to develop NRF2 activators by reversible binding of Kelch-like ECH-associated protein 1 (KEAP1)

Mechanisms that activate innate antioxidant responses, as a way to mitigate oxidative stress at the site of action, hold much therapeutic potential in diseases, such as Parkinsons disease, Alzheimers disease and Huntingtons disease, where the use of antioxidants as monotherapy has not yielded positive results. The nuclear factor NRF2 is a transcription factor whose activity upregulates the expression of cell detoxifying enzymes in response to oxidative stress. NRF2 levels are modulated by KEAP1, a sensor of oxidative stress. KEAP1 binds NRF2 and facilitates its ubiquitination and subsequent degradation. Recently, compounds that reversibly disrupt the NRF2-KEAP1 interaction have been described, opening the field to a new era of safer NRF2 activators. This paper describes a set of new, robust and informative biochemical assays that enable the selection and optimization of non-covalent KEAP1 binders. These include a time-resolved fluorescence resonance energy transfer (TR-FRET) primary assay with high modularity and robustness, a surface plasmon resonance (SPR) based KEAP1 direct binding assay that enables the quantification and analysis of full kinetic binding parameters and finally a 1H-15N heteronuclear single quantum coherence (HSQC) NMR assay suited to study the interaction surface of KEAP1 with residue-specific information to validate the interaction of ligands in the KEAP1 binding site.

A new member of the ribbon-helix-helix transcription factor superfamily from the plant pathogen Xanthomonas axonopodis pv. citri

XACb0070 is an uncharacterized protein coded by the two large plasmids isolated from Xanthomonas axonopodis pv. citri, the agent of citrus canker and responsible for important economical losses in citrus world production. XACb0070 presents sequence homology only with other hypothetical proteins belonging to plant pathogens, none of which have their structure determined. The NMR-derived solution structure reveals this protein is a homodimer in which each monomer presents two domains with different structural and dynamic properties: a folded N-terminal domain with beta alpha alpha topology which mediates dimerization and a long disordered C-terminal tail. The folded domain shows high structural similarity to the ribbon-helix-helix transcriptional repressors, a family of DNA-binding proteins of conserved 3D fold but low sequence homology: indeed XACb0070 binds DNA. Primary sequence and fold comparison of XACb0070 with other proteins of the ribbon-helix-helix family together with examination of the genes in the vicinity of xacb0070 suggest the protein might be the component of a toxin-antitoxin system.

N-Lobe Dynamics of Myosin Light Chain Dictates Its Mode of Interaction with Myosin V IQ1†

Myosin V motors regulate secretion and cell division in eukaryotes. How MyoV activity is differentially regulated by essential and calmodulin light chain binding remains unclear. We have used NMR spectroscopy to compare the dynamic behavior of Mlc1p, a budding yeast essential light chain, with that of the Xenopus laevis calmodulin. Both proteins have a similar structure and bind similar target proteins but differ in the mechanism by which they interact with the myosin V IQ1. This interaction is essential for MyoV activity. Here, we show that the rigid conformation of the loop connecting the two EF-hand motifs of the Mlc1p N-lobe explains its differential ability to interact with myosin V IQ1. Moreover, we show that the maintenance of the N-lobe structure is required for the essential function of Mlc1p in vivo. These data show that the core characteristics of myosin light chain N-lobes differentiate Mlc1p and calmodulin binding capability.

Insights on the conformational dynamics of human frataxin through modifications of loop-1

Human frataxin (FXN) is a highly conserved mitochondrial protein involved in iron homeostasis and activation of the iron-sulfur cluster assembly. FXN deficiency causes the neurodegenerative disease Friedreichs Ataxia. Here, we investigated the effect of alterations in loop-1, a stretch presumably essential for FXN function, on the conformational stability and dynamics of the native state. We generated four loop-1 variants, carrying substitutions, insertions and deletions. All of them were stable and well-folded proteins. Fast local motions (ps-ns) and slower long-range conformational dynamics (μs-ms) were altered in some mutants as judged by NMR. Particularly, loop-1 modifications impact on the dynamics of a distant region that includes residues from the β-sheet, helix α1 and the C-terminal. Remarkably, all the mutants retain the ability to activate cysteine desulfurase, even when two of them exhibit a strong decrease in iron binding, revealing a differential sensitivity of these functional features to loop-1 perturbation. Consequently, we found that even for a small and relatively rigid protein, engineering a loop segment enables to alter conformational dynamics through a long-range effect, preserving the native-state structure and important aspects of function.