Remodelling the surface of thioredoxin from Escherichia coli by grafting an iron-binding site from the CyaY protein family.

In this work, we have designed and generated a Fe(III)-binding protein with thiol oxidoreductase activity. The consensus iron-binding motif EExxED from the frataxin protein family was grafted on a model peptide and on the surface of thioredoxin (TRX) from E. coli. We investigated metal interactions with a family of peptides containing the motif EExxED or altered versions obtained by removing negatively charged residues: EExxEx, xExxED, and xExxEx. The interaction of the metal ion with the peptides was studied by circular dichroism, and our results indicated that the motif EExxED retained its functional properties and also that this motif is able to bind Ga(III) and Al(III). The interaction of the grafted TRX with iron(III) was investigated by NMR, showing that the motif was functional in the context of the protein structure, and also the binding of two equivalents of Fe(III) per TRX molecule was stable in a non-chelating neutral buffer. Protein conformation, stability, and enzymatic activity were studied by applying experimental and computational approaches. Interestingly, the thiol oxidoreductase activity was modulated by interaction with Ga(III), a Fe(III) mimetic ion. Furthermore, the design of functional proteins with both functions, oxidoreductase activity and metal-ion binding ability, should consider the reorganisation of the electrostatic network. Similarly, studying the crosstalk and electrostatic balance among different metal-binding sites may be critical.

Structural Modelling of KCNQ1 and KCNH2 Double Mutant Proteins, Identified in Two Severe Long QT Syndrome Cases, Reveals New Insights into Cardiac Channelopathies.

Congenital long QT syndrome (LQTS) is a cardiac channelopathy characterized by a prolongation of the QT interval and T-wave abnormalities, caused, in most cases, by mutations in KCNQ1, KCNH2, and SCN5A. Although the predominant pattern of LQTS inheritance is autosomal dominant, compound heterozygous mutations in genes encoding potassium channels have been reported, often with early disease onset and more severe phenotypes. Since the molecular mechanisms underlying severe phenotypes in carriers of compound heterozygous mutations are unknown, it is possible that these compound mutations lead to synergistic or additive alterations to channel structure and function. In this study, all-atom molecular dynamic simulations of KCNQ1 and hERG channels were carried out, including wild-type and channels with compound mutations found in two patients with severe LQTS phenotypes and limited family history of the disease. Because channels can likely incorporate different subunit combinations from different alleles, there are multiple possible configurations of ion channels in LQTS patients. This analysis allowed us to establish the structural impact of different configurations of mutant channels in the activated/open state. Our data suggest that channels with these mutations show moderate changes in folding energy (in most cases of stabilizing character) and changes in channel mobility and volume, differentiating them from each other and from WT. This would indicate possible alterations in K+ ion flow. Hetero-tetrameric mutant channels showed intermediate structural and volume alterations vis-à-vis homo-tetrameric channels. These findings support the hypothesis that hetero-tetrameric channels in patients with compound heterozygous mutations do not necessarily lead to synergistic structural alterations.

MHCBI: a pipeline for calculating peptide-MHC binding energy using semi-empirical quantum mechanical methods with explicit/implicit solvent models.

Experimentally estimating peptide-major histocompatibility complex (pMHC) binding affinity has been quite challenging due to the many receptors and the many potential ligands implicated in it. We have thus proposed a straightforward computational methodology considering the different mechanisms involved in pMHC binding to facilitate studying such receptor-ligand interactions. We have developed a pipeline using semi-empirical quantum mechanical methods for calculating pMHC class I and II molecules' binding energy (BE). This pipeline can systematize the methodology for calculating pMHC system BE, enabling the rational design of T-cell epitopes to be used as pharmaceuticals and vaccines.

Exploring the conformational transition between the fully folded and locally unfolded substates of Escherichia coli thiol peroxidase.

Thiol peroxidase from Escherichia coli (EcTPx) is a peroxiredoxin that catalyzes the reduction of different hydroperoxides. During the catalytic cycle of EcTPx, the peroxidatic cysteine (CP) is oxidized to a sulfenic acid by peroxide, then the resolving cysteine (CR) condenses with the sulfenic acid of CP to form a disulfide bond, which is finally reduced by thioredoxin. Purified EcTPx as dithiol and disulfide behaves as a monomer under near physiological conditions. Although secondary structure rearrangements are present when comparing different redox states of the enzyme, no significant differences in unfolding free energies are observed under reducing and oxidizing conditions. A conformational change denominated fully folded (FF) to locally unfolded (LU) transition, involving a partial unfolding of αH2 and αH3, must occur to enable the formation of the disulfide bond since the catalytic cysteines are 12 Å apart in the FF conformation of EcTPx. To explore this process, the FF → LU and LU → FF transitions were studied using conventional molecular dynamics simulations and an enhanced conformational sampling technique for different oxidation and protonation states of the active site cysteine residues CP and CR. Our results suggest that the FF → LU transition has a higher associated energy barrier than the refolding LU → FF process in agreement with the relatively low experimental turnover number of EcTPx. Furthermore, in silico designed single-point mutants of αH3 enhanced locally unfolding events, suggesting that the native FF interactions in the active site are not evolutionarily optimized to fully speed-up the conformational transition of wild-type EcTPx.

Assessing Peptide Binding to MHC II: An Accurate Semiempirical Quantum Mechanics Based Proposal.

Estimating peptide-major histocompatibility complex (pMHC) binding using structural computational methods has an impact on understanding overall immune function triggering adaptive immune responses in MHC class II molecules. We developed a strategy for optimizing pMHC structure interacting with water molecules and for calculating the binding energy of receptor + ligand systems, such as HLA-DR1 + HA, HLA-DR1 + CLIP, HLA-DR2 + MBP, and HLA-DR3 + CLIP, as well as a monosubstitution panel. Taking pMHC's structural properties, we assumed that ΔH ≫ -TΔS would generate a linear model for estimating relative free energy change, using three semiempirical quantum methods (PM6, PM7, and FMO-SCC-DFTB3) along with the implicit solvent models, and considering proteins in neutral and charged states. Likewise, we confirmed our approach's effectiveness in calculating binding energies having high correlation with experimental data and low root-mean-square error (<2 kcal/mol). All in all, our pipeline differentiates weak from strong peptide binders as a reliable method for studying pMHC interactions.

Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants.

Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity.

A helix-coil transition induced by the metal ion interaction with a grafted iron-binding site of the CyaY protein family.

Iron-protein interactions are involved in electron transfer reactions. Alterations of these processes are present in a number of human pathologies; among them, in Friedreich's ataxia, in which a deficiency of functional frataxin, an iron-binding protein, leads to progressive neuromuscular degenerative disease. The putative iron-binding motif of acidic residues EExxED was selected from the first α-helical stretch of the frataxin protein family and grafted onto a foreign peptide scaffold corresponding to the C-terminal α-helix from E. coli thioredoxin. The resulting grafted peptide named GRAP was studied by applying experimental (circular dichroism, isothermal titration calorimetry, capillary zone electrophoresis, thermal denaturation, NMR) and computational approaches (docking, molecular dynamics simulations). Although isolated GRAP lacks a stable secondary structure in solution, when iron is added, the peptide acquires an α-helical structure. Here we have shown that the designed peptide is able to specifically bind Fe(3+) with a moderate affinity (KD = 1.9 ± 0.2 µM) and a 1 : 1 stoichiometry. Remarkably, the GRAP/Fe(3+) interaction is entropically driven (ΔH° = -1.53 ± 0.03 kcal mol(-1) and TΔS° = 6.26 kcal mol(-1)). Experiments and simulations indicate that Fe(3+) interacts with the peptide through three acidic side chains, inducing an α-helical conformation of the grafted motif. In addition, the acidic side chains involved undergo significant conformational rearrangements upon binding, as judged by the analysis of MDs. Altogether, these results contribute to an understanding of the iron-binding mechanisms in proteins and, in particular, in the case of human frataxin.

Electrostatic potential as a tool to understand interactions between malaria vaccine candidate peptides and MHC II molecules.

One of the most important problems in vaccine development consists in understanding receptor-ligand interactions between Class II Major Histocompatibility Complex molecules (MHC II) and antigenic peptides involved in inducing an appropriate immune response. In this study, we used X-ray crystallography structural data provided by the HLA-DRß1*0301-CLIP peptide interaction to compare native non-immunogenic and specifically-modified immunogenic peptides derived from the malarial SALSA protein, by analyzing molecular electrostatic potential surfaces on the most important regions of the peptide binding groove (Pockets 1, 4, 6 and 9). Important differences were found on the electrostatic potential induced by these peptides, particularly in MHC II conserved residues: Qα9, Sα53, Nα62, Nα69, Yß30, Yß60, Wß61, Qß70, Kß71 and Vß86, the same ones involved in establishing hydrogen bonds between Class II molecule-peptide and the recognition by T cell receptor, it correlating well with the change in their immunological properties. The results clearly suggest that modifications done on the electrostatic potential of these amino acids could favor the induction of different immune responses and therefore, their identification could allow modifying peptides a priori and in silico, so as to render them into immunogenic and protection-inducers and hence suitable components of a chemically-synthesized, multi-antigenic, minimal subunit based vaccine.

Variations in the electrostatic landscape of class II human leukocyte antigen molecule induced by modifications in the myelin basic protein peptide: a theoretical approach.

The receptor-ligand interactions involved in the formation of the complex between Class II Major Histocompatibility Complex molecules and antigenic peptides, which are essential for establishing an adaptive immunological response, were analyzed in the Class II Human Leukocyte Antigen (HLA)--Myelin Basic Protein (MBP) peptide complex (HLA-DRbeta1*1501-MBP) using a multipolar molecular electrostatic potential approach. The Human Leukocyte Antigen--peptide complex system was divided into four pockets together with their respective peptide fragment and the corresponding occupying amino acid was replaced by each of the remaining 19 amino acids. Partial atomic charges were calculated by a quantum chemistry approach at the Hatree Fock/3-21*G level, to study the behavior of monopole, dipole and quadrupole electrostatic multipolar moments. Two types of electrostatic behavior were distinguished in the pockets' amino acids: "anchoring" located in Pocket 1 and 4, and "recognition" located in Pocket 4 and 7. According to variations in the electrostatic landscape, pockets were ordered as: Pocket 1>Pocket 9>>Pocket 4 approximately Pocket 7 which is in agreement with the binding ability reported for Class II Major Histocompatibility Complex pockets. In the same way, amino acids occupying the polymorphic positions beta13R, beta26F, beta28D, beta9W, beta74A, beta47F and beta57D were shown to be key for this Receptor-Ligand interaction. The results show that the multipolar molecular electrostatic potential approach is appropriate for characterizing receptor-ligand interactions in the MHC-antigenic peptide complex, which could have potential implications for synthetic vaccine design.

Radial scan of the molecular electrostatic potential of RNA double helices: an application to the enzyme-tRNA recognition.

We introduced a method to characterize quantitatively the molecular electrostatic potential (MEP) of the minor and major grooves of base pairs located at nucleic acid double helices. By means of a radial MEP scan, we obtained a n-tuple of potential values corresponding to each groove, which can be analyzed by plotting the MEP values as a function of the angle in the radial scan. We studied base pairs of two different tRNAs, relevant in the recognition process with their cognate aminoacyl tRNA synthetases (aaRSs), in order to correlate their electrostatic behavior with the corresponding aminoacylation activity. We analyzed the first three base pairs of the Escherichia coli tRNA(Ala) acceptor stem, finding several cases where the MEP profiles obtained from the plots are in agreement with the reported aminoacylation activities. Additionally, a non-hierarchical clustering performed over the MEP n-tuples resulted in meaningful classifications that correlate with the activity and with the predicted stereochemistry of the reaction. We also studied the first two base pairs of the E. coli tRNA(Thr) acceptor stem but constraining the analysis to the angle intervals that seem relevant for the binding sites of the enzyme. These intervals were deduced from the ThrRS-tRNA(Thr) complex crystal structure. In this case, we also found a good agreement between the MEP profiles and the activity, supporting the idea that the tRNA identity elements function is to allow an optimal electrostatic complementarity between the aminoacyl-tRNA synthetase and the tRNA.