DynaDom: structure-based prediction of T cell receptor inter-domain and T cell receptor-peptide-MHC (class I) association angles

Table S3. Per residue flip states using Reduce, Protoss and DynaDom comparing single domains and TCR complexes. (PDF 145 kb

Predicting MHC class I epitopes in large datasets

BACKGROUND: Experimental screening of large sets of peptides with respect to their MHC binding capabilities is still very demanding due to the large number of possible peptide sequences and the extensive polymorphism of the MHC proteins. Therefore, there is significant interest in the development of computational methods for predicting the binding capability of peptides to MHC molecules, as a first step towards selecting peptides for actual screening. RESULTS: We have examined the performance of four diverse MHC Class I prediction methods on comparatively large HLA-A and HLA-B allele peptide binding datasets extracted from the Immune Epitope Database and Analysis resource (IEDB). The chosen methods span a representative cross-section of available methodology for MHC binding predictions. Until the development of IEDB, such an analysis was not possible, as the available peptide sequence datasets were small and spread out over many separate efforts. We tested three datasets which differ in the IC50 cutoff criteria used to select the binders and non-binders. The best performance was achieved when predictions were performed on the dataset consisting only of strong binders (IC50 less than 10 nM) and clear non-binders (IC50 greater than 10,000 nM). In addition, robustness of the predictions was only achieved for alleles that were represented with a sufficiently large (greater than 200), balanced set of binders and non-binders. CONCLUSIONS: All four methods show good to excellent performance on the comprehensive datasets, with the artificial neural networks based method outperforming the other methods. However, all methods show pronounced difficulties in correctly categorizing intermediate binders

Thiazoline-specific amidohydrolase PurAH is the gatekeeper of bottromycin biosynthesis

The ribosomally synthesized and post-translationally modified peptide (RiPP) bottromycin A2 possesses potent antimicrobial activity. Its biosynthesis involves the enzymatic formation of a macroamidine, a process previously suggested to require the concerted efforts of a YcaO enzyme (PurCD) and an amidohydrolase (PurAH) in vivo. In vitro, PurCD alone is sufficient to catalyze formation of the macroamidine, but the process is reversible. We set out to probe the role of PurAH in macroamidine formation in vitro. We demonstrate that PurAH is highly selective for macroamidine-containing precursor peptides and cleaves C-terminal of a thiazoline, thus removing the follower peptide. After follower cleavage, macroamidine formation is irreversible, indicating PurAH as the gatekeeper of bottromycin biosynthesis. The structure of PurAH suggests residues involved in catalysis, which were probed through mutagenesis

Systematic analysis of the binding behaviour of UHRF1 towards different methyl- and carboxylcytosine modification patterns at CpG dyads

The multi-domain protein UHRF1 is essential for DNA methylation maintenance and binds DNA via a base-flipping mechanism with a preference for hemi-methylated CpG sites. We investigated its binding to hemi- and symmetrically modified DNA containing either 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), or 5-carboxylcytosine (caC). Our experimental results indicate that UHRF1 binds symmetrically carboxylated and hybrid methylated/carboxylated CpG dyads in addition to its previously reported substrates. Complementary molecular dynamics simulations provide a possible mechanistic explanation of how the protein could differentiate between modification patterns. First, we observe different local binding modes in the nucleotide binding pocket as well as the protein's NKR finger. Second, both DNA modification sites are coupled through key residues within the NKR finger, suggesting a communication pathway affecting protein-DNA binding for carboxylcytosine modifications. Our results suggest a possible additional function of the hemi-methylation reader UHRF1 through binding of carboxylated CpG sites. This opens the possibility of new biological roles of UHRF1 beyond DNA methylation maintenance and of oxidised methylcytosine derivates in epigenetic regulation

Molecular basis of telaprevir resistance due to V36 and T54 mutations in the NS3-4A protease of the hepatitis C virus

Structural analysis of the inhibitor Telaprevir (VX-950) of the hepatitis C virus (HCV) protease NS3-4A shows that mutations at V36 and/or T54 result in impaired interaction with VX-950, explaining the development of viral breakthrough variants

Ketoamide Resistance and Hepatitis C Virus Fitness in Val55 Variants of the NS3 Serine Protease

ABSTRACT Drug-resistant viral variants are a major issue in the use of direct-acting antiviral agents in chronic hepatitis C. Ketoamides are potent inhibitors of the NS3 protease, with V55A identified as mutation associated with resistance to boceprevir. Underlying molecular mechanisms are only partially understood. We applied a comprehensive sequence analysis to characterize the natural variability at Val55 within dominant worldwide patient strains. A residue-interaction network and molecular dynamics simulation were applied to identify mechanisms for ketoamide resistance and viral fitness in Val55 variants. An infectious H77S.3 cell culture system was used for variant phenotype characterization. We measured antiviral 50% effective concentration (EC 50 ) and fold changes, as well as RNA replication and infectious virus yields from viral RNAs containing variants. Val55 was found highly conserved throughout all hepatitis C virus (HCV) genotypes. The conservative V55A and V55I variants were identified from HCV genotype 1a strains with no variants in genotype 1b. Topology measures from a residue-interaction network of the protease structure suggest a potential Val55 key role for modulation of molecular changes in the protease ligand-binding site. Molecular dynamics showed variants with constricted binding pockets and a loss of H-bonded interactions upon boceprevir binding to the variant proteases. These effects might explain low-level boceprevir resistance in the V55A variant, as well as the Val55 variant, reduced RNA replication capacity. Higher structural flexibility was found in the wild-type protease, whereas variants showed lower flexibility. Reduced structural flexibility could impact the Val55 variant's ability to adapt for NS3 domain-domain interaction and might explain the virus yield drop observed in variant strains

1,4-Disubstituted 1H-1,2,3-Triazole Containing Peptidotriazolamers: A New Class of Peptidomimetics With Interesting Foldamer Properties

Schröder DC, Kracker O, Fröhr T, et al. 1,4-Disubstituted 1H-1,2,3-Triazole Containing Peptidotriazolamers: A New Class of Peptidomimetics With Interesting Foldamer Properties. Frontiers in Chemistry. 2019;7: 155.Peptidotriazolamers are hybrid foldamers with features of peptides and triazolamers, containing alternation of amide bonds and 1,4-disubstituted 1H-1,2,3-triazoles with conservation of the amino acid side chains. We report on the synthesis of a new class of peptidomimetics, containing 1,4-disubstituted 1H-1,2,3-triazoles in alternation with amide bonds and the elucidation of their conformational properties in solution. Based on enantiomerically pure propargylamines bearing the stereogenic center in the propargylic position and α-azido esters, building blocks were obtained by copper-catalyzed azide-alkyne cycloaddition. With these building blocks the peptidotriazolamers were readily available by solution phase synthesis. A panel of homo- and heterochiral tetramers, hexamers, and heptamers was synthesized and the heptamer Boc-Ala-Val-Ψ[4Tz]Phe-LeuΨ[4Tz]Phe-LeuΨ[4Tz]Val-OAll as well as an heterochiral and a Gly-containing equivalent were structurally characterized by NMR-based molecular dynamics simulations using a specifically tailored force field to determine their conformational and solvation properties. All three variants adopt a compact folded conformation in DMSO as well as in water. In addition to the heptamers we predicted the conformational behavior of similar longer oligomers i.e., Boc-Ala-(AlaΨ[4Tz]Ala)6-OAll as well as Boc-Ala-(d-AlaΨ[4Tz]Ala)6-OAll and Boc-Ala-(GlyΨ[4Tz]Ala)6-OAll. Our calculations predict a clear secondary structure of the first two molecules in DMSO that collapses in water due to the hydrophobic character of the side chains. The homochiral compound folds into a regular helical structure and the heterochiral one shows a twisted “S”-shape, while the Gly variant exhibits no clear secondary structure

Recognition of 5-Hydroxymethylcytosine by the Uhrf1 SRA Domain

Recent discovery of 5-hydroxymethylcytosine (5hmC) in genomic DNA raises the question how this sixth base is recognized by cellular proteins. In contrast to the methyl-CpG binding domain (MBD) of MeCP2, we found that the SRA domain of Uhrf1, an essential factor in DNA maintenance methylation, binds 5hmC and 5-methylcytosine containing substrates with similar affinity. Based on the co-crystal structure, we performed molecular dynamics simulations of the SRA:DNA complex with the flipped cytosine base carrying either of these epigenetic modifications. Our data indicate that the SRA binding pocket can accommodate 5hmC and stabilizes the flipped base by hydrogen bond formation with the hydroxyl group