Benchmarking biomolecular force field-based Zn for mono- and bimetallic ligand binding sites.

Zn is one of the most versatile biologically available metal ions, but accurate modeling of Zn -containing metalloproteins at the biomolecular force field level can be challenging. Since most Zn models are parameterized in bulk solvent, in-depth knowledge about their performance in a protein environment is limited. Thus, we systematically investigate here the behavior of non-polarizable Zn models for their ability to reproduce experimentally determined metal coordination and ligand binding in metalloproteins.

Distinct conformational and energetic features define the specific recognition of (di)aromatic peptide motifs by PEX14.

The cycling import receptor PEX5 and its membrane-located binding partner PEX14 are key constituents of the peroxisomal import machinery. Upon recognition of newly synthesized cargo proteins carrying a peroxisomal targeting signal type 1 (PTS1) in the cytosol, the PEX5/cargo complex docks at the peroxisomal membrane by binding to PEX14. The PEX14 N-terminal domain (NTD) recognizes (di)aromatic peptides, mostly corresponding to Wxxx(F/Y)-motifs, with nano-to micromolar affinity.

Comparison of allosteric signaling in DnaK and BiP using mutual information between simulated residue conformations.

The heat shock protein 70 kDa (Hsp70) chaperone system serves as a critical component of protein quality control across a wide range of prokaryotic and eukaryotic organisms. Divergent evolution and specialization to particular organelles have produced numerous Hsp70 variants which share similarities in structure and general function, but differ substantially in regulatory aspects, including conformational dynamics and activity modulation by cochaperones. The human Hsp70 variant BiP (also known as GRP78 or HSPA5) is of therapeutic interest in the context of cancer, neurodegenerative diseases, and viral infection, including for treatment of the pandemic virus SARS-CoV-2.

Dynamic Docking of Macrocycles in Bound and Unbound Protein Structures with DynaDock.

Macrocycles are interesting molecules with unique features due to their conformationally constrained yet flexible ring structure. This characteristic poses a difficult challenge for computational modeling studies since they rely on accurate structural descriptions. In particular, molecular docking calculations suffer from the lack of ring flexibility during pose generation, which is often compensated by using pregenerated ligand conformer ensembles.

Investigation of the pH-dependent aggregation mechanisms of GCSF using low resolution protein characterization techniques and advanced molecular dynamics simulations.

Granulocyte-colony stimulating factor (GCSF) is a widely used therapeutic protein to treat neutropenia. GCSF has an increased propensity to aggregate if the pH is increased above 5.0.

SenseNet, a tool for analysis of protein structure networks obtained from molecular dynamics simulations.

Computational methods play a key role for investigating allosteric mechanisms in proteins, with the potential of generating valuable insights for innovative drug design. Here we present the SenseNet ("Structure ENSEmble NETworks") framework for analysis of protein structure networks, which differs from established network models by focusing on interaction timelines obtained by molecular dynamics simulations. This approach is evaluated by predicting allosteric residues reported by NMR experiments in the PDZ2 domain of hPTP1e, a reference system for which previous computational predictions have shown considerable variance.

Structure-Guided Modulation of the Catalytic Properties of [2Fe-2S]-Dependent Dehydratases.

The FeS cluster-dependent dihydroxyacid dehydratases (DHADs) and sugar acid-specific dehydratases (DHTs) from the ilvD/EDD superfamily are key enzymes in the bioproduction of a wide variety of chemicals. We analyzed [2Fe-2S]-dependent dehydratases in silico and in vitro, deduced functionally relevant sequence, structure, and activity relationships within the ilvD/EDD superfamily, and we propose a new classification based on their evolutionary relationships and substrate profiles. In silico simulations and analyses identified several key positions for specificity, which were experimentally investigated with site-directed and saturation mutagenesis.

The Q41R mutation in the HCV-protease enhances the reactivity towards MAVS by suppressing non-reactive pathways.

Recent experimental findings pointed out a new mutation in the HCV protease, Q41R, responsible for a significant enhancement of the enzyme's reactivity towards the mitochondrial antiviral-signaling protein (MAVS). The Q41R mutation is located rather far from the active site, and its involvement in the overall reaction mechanism is thus unclear. We used classical molecular dynamics and QM/MM to study the acylation reaction of HCV NS3/4A protease variants bound to MAVS and the NS4A/4B substrate and uncovered the indirect mechanism by which the Q41R mutation plays a critical role in the efficient cleavage of the substrate.

Functional analysis of peripheral and intratumoral neoantigen-specific TCRs identified in a patient with melanoma.

Background: Neoantigens derived from somatic mutations correlate with therapeutic responses mediated by treatment with immune checkpoint inhibitors. Neoantigens are therefore highly attractive targets for the development of therapeutic approaches in personalized medicine, although many aspects of their quality and associated immune responses are not yet well understood. In a case study of metastatic malignant melanoma, we aimed to perform an in-depth characterization of neoantigens and respective T-cell responses in the context of immune checkpoint modulation.

Turning the Actin Nucleating Compound Miuraenamide into Nucleation Inhibitors.

Natural compounds that either increase or decrease polymerization of actin into filaments have become indispensable tools for cell biology. However, to date, it was not possible to use them as therapeutics due to their overall cytotoxicity and their unfavorable pharmacokinetics. Furthermore, their synthesis is in general quite complicated.

DynaBiS: A hierarchical sampling algorithm to identify flexible binding sites for large ligands and peptides.

Knowing the ligand or peptide binding site in proteins is highly important to guide drug discovery, but experimental elucidation of the binding site is difficult. Therefore, various computational approaches have been developed to identify potential binding sites in protein structures. However, protein and ligand flexibility are often neglected in these methods due to efficiency considerations despite the recognition that protein-ligand interactions can be strongly affected by mutual structural adaptations.

Extended interaction networks with HCV protease NS3-4A substrates explain the lack of adaptive capability against protease inhibitors.

Inhibitors against the NS3-4A protease of hepatitis C virus (HCV) have proven to be useful drugs in the treatment of HCV infection. Although variants have been identified with mutations that confer resistance to these inhibitors, the mutations do not restore replicative fitness and no secondary mutations that rescue fitness have been found. To gain insight into the molecular mechanisms underlying the lack of fitness compensation, we screened known resistance mutations in infectious HCV cell culture with different genomic backgrounds.

A modular approach to the bisbenzylisoquinoline alkaloids tetrandrine and isotetrandrine.

An efficient racemic total synthesis of the bisbenzylisoquinoline alkaloids tetrandrine and isotetrandrine in four different routes is reported herein. Key steps of the synthesis include N-acyl Pictet-Spengler condensations to access the tetrahydroisoquinoline moieties, as well as copper-catalyzed Ullmann couplings for diaryl ether formation. Starting from commercially available building blocks tetrandrine and isotetrandrine are accessed in 12 steps.

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.

Repurposing human kinase inhibitors to create an antibiotic active against drug-resistant Staphylococcus aureus, persisters and biofilms.

New drugs are desperately needed to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we report screening commercial kinase inhibitors for antibacterial activity and found the anticancer drug sorafenib as major hit that effectively kills MRSA strains. Varying the key structural features led to the identification of a potent analogue, PK150, that showed antibacterial activity against several pathogenic strains at submicromolar concentrations.

Acyldepsipeptide Probes Facilitate Specific Detection of Caseinolytic Protease P Independent of Its Oligomeric and Activity State.

Caseinolytic protease P (ClpP) is a tetradecameric peptidase that assembles with chaperones such as ClpX to gain proteolytic activity. Acyldepsipeptides (ADEPs) are small-molecule mimics of ClpX that bind into hydrophobic pockets on the apical site of the complex, thereby activating ClpP. Detection of ClpP has so far been facilitated with active-site-directed probes which depend on the activity and oligomeric state of the complex.

Key Features Relevant to Select Antigens and TCR From the MHC-Mismatched Repertoire to Treat Cancer.

Adoptive transfer of T cells transgenic for tumor-reactive T-cell receptors (TCR) is an attractive immunotherapeutic approach. However, clinical translation is so far limited due to challenges in the identification of suitable target antigens as well as TCRs that are concurrent safe and efficient. Definition of key characteristics relevant for effective and specific tumor rejection is essential to improve current TCR-based adoptive T-cell immunotherapies.

Actin stabilizing compounds show specific biological effects due to their binding mode.

Actin binding compounds are widely used tools in cell biology. We compare the biological and biochemical effects of miuraenamide A and jasplakinolide, a structurally related prototypic actin stabilizer. Though both compounds have similar effects on cytoskeletal morphology and proliferation, they affect migration and transcription in a distinctive manner, as shown by a transcriptome approach in endothelial cells.

Predicting the bioactive conformations of macrocycles: a molecular dynamics-based docking procedure with DynaDock.

Macrocyclic compounds are of growing interest as a new class of therapeutics, especially as inhibitors binding to protein-protein interfaces. As molecular modeling is a well-established complimentary tool in modern drug design, the number of attempts to develop reliable docking strategies and algorithms to accurately predict the binding mode of macrocycles is rising continuously. Standard molecular docking approaches need to be adapted to this application, as a comprehensive yet efficient sampling of all ring conformations of the macrocycle is necessary.

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.

A combined in silico and in vitro study on mouse Serpina1a antitrypsin-deficiency mutants.

Certain point-mutations in the human SERPINA1-gene can cause severe α1-antitrypsin-deficiency (A1AT-D). Affected individuals can suffer from loss-of-function lung-disease and from gain-of-function liver-disease phenotypes. However, age of onset and severity of clinical appearance is heterogeneous amongst carriers, suggesting involvement of additional genetic and environmental factors.

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

Peptidotriazolamers are hybrid foldamers with features of peptides and triazolamers, containing alternation of amide bonds and 1,4-disubstituted 1-1,2,3-triazoles with conservation of the amino acid side chains. We report on the synthesis of a new class of peptidomimetics, containing 1,4-disubstituted 1-1,2,3-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.

Chemical Cross-Linking Enables Drafting ClpXP Proximity Maps and Taking Snapshots of In Situ Interaction Networks.

Detection of dynamic protein-protein interactions within complexes and networks remains a challenging task. Here, we show by the example of the proteolytic ClpXP complex the utility of combined chemical cross-linking and mass spectrometry (XL-MS) to map interactions within ClpP and ClpX as well as across the enigmatic ClpX hexamer-ClpP heptamer interface. A few hot-spot lysines located in signature loops in ClpX were shown to be in proximity to several structural regions of ClpP providing an initial draft of the ClpX-ClpP interaction.

Ca2+ binding induced sequential allosteric activation of sortase A: An example for ion-triggered conformational selection.

The allosteric activation of the intrinsically disordered enzyme Staphylococcus aureus sortase A is initiated via binding of a Ca2+ ion. Although Ca2+ binding was shown to initiate structural changes inducing disorder-to-order transitions, the details of the allosteric activation mechanism remain elusive. We performed long-term molecular dynamics simulations of sortase A without (3 simulations of 1.