Target-Templated Construction of Functional Proteomimetics Using Photo-Foldamer Libraries.

Current methods for proteomimetic engineering rely on structure-based design. Here we describe a design strategy that allows the construction of proteomimetics against challenging targets without a priori characterization of the target surface. Our approach employs (i) a 100-membered photoreactive foldamer library, the members of which act as local surface mimetics, and (ii) the subsequent affinity maturation of the primary hits using systems chemistry.

Solvent induced amyloid polymorphism and the uncovering of the elusive class 3 amyloid topology.

Aggregation-prone-motifs (APRs) of proteins are short segments, which - as isolated peptides - form diverse amyloid-like crystals. We introduce two APRs - designed variants of the incretin mimetic Exendin-4 - that both display crystal-phase polymorphism. Crystallographic and spectroscopic analysis revealed that a single amino-acid substitution can greatly reduce topological variability: while LYIQWL can form both parallel and anti-parallel β-sheets, LYIQNL selects only the former.

LoCoHD: a metric for comparing local environments of proteins.

Protein folds and the local environments they create can be compared using a variety of differently designed measures, such as the root mean squared deviation, the global distance test, the template modeling score or the local distance difference test. Although these measures have proven to be useful for a variety of tasks, each fails to fully incorporate the valuable chemical information inherent to atoms and residues, and considers these only partially and indirectly. Here, we develop the highly flexible local composition Hellinger distance (LoCoHD) metric, which is based on the chemical composition of local residue environments.

Polymorphic amyloid nanostructures of hormone peptides involved in glucose homeostasis display reversible amyloid formation.

A large group of hormones are stored as amyloid fibrils in acidic secretion vesicles before they are released into the bloodstream and readopt their functional state. Here, we identify an evolutionarily conserved hexapeptide sequence as the major aggregation-prone region (APR) of gastrointestinal peptides of the glucagon family: xFxxWL. We determine nine polymorphic crystal structures of the APR segments of glucagon-like peptides 1 and 2, and exendin and its derivatives.

A carbapenem antibiotic inhibiting a mammalian serine protease: structure of the acylaminoacyl peptidase-meropenem complex.

The structure of porcine AAP (pAAP) in a covalently bound complex with meropenem was determined by cryo-EM to 2.1 Å resolution, showing the mammalian serine-protease inhibited by a carbapenem antibiotic. AAP is a modulator of the ubiquitin-proteasome degradation system and the site of a drug-drug interaction between the widely used antipsychotic, valproate and carbapenems.

Omicron Binding Mode: Contact Analysis and Dynamics of the Omicron Receptor-Binding Domain in Complex with ACE2.

On 26 November 2021, the WHO classified the Omicron variant of the SARS-CoV-2 virus (B.1.1.

Cryo-EM structure of acylpeptide hydrolase reveals substrate selection by multimerization and a multi-state serine-protease triad.

The first structure of tetrameric mammalian acylaminoacyl peptidase, an enzyme that functions as an upstream regulator of the proteasome through the removal of terminal -acetylated residues from its protein substrates, was determined by cryo-EM and further elucidated by MD simulations. Self-association results in a toroid-shaped quaternary structure, guided by an amyloidogenic β-edge and unique inserts. With a Pro introduced into its central β-sheet, sufficient conformational freedom is awarded to the segment containing the catalytic Ser587 that the serine protease catalytic triad alternates between active and latent states.

The Importance of Mg -Free State in Nucleotide Exchange of Oncogenic K-Ras Mutants.

For efficient targeting of oncogenic K-Ras interaction sites, a mechanistic picture of the Ras-cycle is necessary. Herein, we used NMR relaxation techniques and molecular dynamics simulations to decipher the role of slow dynamics in wild-type and three oncogenic P-loop mutants of K-Ras. Our measurements reveal a dominant two-state conformational exchange on the ms timescale in both GDP- and GTP-bound K-Ras.

Directed Evolution-Driven Increase of Structural Plasticity Is a Prerequisite for Binding the Complement Lectin Pathway Blocking MASP-Inhibitor Peptides.

MASP-1 and MASP-2 are key activator proteases of the complement lectin pathway. The first specific mannose-binding lectin-associated serine protease (MASP) inhibitors had been developed from the 14-amino-acid sunflower trypsin inhibitor (SFTI) peptide by phage display, yielding SFTI-based MASP inhibitors, SFMIs. Here, we present the crystal structure of the MASP-1/SFMI1 complex that we analyzed in comparison to other existing MASP-1/2 structures.

Structural impact of GTP binding on downstream KRAS signaling.

Oncogenic RAS proteins, involved in ∼30% of human tumors, are molecular switches of various signal transduction pathways. Here we apply a new protocol for the NMR study of KRAS in its (inactive) GDP- and (activated) GTP-bound form, allowing a comprehensive analysis of the backbone dynamics of its WT-, G12C- and G12D variants. We found that Tyr32 shows opposite mobility with respect to the backbone of its surroundings: it is more flexible in the GDP-bound form while more rigid in GTP-complexes (especially in WT- and G12D-GTP).

Dynamically encoded reactivity of Ras enzymes: opening new frontiers for drug discovery.

Decoding molecular flexibility in order to understand and predict biological processes-applying the principles of dynamic-structure-activity relationships (DSAR)-becomes a necessity when attempting to design selective and specific inhibitors of a protein that has overlapping interaction surfaces with its upstream and downstream partners along its signaling cascade. Ras proteins are molecular switches that meet this definition perfectly. The close-lying P-loop and the highly flexible switch I and switch II regions are the site of nucleotide-, assisting-, and effector-protein binding.

Gas Sensing by Bacterial H-NOX Proteins: An MD Study.

Gas sensing is crucial for both prokaryotes and eukaryotes and is primarily performed by heme-based sensors, including H-NOX domains. These systems may provide a new, alternative mode for transporting gaseous molecules in higher organisms, but for the development of such systems, a detailed understanding of the ligand-binding properties is required. Here, we focused on ligand migration within the protein matrix: we performed molecular dynamics simulations on three bacterial (Ka, Ns and Cs) H-NOX proteins and studied the kinetics of CO, NO and O diffusion.

Pseudouridylation defect due to and mutations causes nephrotic syndrome with cataracts, hearing impairment, and enterocolitis.

RNA modifications play a fundamental role in cellular function. Pseudouridylation, the most abundant RNA modification, is catalyzed by the H/ACA small ribonucleoprotein (snoRNP) complex that shares four core proteins, dyskerin (DKC1), NOP10, NHP2, and GAR1. Mutations in , , or cause dyskeratosis congenita (DC), a disorder characterized by telomere attrition.

Tissue-Specific Requirement for the GINS Complex During Zebrafish Development.

Efficient and accurate DNA replication is particularly critical in stem and progenitor cells for successful proliferation and survival. The replisome, an amalgam of protein complexes, is responsible for binding potential origins of replication, unwinding the double helix, and then synthesizing complimentary strands of DNA. According to current models, the initial steps of DNA unwinding and opening are facilitated by the CMG complex, which is composed of a GINS heterotetramer that connects Cdc45 with the mini-chromosome maintenance (Mcm) helicase.

Tumor-Suppressor p53TAD Forms a Fuzzy Complex with Metastasis-Associated S100A4: Structural Insights and Dynamics by an NMR/MD Approach.

Conformationally flexible protein complexes represent a major challenge for structural and dynamical studies. We present herein a method based on a hybrid NMR/MD approach to characterize the complex formed between the disordered p53TAD and the metastasis-associated S100A4. Disorder-to-order transitions of both TAD1 and TAD2 subdomains upon interaction is detected.

Configuration-Controlled Crystal and/or Gel Formation of Protected d-Glucosamines Supported by Promiscuous Interaction Surfaces and a Conformationally Heterogeneous Solution State.

The configuration-dependent self-association mode of the two anomers of O-Ac,N-Fmoc-d-glucosamine, a foldamer building block, leading to gel and/or single crystal formation is described. The β-anomer of the sugar amino acid (2) forms a gel from various solvents (confirmed by SEM, rheology measurements, NMR, and ECD spectroscopy), whereas the α-anomer (1) does not form a gel with any solvent tested. Transition from the solution state to a gel is coupled to a concurrent shift of the Fmoc-groups: from a freely rotating (almost symmetrical) to a specific, asymmetric orientation.

Off-pathway 3D-structure provides protection against spontaneous Asn/Asp isomerization: shielding proteins Achilles heel.

Spontaneous deamidation prompted backbone isomerization of Asn/Asp residues resulting in - most cases - the insertion of an extra methylene group into the backbone poses a threat to the structural integrity of proteins. Here we present a systematical analysis of how temperature, pH, presence of charged residues, but most importantly backbone conformation and dynamics affect isomerization rates as determined by nuclear magnetic resonance in the case of designed peptide-models. We demonstrate that restricted mobility (such as being part of a secondary structural element) may safeguard against isomerization, but this protective factor is most effective in the case of off-pathway folds which can slow the reaction by several magnitudes compared to their on-pathway counterparts.

The Route from the Folded to the Amyloid State: Exploring the Potential Energy Surface of a Drug-Like Miniprotein.

Invited for the cover of this issue is the group of András Perczel at Eötvös Loránd University, Budapest, Hungary and colleagues from Osaka University, Japan. The image depicts the amyloid buildup of an Exenatide derivate miniprotein (E5) monitored on a simplified hyperspace. Read the full text of the article at 10.

The Route from the Folded to the Amyloid State: Exploring the Potential Energy Surface of a Drug-Like Miniprotein.

The amyloid formation of the folded segment of a variant of Exenatide (a marketed drug for type-2 diabetes mellitus) was studied by electronic circular dichroism (ECD) and NMR spectroscopy. We found that the optimum temperature for E5 protein amyloidosis coincides with body temperature and requires well below physiological salt concentration. Decomposition of the ECD spectra and its barycentric representation on the folded-unfolded-amyloid potential energy surface allowed us to monitor the full range of molecular transformation of amyloidogenesis.

Achieving Functionality Through Modular Build-up: Structure and Size Selection of Serine Oligopeptidases.

Enzymes of the prolyl oligopeptidase family (S9 family) recognize their substrates not only by the specificity motif to be cleaved but also by size - they hydrolyze oligopeptides smaller than 30 amino acids. They belong to the serine-protease family, but differ from classical serine-proteases in size (80 kDa), structure (two domains) and regulation system (size selection of substrates). This group of enzymes is an important target for drug design as they are linked to amnesia, schizophrenia, type 2 diabetes, trypanosomiasis, periodontitis and cell growth.

Protein Aggregation in a Nutshell: The Splendid Molecular Architecture of the Dreaded Amyloid Fibrils.

The recent high-resolution structures of amyloid fibrils show that the organization of peptide segments into amyloid aggregate architecture is a general process, though the morphology is more complex and intricate than suspected previously. The amyloid fibrils are often cytotoxic, accumulating as intracellular inclusions or extracellular plaques and have the ability to interfere with cellular physiology causing various cellular malfunctions. At the same time, the highly ordered amyloid structures also present an opportunity for nature to store and protect peptide chains under extreme conditions - something that might be used for designing storage, formulation, and delivery of protein medications or for contriving bio-similar materials of great resistance or structure-ordering capacity.

Assignment of Vibrational Circular Dichroism Cross-Referenced Electronic Circular Dichroism Spectra of Flexible Foldamer Building Blocks: Towards Assigning Pure Chiroptical Properties of Foldamers.

Assignment of the most established electronic circular dichroism (ECD) spectra of polypeptides and foldamers is either "evidence based" or relies on the 3D structures of longer oligomers of limited internal dynamics, which are derived from NMR spectroscopy (or X-ray) data. Critics warn that the use of NMR spectroscopy and ECD side by side has severe limitations for flexible molecules because explicit knowledge of conformational ensembles is a challenge. Herein, an old-new method of comparing ab initio computed and measured vibrational circular dichroism (VCD) data is presented to validate both the structures (conf(i)) and their relative weights (c(i)) that make up the conformational ensemble.

Hydration shell differentiates folded and disordered states of a Trp-cage miniprotein, allowing characterization of structural heterogeneity by wide-line NMR measurements.

Hydration properties of folded and unfolded/disordered miniproteins were monitored in frozen solutions by wide-line H-NMR. The amount of mobile water as function of T (-80 °C < T < 0 °C) was found characteristically different for folded (TC5b), semi-folded (pH < 3, TCb5(H+)) and disordered (TC5b_N1R) variants. Comparing results of wide-line H-NMR and molecular dynamics simulations we found that both the amount of mobile water surrounding proteins in ice, as well as their thaw profiles differs significantly as function of the compactness and conformational heterogeneity of their structure.

The mutation-dependent pathogenicity of NPHS2 p.R229Q: A guide for clinical assessment.

NPHS2, encoding podocin, is the major gene implicated in steroid-resistant nephrotic syndrome. Its c.686G>A, p.