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. By comparing the structure of various members of the family we show that the most important features contributing to selectivity and efficiency are: (i) whether the interactions weaving the two domains together play a role in stabilizing the catalytic triad and thus their absence may provide for its deactivation: these oligopeptidases can screen their substrates by opening up, and (ii) whether the interaction-prone β-edge of the hydrolase domain is accessible and thus can guide a multimerization process that creates shielded entrance or intricate inner channels for the size-based selection of substrates. These cornerstones can be used to estimate the multimeric state and selection strategy of yet undetermined structures

Assignment of vibrational circular dichroism cross-referenced electronic circular dichroism spectra of flexible foldamer building blocks: towards assigning foldamers’ pure chiroptical properties

The assignment of the most established ECD spectra of polypeptides and foldamers is either “evidence based” or rely on the 3D-structures of longer oligomers of limited internal dynamics, derived from NMR (or X-ray) data. Critics warn that using NMR and ECD side by side have severe limitations for flexible molecules as the explicit knowledge of the conformational ensembles is a challenge. Herein we present the old-new method of comparing ab initio computed and measured VCD data to validate both structures and, conf(i), and their relative weights, c(i), making up the conformational ensemble. Based on the array of {conf(i), c(i)} the pure ECD spectra, g(i)conf(i), can be ab initio calculated. The reconstructed spectrum Σc(i)*g(i)conf(i) can thus help to assign any experimental ECD counterpart. Here we present such a protocol successfully applied for flexible foldamer building blocks of sugar β-amino acid diamides. The epimeric pair of our model system was selected because these molecules are conformationally tunable by simple chemical modification (N-methylation) and thus, the robustness of our current approach could be probed. The initial H-bond (NH..O) eliminated by N-methylation reorients the amide plain influencing the chiroptical properties of the foldamer building block, a structural change successfully monitored by the VCD- and ECD-transition changes now assigned to pure conformers. The current method seems general and effective without requiring extensive CPU and spectroscopic resources

First principles calculation of the reaction rates for ligand binding to myoglobin: the cases of NO and CO

Ligand binding by proteins is among the most fundamental processes in nature. Among these processes the binding of small gas molecules, such as O2, CO and NO to heme proteins has traditionally received vivid interest, which was further boosted by their recently recognized significant role in gas sensing in the body. At the heart of the binding of these ligands to the heme group is the spin-forbidden reaction between high-spin iron(II) and the ligand yielding a low spin adduct. We use computational means to address the complete mechanism of CO and NO binding by myoglobin. As it involves several steps occurring on different time-scales, molecular dynamics simulations were performed to address the diffusion of the ligand through the enzyme, and DFT calculations in combination with statistical rate calculation to investigate the spin-forbidden reaction. The calculations yielded rate constants in qualitative agreement with experiment and revealed that the bottle-neck of NO and CO binding is different: for NO diffusion was found to be rate-limiting, while for CO the spin-forbidden step is the slowest

Predictable Conformational Diversity in Foldamers of Sugar Amino Acids

Systematic conformational search was carried out for monomers and homohexamers of furanoid β-amino acids: cis-(S,R) and trans-(S,S) stereoisomers of aminocyclopentane carboxylic acid (ACPC), two different aminofuranuronic-acids (AFU(alpha) and AFUβ), their isopropylidene derivatives (AFU(ip)) as well as the key intermediate β-aminotetrahydrofurancarboxylic acid (ATFC). Stereochemistry of the building blocks was chosen to match with that of natural sugar amino acid (xylose and ribose) precursors (XylAFU and RibAFU). Results show that hexamers of cis furanoid beta-amino acids show great variability: while hydrophobic cyclopentane (cis(ACPC)6), and hydrophilic (XylAFU(alpha)/(beta))6 foldamers favor two different zigzagged conformation as hexamers, the backbone fold turns into a helix in case of (cisATFC)6 (10-helix) and (XylAFU(ip))6 (14-helix). Trans stereochemistry resulted in hexamers exclusively of right-handed helix conformation, (H12P)6, regardless of their polarity. We found that the preferred oligomeric structure of XylAFU(alpha)/(beta) is conformationally compatible with beta-pleated sheets, while that of the trans/(S,S) units match with alpha-helices of proteins

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 ECD and NMR. 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. We identified points of no return ( e.g. T =37°C, pH =4.1, c E5 =250µM, c NaCl =50mM, t >4-6 h) which will inevitably gravitate into the amyloid-state. The strong B-type FUV-ECD spectra and an unexpectedly strong NUV-ECD signal (Θ ~275-285nm ) indicate that the amyloid phase of E5 is built from monomers of quasi -elongated backbone structure ( φ ~-145°, ψ ~+145°) with strong interstrand Tyr↔Trp interaction. Misfolded intermediers and the buildup of "toxic" early-stage oligomers leading to self-association were identified and monitored as function of time. Results indicate that the amyloid transition is triggered by subtle misfolding of the α-helix exposing aromatic and hydrophobic side chains that may provide the first centers for an intermolecular reorganization. These initial clusters provide the spatial closeness and sufficient time for a transition to the β-structured amyloid nucleus thus the process follows a nucleated growth mechanism

A self-compartmentalizing hexamer serine protease from Pyrococcus Horikoshii: Substrate selection achieved through multimerization

Oligopeptidases impose a size limitation on their substrates, the mechanism of which has long been in debate. Here we present the structure of a hexameric serine protease, an oligopeptidase from Pyrococcus horikoshii (PhAAP), revealing a complex, self-compartmentalized inner space, where substrates may access the monomer active sites passing through a double-gated "check-in" system: first passing through a pore on the hexamer surface, then turning to enter through an even smaller opening at the monomers' domain-interface. This substrate screening strategy is unique within the family. We found that among oligopeptidases a member of catalytic apparatus is positioned near an amylogenic beta-edge, which needs to be protected to prevent aggregation and found different strategies applied to such end. We propose that self-assembly within the family results in characteristically different substrate selection mechanisms coupled to different multimerization states