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. Rigidified backbone structure has long been accepted as a structural prerequisite for peptide inhibitors of proteases. We found that a hydrophobic cluster organized around the P2 Thr residue is essential for the structural stability of wild-type SFTI. We also found that the same P2 Thr prevents binding of the rigid SFTI-like peptides to the substrate-binding cleft of both MASPs as the cleft is partially blocked by large gatekeeper enzyme loops. Directed evolution removed this obstacle by replacing the P2 Thr with a Ser, providing the SFMIs with high-degree structural plasticity, which proved to be essential for MASP inhibition. To gain more insight into the structural criteria for SFMI-based MASP-2 inhibition, we systematically modified MASP-2-specific SFMI2 by capping its two termini and by replacing its disulfide bridge with varying length thioether linkers. By doing so, we also aimed to generate a versatile scaffold that is resistant to reducing environment and has increased stability in exopeptidase-containing biological environments. We found that the reduction-resistant disulfide-substituted l-2,3-diaminopropionic acid (Dap) variant possessed near-native potency. As MASP-2 is involved in the life-threatening thrombosis in COVID-19 patients, our synthetic, selective MASP-2 inhibitors could be relevant coronavirus drug candidates.

Occurrence of Ordered and Disordered Structural Elements in Postsynaptic Proteins Supports Optimization for Interaction Diversity.

The human postsynaptic density is an elaborate network comprising thousands of proteins, playing a vital role in the molecular events of learning and the formation of memory. Despite our growing knowledge of specific proteins and their interactions, atomic-level details of their full three-dimensional structure and their rearrangements are mostly elusive. Advancements in structural bioinformatics enabled us to depict the characteristic features of proteins involved in different processes aiding neurotransmission. We show that postsynaptic protein-protein interactions are mediated through the delicate balance of intrinsically disordered regions and folded domains, and this duality is also imprinted in the amino acid sequence. We introduce Diversity of Potential Interactions (DPI), a structure and regulation based descriptor to assess the diversity of interactions. Our approach reveals that the postsynaptic proteome has its own characteristic features and these properties reliably discriminate them from other proteins of the human proteome. Our results suggest that postsynaptic proteins are especially susceptible to forming diverse interactions with each other, which might be key in the reorganization of the postsynaptic density (PSD) in molecular processes related to learning and memory.

Ensemble-based interpretations of NMR structural data to describe protein internal dynamics.

NMR spectroscopy is the leading technique to characterize protein internal dynamics at the atomic level and on multiple time scales. However, the structural interpretation of the observables obtained by various measurements is not always straightforward and in many cases dynamics-related parameters are only used to "decorate" static structural models without offering explicit description of conformational heterogeneity. To overcome such limitations, several computational techniques have been developed to generate ensemble-based representations of protein structure and dynamics with the use of NMR-derived data. An important common aspect of the methods is that NMR observables and derived parameters are interpreted as properties of the ensemble instead of individual conformers. The resulting ensembles reflect the experimentally determined internal mobility of proteins at a given time scale and can be used to understand the role of internal motions in biological processes at atomic detail. In this review we provide an overview of the calculation methods currently available and examples of biological insights obtained by the ensemble-based models of the proteins investigated.

Estimating intrinsic structural preferences of de novo emerging random-sequence proteins: is aggregation the main bottleneck?

Present-day proteins are believed to have evolved features to reduce the risk of aggregation. However, proteins can emerge de novo by translation of non-coding DNA segments. In this study we assess the aggregation, disorder and transmembrane propensity of protein sequences generated by translating random nucleotide sequences of varying GC-content. Potential de novo random-sequence proteins translated from regions with GC content between 40% and 60% do not show stronger aggregation propensity than existing ones and exhibit similar tendency to be disordered. We suggest that de novo emerging proteins do not mean an unavoidable aggregation threat to evolving organisms.

CoNSEnsX: an ensemble view of protein structures and NMR-derived experimental data.

BACKGROUND: In conjunction with the recognition of the functional role of internal dynamics of proteins at various timescales, there is an emerging use of dynamic structural ensembles instead of individual conformers. These ensembles are usually substantially more diverse than conventional NMR ensembles and eliminate the expectation that a single conformer should fulfill all NMR parameters originating from 10(16) - 10(17) molecules in the sample tube. Thus, the accuracy of dynamic conformational ensembles should be evaluated differently to that of single conformers. RESULTS: We constructed the web application CoNSEnsX (Consistency of NMR-derived Structural Ensembles with eXperimental data) allowing fast, simple and convenient assessment of the correspondence of the ensemble as a whole with diverse independent NMR parameters available. We have chosen different ensembles of three proteins, human ubiquitin, a small protease inhibitor and a disordered subunit of cGMP phosphodiesterase 5/6 for detailed evaluation and demonstration of the capabilities of the CoNSEnsX approach. CONCLUSIONS: Our results present a new conceptual method for the evaluation of dynamic conformational ensembles resulting from NMR structure determination. The designed CoNSEnsX approach gives a complete evaluation of these ensembles and is freely available as a web service at http://consensx.chem.elte.hu.

Probing dynamic protein ensembles with atomic proximity measures.

The emerging role of internal dynamics in protein fold and function requires new avenues of structure analysis. We analyzed the dynamically restrained conformational ensemble of ubiquitin generated from residual dipolar coupling data, in terms of protruding and buried atoms as well as interatomic distances, using four proximity-based algorithms, CX, DPX, PRIDE and PRIDE-NMR (http://hydra.icgeb.trieste.it/protein/). We found that Ubiquitin, this relatively rigid molecule has a highly diverse dynamic ensemble. The environment of protruding atoms is highly variable across conformers, on the other hand, only a part of buried atoms tends to fluctuate. The variability of the ensemble cautions against the use of single conformers when explaining functional phenomena. We also give a detailed evaluation of PRIDE-NMR on a wide dataset and discuss its usage in the light of the features of available NMR distance restraint sets in public databases.

Fast protein fold estimation from NMR-derived distance restraints.

UNLABELLED: PRIDE-NMR is a fast novel method to relate known protein folds to NMR distance restraints. It can be used to obtain a first guess about a structure being determined, as well as to estimate the completeness or verify the correctness of NOE data. AVAILABILITY: The PRIDE-NMR server is available at http://www.icgeb.org/pride