Distinct 3D architecture and dynamics of the human HtrA2(Omi) protease and its mutated variants

HtrA2(Omi) protease controls protein quality in mitochondria and plays a major role in apoptosis. Its HtrA2S306A mutant (with the catalytic serine routinely disabled for an X-ray study to avoid self-degradation) is a homotrimer whose subunits contain the serine protease domain (PD) and the regulatory PDZ domain. In the inactive state, a tight interdomain interface limits penetration of both PDZ-activating ligands and PD substrates into their respective target sites. We successfully crystalized HtrA2V226K/S306A, whose active counterpart HtrA2V226K has had higher proteolytic activity, suggesting higher propensity to opening the PD-PDZ interface than that of the wild type HtrA2. Yet, the crystal structure revealed the HtrA2V226K/S306A architecture typical of the inactive protein. To get a consistent interpretation of crystallographic data in the light of kinetic results, we employed molecular dynamics (MD). V325D inactivating mutant was used as a reference. Our simulations demonstrated that upon binding of a specific peptide ligand NH2-GWTMFWV-COOH, the PDZ domains open more dynamically in the wild type protease compared to the V226K mutant, whereas the movement is not observed in the V325D mutant. The movement relies on a PDZ vs. PD rotation which opens the PD-PDZ interface in a lid-like (budding flower-like in trimer) fashion. The noncovalent hinges A and B are provided by two clusters of interfacing residues, harboring V325D and V226K in the C- and N-terminal PD barrels, respectively. The opening of the subunit interfaces progresses in a sequential manner during the 50 ns MD simulation. In the systems without the ligand only minor PDZ shifts relative to PD are observed, but the interface does not open. Further activation-associated events, e.g. PDZ-L3 positional swap seen in any active HtrA protein (vs. HtrA2), were not observed. In summary, this study provides hints on the mechanism of activation of wtHtrA2, the dynamics of the inactive HtrA2V325D, but does not allow to explain an increased activity of HtrA2V226K

Discovery of inhibitory fragments that selectively target Spire2−FMN2 interaction

Here, we report the fragment-based drug discovery of potent and selective fragments that disrupt the Spire2–FMN2 but not the Spire1–FMN2 interaction. Hit fragments were identified in a differential scanning fluorimetry-based screen of an in-house library of 755 compounds and subsequently validated in multiple orthogonal biophysical assays, including fluorescence polarization, microscale thermophoresis, and 1H–15N HSQC nuclear magnetic resonance. Extensive structure–activity relationships combined with molecular docking followed by chemical optimization led to the discovery of compound 13, which exhibits micromolar potency and high ligand efficiency (LE = 0.38). Therefore, this fragment represents a validated starting point for the future development of selective chemical probes targeting the Spire2–FMN2 interaction

The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal \beta-sandwich domain

In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway

A multi-country test of brief reappraisal interventions on emotions during the COVID-19 pandemic.

The COVID-19 pandemic has increased negative emotions and decreased positive emotions globally. Left unchecked, these emotional changes might have a wide array of adverse impacts. To reduce negative emotions and increase positive emotions, we tested the effectiveness of reappraisal, an emotion-regulation strategy that modifies how one thinks about a situation. Participants from 87 countries and regions (n = 21,644) were randomly assigned to one of two brief reappraisal interventions (reconstrual or repurposing) or one of two control conditions (active or passive). Results revealed that both reappraisal interventions (vesus both control conditions) consistently reduced negative emotions and increased positive emotions across different measures. Reconstrual and repurposing interventions had similar effects. Importantly, planned exploratory analyses indicated that reappraisal interventions did not reduce intentions to practice preventive health behaviours. The findings demonstrate the viability of creating scalable, low-cost interventions for use around the world

Crystallization of a serine protease SplB, from Staphylococcus aureus, complexed with it's inhibitor - s3zw3

Staphylococcus aureus is a leading cause of hospital associated infections. It is observed that multi-drug resistance among staphylococci is growing rapidly. Together with the fact that Staphylococcus aureus is able to produce a variety of virulence factors it is becoming life-threatening also for healthy individuals. Among Staphylococcal virulence factors there occur it’s polysaccharide capsule, as well as complement inhibitors, protein A, coagulase and many proteases. The later group might be divided into three subgroups: cysteine proteases (staphopain A), metaloproteases (aureolysin) and serine proteases, among which SplA-F are recognized.In former studies on the structure of SplB protease it was shown that it’s unusual active site conformation should abolish any proteolytic activity of this protein. However, the above is in contradiction with a finding that SplB is able to cleave a synthetic substrate – Ac-VEID-MCA. Together this data suggested that due to substrate binding conformational changes occur leading to active site formation. To verify this hypothesis it was suggested to investigate structure of SplB protease complexed with it’s transition-state analogue which is the topic of the current studyIn this study it is demonstrated that s3zw3 compound is a potent inhibitor of SplB protease. Having formed a stable protein-inhibitor complex, it appears as a good target for crystallization trails of such complex. The crystallization conditions are reported which shall in the future allow to solve the structure of the protease and verify the assumptions of substrate induced active site rearrangements.Bakterie z gatunku Staphylococcus aureus są jedną z najczęstszych przyczyn zakażeń szpitalnych. Jednakże, mając na uwadze rosnącą ich wielolekową oporność, jak i mnogość produkowanych czynników wirulencji stanowią także zagrożenie dla zdrowych osobników. Do czynników wirulencji Staphylococcus aureus zaliczyć należy wytwarzaną przez ten gatunek mikroorganizmu otoczkę polisacharydową, inhibitory układu dopełniacza, białko A – wiążące immunoglobuliny, koagulazy, a także liczne proteazy. Do ostatniej z tych grup zaliczane są: proteazy cysteinowe (np. stafopaina A), metaloproteinazy (np. aureolizyna) oraz proteazy serynowe, do których zaliczane są proteazy SplA-F, w tym proteaza SplB stanowiąca temat niniejszej rozprawy.W uprzednio opublikowanym artykule, ukazującym strukturę proteazy SplB, zostało wykazane, iż białko to posiada zdeformowane miejsce aktywne, która to deformacja powinna skutkować brakiem aktywności tej proteazy. Teza ta kłóci nie jest jednak zgodna z faktem, iż proteaza SplB, wykazuje aktywność enzymatyczną względem syntetycznego substratu Ac-VEID-MCA. Postuluje Zapostulowano zatem, iż wiązanie substratu przez proteazę SplB powoduje zmiany konformacyjne białka, skutkujące rearanżacją miejsca aktywnego i utworzeniem jego aktywnej formy. By udowodnić tę hipotezę postanowiono zbadać strukturę proteazy SplB w kompleksie z inhibitorem, będącym analogiem stanu przejściowego substratu występującego podczas katalizy enzymatycznej. W poniższej pracy zaprezentowano, iż związek s3zw3, hamuje aktywność proteazy SplB, tworząc stabilny kompleks białko-inhibitor, co pozwala na wykrystalizowanie tego kompleksu. Tym samym będzie możliwe określenie struktury przestrzennej kompleksu a tym samym określenie szczegółów budowy centrum aktywnego z rozdzielczością atomową. W założeniu dane te powinny pozwolić potwierdzić lub obalić postawioną wyżej hipotezę dotyczącą rearanżacji miejsca katalitycznego w obecności substratu

Structural basis of GD2 ganglioside and mimetic peptide recognition by 14G2a antibody

Monoclonal antibodies targeting GD2 ganglioside (GD2) have recently been approved for the treatment of high risk neuroblastoma and are extensively evaluated in clinics in other indications. This study illustrates how a therapeutic antibody distinguishes between different types of gangliosides present on normal and cancer cells and informs how synthetic peptides can imitate ganglioside in its binding to the antibody. Using high resolution crystal structures we demonstrate that the ganglioside recognition by a model antibody (14G2a) is based primarily on an extended network of direct and water molecule mediated hydrogen bonds. Comparison of the GD2-Fab structure with that of a ligand free antibody reveals an induced fit mechanism of ligand binding. These conclusions are validated by directed mutagenesis and allowed structure guided generation of antibody variant with improved affinity toward GD2. Contrary to the carbohydrate, both evaluated mimetic peptides utilize a "key and lock" interaction mechanism complementing the surface of the antibody binding groove exactly as found in the empty structure. The interaction of both peptides with the Fab relies considerably on hydrophobic contacts however, the detailed connections differ significantly between the peptides. As such, the evaluated peptide carbohydrate mimicry is defined primarily in a functional and not in structural manner

Atomic resolution crystal structure of HV-BBI protease inhibitor from amphibian skin in complex with bovine trypsin

Protease inhibitors of the Bowman-Birk (BBI) family are commonly found in plants and animals where they play a protective role against invading pathogens. Here, we report an atomic resolution (1Å) crystal structure of a peptide inhibitor isolated from a skin secretion of a Chinese bamboo odorous frog Huia versabilis (HV-BBI) in complex with trypsin. HV-BBI shares significant similarities in sequence with a previously described inhibitor from a diskless-fingered odorous frog Odorrana graham (ORB). However, the latter is characterized by more than a 16,000 fold higher K$_i$ against trypsin than HV-BBI. Comparative analysis of trypsin cocrystal structures of HV-BBI and ORB and additionally that of Sunflower Trypsin Inhibitor (SFTI-1) together with accessory information on the affinities of inhibitor variants allowed us to pinpoint the inhibitor moiety responsible for the observed large difference in activity and also to define the extent of modifications permissible within the common protease-binding loop scaffold of BBI inhibitors. We suggest that modifications outside of the inhibitory loop permit the evolution of specificity toward different enzymes characterized by trypsin-like specificity. Proteins 2015; 83:582–589. © 2014 Wiley Periodicals, Inc

Structural characterization of human coronavirus NL63 N protein

ABSTRACT Coronaviruses are responsible for upper and lower respiratory tract infections in humans. It is estimated that 1 to 10% of the population suffers annually from cold-like symptoms related to infection with human coronavirus NL63 (HCoV-NL63), an alphacoronavirus. The nucleocapsid (N) protein, the major structural component of the capsid, facilitates RNA packing, links the capsid to the envelope, and is also involved in multiple other processes, including viral replication and evasion of the immune system. Although the role of N protein in viral replication is relatively well described, no structural data are currently available regarding the N proteins of alphacoronaviruses. Moreover, our understanding of the mechanisms of RNA binding and nucleocapsid formation remains incomplete. In this study, we solved the crystal structures of the N- and C-terminal domains (NTD, residues 10 to 140, and CTD, residues 221 to 340, respectively) of the N protein of HCoV-NL63, both at a 1.5-Å resolution. Based on our structure of NTD solved here, we proposed and experimentally evaluated a model of RNA binding. The structure of the CTD reveals the mode of N protein dimerization. Overall, this study expands our understanding of the initial steps of N protein-nucleic acid interaction and may facilitate future efforts to control the associated infections. IMPORTANCE Coronaviruses are responsible for the common cold and other respiratory tract infections in humans. According to multiple studies, 1 to 10% of the population is infected each year with HCoV-NL63. Viruses are relatively simple organisms composed of a few proteins and the nucleic acids that carry the information determining their composition. The nucleocapsid (N) protein studied in this work protects the nucleic acid from the environmental factors during virus transmission. This study investigated the structural arrangement of N protein, explaining the first steps of its interaction with nucleic acid at the initial stages of virus structure assembly. The results expand our understanding of coronavirus physiology and may facilitate future efforts to control the associated infections. </jats:p