Random coil conformation of a Gly/Ala-rich insert in IκBα excludes structural stabilization as the mechanism for protection against proteasomal degradation

AbstractPeptide segments of multiple glycine and alanine residues prevent the proteolytic degradation of ubiquitinated proteins by the proteasome. The structure of a Gly/Ala-rich insert in IκBα was probed by nuclear magnetic resonance (NMR) spectroscopy, comparing IκBα samples with and without Gly/Ala-rich insert. Narrow 1H-NMR resonances at chemical shifts indicative of random coil conformations were observed in the difference spectrum. circular dichroism (CD) measurements further confirm that the mechanism of protection against proteolytic degradation is not based on structural transition or stabilization caused by the Gly/Ala-rich segment. In addition, most of the N- and C-terminal residues outside the ankyrin repeats in wild-type IκBα were found to be flexibly disordered

Novel Sortase A Inhibitors to Counteract Gram-Positive Bacterial Biofilms

Sortase A (SrtA) is a membrane enzyme responsible for the covalent anchoring of surface proteins on the cell wall of Gram-positive bacteria. Nowadays it is considered an interesting target for the development of new anti-infective drugs which aim to interfere with important Gram-positive virulence mechanisms. Along the years, we studied the anti-staphylococcal and anti-biofilm activity of some natural and synthetic polyhalogenated pyrrolic compounds, called pyrrolomycins. Some of them were active on Gram-positive pathogens at a μg/mL range of concentration (1.5-0.045 μg/mL) and showed a biofilm inhibition in the range of 50-80%. [1-3] In light of these encouraging results, herein we present our efforts in the design and synthesis of novel pyrrolomycins. To dispose of sufficient amount for the in-depth in vitro investigation, we developed an efficient and easy-to-use microwave synthetic methodology. All compounds showed a good inhibitory activity toward SrtA, in accordance with the molecular modelling studies, having IC50 values ranging from 130 to 300 µM comparable to berberine hydrochloride, our reference compound. Particularly, the pentabromo-derivative exhibited the highest capability to interfere with biofilm formation of S. aureus with an IC50 of 3.4 nM. This compound was also effective in altering S. aureus murein hydrolase activity, responsible for degradation, turnover, and maturation of bacterial peptidoglycan and involved in the initial stages of S. aureus biofilm formation. [4

Crystal structure of c5321 : a protective antigen present in uropathogenic Escherichia coli strains displaying an SLR fold

Background: Increasing rates of antimicrobial resistance among uropathogens led, among other efforts, to the application of subtractive reverse vaccinology for the identification of antigens present in extraintestinal pathogenic E. coli (ExPEC) strains but absent or variable in non-pathogenic strains, in a quest for a broadly protective Escherichia coli vaccine. The protein coded by locus c5321 from CFT073 E. coli was identified as one of nine potential vaccine candidates against ExPEC and was able to confer protection with an efficacy of 33% in a mouse model of sepsis. c5321 (known also as EsiB) lacks functional annotation and structurally belongs to the Sel1-like repeat (SLR) family. Herein, as part of the general characterization of this potential antigen, we have focused on its structural properties. Results: We report the 1.74 Å-resolution crystal structure of c5321 from CFT073 E. coli determined by Se-Met SAD phasing. The structure is composed of 11 SLR units in a topological organisation that highly resembles that found in HcpC from Helicobacter pylori, with the main difference residing in how the super-helical fold is stabilised. The stabilising effect of disulfide bridges in HcpC is replaced in c5321 by a strengthening of the inter-repeat hydrophobic core. A metal-ion binding site, uncharacteristic of SLR proteins, is detected between SLR units 3 and 4 in the region of the inter-repeat hydrophobic core. Crystal contacts are observed between the C-terminal tail of one molecule and the C-terminal amphipathic groove of a neighbouring one, resembling interactions between ligand and proteins containing tetratricopeptide-like repeats. Conclusions: The structure of antigen c5321 presents a mode of stabilization of the SLR fold different from that observed in close homologs of known structure. The location of the metal-ion binding site and the observed crystalcontacts suggest a potential role in regulation of conformational flexibility and interaction with yet unidentified target proteins, respectively. These findings open new perspectives in both antigen design and for the identification of a functional role for this protective antigen

Regulation of protein degradation by virus derived repeated amino acid sequences

The ubiquitin-proteasome pathway plays a central role in the controlled degradation of shortlived and regulatory proteins important m a variety of cellular processes, including antigen presentation and combating viral infections. The presentation of antigenic peptides derived from ubiquitin-proteasome dependent degradation of vital proteins to MHC class I restricted cytotoxic T cells is a central component of antiviral responses. It is therefore not surprising that viruses have developed strategies to block this proteolytic machinery m order to escape detection by the host immune system. A particularly interesting example of viral protein that interferes with the ubiquitinproteasome pathway is the Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) that contains a long internal glycine-alanine repeat (GAr) and generates a cis-acting inhibitory signal that interferes with antigen presentation. The aim of this study was to investigate the mechanism by which the GAr interferes with the ubiquitin-proteasome pathway in protecting proteins from proteolysis. We used the capacity of GAr to act as a transferable element by inhibiting the processing of a wide variety of proteasomal substrates. Four main aspects were explored in the present study. We studied the influence of the GAr on the proteasomal degradation of different viral and cellular proteins. We also analyzed the conformational stability of the chimeric protein. Furthermore, we investigated the impact of amino acid composition and trans-inhibitory effect of the GAr on the ubiquitin-dependent degradation. First, we compared the proteasomal degradation of native EBNA1, GAr deleted EBNA1 and an EBNA4 chimeric protein. EBNA4 was efficiently degraded whereas EBNA1 was resistant to degradation. EBNA1 degradation was restored by deletion of the GAr, both in vitro and in vivo, whereas insertion of the GAr of various lengths and in different positions prevented the degradation of EBNA4 without considerable effect on ubiquitination. These results proposed that the GAr might affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. Next, we investigated the impact of the GAr in degradation of a well-known target of the ubiquitin-proteasome pathway, inhibitor of nuclear factor kappa B, I kappa B alpha in vivo. We found that insertion of octamer repeat GGAGAGAG with three alanines was sufficient to block I kappa B alpha from signal-induced degradation. This study demonstrated that chimeras were phosphorylated and ubiquitinated in response to TNF alpha and retained the capacity to bind NF-kappa B, but then were released from NF-kappa B and failed to associate with the proteasome. It suggested that the repeat allects ubiquitin-proteasome pathway at a step that lies downstream of ubiquitination and upstream of the interaction of ubiquitinated proteins with the proteasome. We also used p53 to generate a set of chimeras with different length and site of insertion of the GAr to monitore Mdm2- and E6-induced degradation. p53 chimeras were ubiquitinated and protected from proteolysis, retained the capacity to interact with the S5a ubiquitin-binding subunit of the proteasome and maintained the functional properties of wild-type p53. Previous work raised a possibility that insertion of the GAr domain in target protein may interfere with direct masking of a putative binding site or increase the stability of chimeric protein against unfolding activity of molecular chaperones or the 19S proteasome. The NMR and CD spectroscopy data demonstrated that glycine-alanine insert assumed a flexible random coil conformation and its presence did not increase the stability of I kappa B alpha against denaturation. These results suggested that protection against proteolytic degradation may not be based on structural differences or stabilization caused by the GAr and excluded a structural effect of the Gly-Ala segment. Next aspect of this study deals with the relationship between the inhibitory activity and the amino acid composition of the repeat. Inhibition of TNF alpha- induced degradation was achieved by insertion of different octamers containing three alanines interspersed by no more then three consecutive glycines. We also found that inhibitory activity of the GAr was abolished when the length of the glycine spacer was increased and when a single alanine residue of the octamer was substituted with a polar or charged amino acid. A substitution of alanines with valine resulted in the maintenance of the inhibitory effect. These findings suggested a model where inhibition requires an interaction of at least three alanine residues of the GAr in a beta-strand conformation with adjacent hydrophobic binding pocket of a putative receptor. We also studied in trans inhibition of degradation of proteasomal substrate lysozyme in the presence of the GAr oligopeptide that mimics a sequence of the repeat. The presence of peptide did not affect the ubiquitination of lysozyme, hydrolysis of fluorogenic substrate or binding of a synthetic tetra-ubiquitin complex to the S5a subunit. Our findings therefore propose that the GAr domain may act by weakening the interaction of ubiquitinated substrates with the 19S proteasome regulator and promote the premature release of the substrate

Staphylococcus aureus sortase A cyclization and evaluation of enzymatic activity in vitro

Abstract Protein cyclization has been shown to increase the stability of molecule structure by reducing the number of possible conformation variations. Cyclization of an enzyme can significantly enlarge the spectrum of its application in specific conditions, including high temperature, presence of a chaotropic agent, and denaturing environment. In this study the cyclic form of Staphylococcus aureus sortase A enzyme (srtA) was obtained by introducing a peptide bond between amino acids at N-and C-termini. The catalytic activity of purified cyclic srtA was tested in vitro by protein-protein ligation reaction. In contrast to the linear srtA, the cyclic form of the enzyme was active in presence of chaotropic agent. This finding suggests the potential use of srtA enzyme in protein engineering techniques

Sorting out the Superbugs: Potential of Sortase A Inhibitors among Other Antimicrobial Strategies to Tackle the Problem of Antibiotic Resistance

Rapid spread of antibiotic resistance throughout the kingdom bacteria is inevitably bringing humanity towards the “post-antibiotic” era. The emergence of so-called “superbugs”—pathogen strains that develop resistance to multiple conventional antibiotics—is urging researchers around the globe to work on the development or perfecting of alternative means of tackling the pathogenic bacteria infections. Although various conceptually different approaches are being considered, each comes with its advantages and drawbacks. While drug-resistant pathogens are undoubtedly represented by both Gram(+) and Gram(−) bacteria, possible target spectrum across the proposed alternative approaches of tackling them is variable. Numerous anti-virulence strategies aimed at reducing the pathogenicity of target bacteria rather than eliminating them are being considered among such alternative approaches. Sortase A (SrtA) is a membrane-associated cysteine protease that catalyzes a cell wall sorting reaction by which surface proteins, including virulence factors, are anchored to the bacterial cell wall of Gram(+) bacteria. Although SrtA inhibition seems perspective among the Gram-positive pathogen-targeted antivirulence strategies, it still remains less popular than other alternatives. A decrease in virulence due to inactivation of SrtA activity has been extensively studied in Staphylococcus aureus, but it has also been demonstrated in other Gram(+) species. In this manuscript, results of past studies on the discovery of novel SrtA inhibitory compounds and evaluation of their potency were summarized and commented on. Here, we discussed the rationale behind the inhibition of SrtA, raised some concerns on the comparability of the results from different studies, and touched upon the possible resistance mechanisms as a response to implementation of such therapy in practice. The goal of this article is to encourage further studies of SrtA inhibitory compounds

Discovery and Structure–Activity Relationship Studies of Irreversible Benzisothiazolinone-Based Inhibitors against Staphylococcus Aureus Sortase A Transpeptidase

Gram-positive bacteria, in general, and staphylococci, in particular, are the widespread cause of nosocomial and community-acquired infections. The rapid evolvement of strains resistant to antibiotics currently in use is a serious challenge. Novel antimicrobial compounds have to be developed to fight these resistant bacteria, and sortase A, a bacterial cell wall enzyme, is a promising target for novel therapies. As a transpeptidase that covalently attaches various virulence factors to the cell surface, this enzyme plays a crucial role in the ability of bacteria to invade the host’s tissues and to escape the immune response. In this study we have screened a small molecule library against recombinant Staphylococcus aureus sortase A using an in vitro FRET-based assay. The selected hits were validated by NMR methods in order to exclude false positives and to analyze the reversibility of inhibition. Further structural and functional analysis of the best hit allowed the identification of a novel class of benzisothiazolinone-based compounds as potent and promising sortase inhibitors

Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein–Barr virus nuclear antigen 1

The Epstein–Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATP-dependent ubiquitination and degradation by the proteasome. We have investigated the influence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP/ubiquitin/proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect

Site-specific Labelling with a Metal Chelator for Protein-structure Refinement

A single free Cys sidechain in the N-terminal domain of the E. coli arginine repressor was covalently derivatized with S-cysteaminyl-EDTA for site-specific attachment of paramagnetic metal ions. The effects of chelated metal ions were monitored with 15N-HSQC spectra. Complexation of Co2+, which has a fast relaxing electron spin, resulted in significant pseudocontact shifts, but also in peak doubling which was attributed to the possibility of forming two different stereoisomers of the EDTA-Co2+ complex. In contrast, complexation of Cu2+ or Mn2+, which have slowly relaxing electron spins, did not produce chemical shift changes and yielded self-consistent sets of paramagnetic relaxation enhancements of the amide protons. T1 relaxation enhancements with Cu2+ combined with T2 relaxation enhancements with Mn2+ are shown to provide accurate distance restraints ranging from 9 to 25 Å. These long-range distance restraints can be used for structural studies inaccessible to NOEs. As an example, the structure of a solvent-exposed loop in the N-terminal domain of the E. coli arginine repressor was refined by paramagnetic restraints. Electronic correlation times of Cu2+ and Mn2+ were determined from a comparison of T1 and T2 relaxation enhancements

Solution Structure of the R3H Domain from Human Sμbp-2

The R3H domain is a conserved sequence motif, identified in over 100 proteins, that is thought to be involved in polynucleotide-binding, including DNA, RNA and single-stranded DNA. In this work the 3D structure of the R3H domain from human Sμbp-2 was de