Hepatitis C virus (HCV) is a major global health burden as the leading causative agent of chronic liver disease and hepatocellular carcinoma. While the main antigenic target for HCV-neutralizing ...antibodies is the membrane-associated E1E2 surface glycoprotein, the development of effective vaccines has been hindered by complications in the biochemical preparation of soluble E1E2 ectodomains. Here, we present a cryo-EM structure of an engineered, secreted E1E2 ectodomain of genotype 1b in complex with neutralizing antibodies AR4A, HEPC74, and IGH520. Structural characterization of the E1 subunit and C-terminal regions of E2 reveal an overall architecture of E1E2 that concurs with that observed for non-engineered full-length E1E2. Analysis of the AR4A epitope within a region of E2 that bridges between the E2 core and E1 defines the structural basis for its broad neutralization. Our study presents the structure of an E1E2 complex liberated from membrane via a designed scaffold, one that maintains all essential structural features of native E1E2. The study advances the understanding of the E1E2 heterodimer structure, crucial for the rational design of secreted E1E2 antigens in vaccine development.
AP endonuclease 1 (APE1) processes DNA lesions including apurinic/apyrimidinic sites and 3´-blocking groups, mediating base excision repair and single strand break repair. Much effort has focused on ...developing specific inhibitors of APE1, which could have important applications in basic research and potentially lead to clinical anticancer agents. We used structural, biophysical, and biochemical methods to characterize several reported inhibitors, including 7-nitroindole-2-carboxylic acid (CRT0044876), given its small size, reported potency, and widespread use for studying APE1. Intriguingly, NMR chemical shift perturbation (CSP) experiments show that CRT0044876 and three similar indole-2-carboxylic acids bind a pocket distal from the APE1 active site. A crystal structure confirms these findings and defines the pose for 5-nitroindole-2-carboxylic acid. However, dynamic light scattering experiments show the indole compounds form colloidal aggregates that could bind (sequester) APE1, causing nonspecific inhibition. Endonuclease assays show the compounds lack significant APE1 inhibition under conditions (detergent) that disrupt aggregation. Thus, binding of the indole-2-carboxylic acids at the remote pocket does not inhibit APE1 repair activity. Myricetin also forms aggregates and lacks APE1 inhibition under aggregate-disrupting conditions. Two other reported compounds (MLS000552981, MLS000419194) inhibit APE1 in vitro with low micromolar IC50 and do not appear to aggregate in this concentration range. However, NMR CSP experiments indicate the compounds do not bind specifically to apo- or Mg2+-bound APE1, pointing to a non-specific mode of inhibition, possibly DNA binding. Our results highlight methods for rigorous interrogation of putative APE1 inhibitors and should facilitate future efforts to discover compounds that specifically inhibit this important repair enzyme.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In this work glycosyl hydrolase (GH) family 30 (GH30) is analyzed and shown to consist of its currently classified member sequences as well as several homologous sequence groups currently assigned ...within family GH5. A large scale amino acid sequence alignment and a phylogenetic tree were generated and GH30 groups and subgroups were designated. A partial rearrangement in the GH30 defining side-associated β-domain contributes to the differentiation of two major groups that contain up to eight subgroups. For this CAZy family of Clan A enzymes the dual domain fold is conserved, suggesting that it may be a requirement for evolved function. This work redefines GH family 30 and serves as a guide for future efforts regarding enzymes classified within this family.
Novel therapeutics are needed to treat pathologies associated with the
binary toxin (CDT), particularly when
infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in ...addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell's cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.
Cytosine methylation at CpG dinucleotides produces m⁵CpG, an epigenetic modification that is important for transcriptional regulation and genomic stability in vertebrate cells. However, m⁵C ...deamination yields mutagenic G·T mispairs, which are implicated in genetic disease, cancer, and aging. Human thymine DNA glycosylase (hTDG) removes T from G·T mispairs, producing an abasic (or AP) site, and follow-on base excision repair proteins restore the G·C pair. hTDG is inactive against normal A·T pairs, and is most effective for G·T mispairs and other damage located in a CpG context. The molecular basis of these important catalytic properties has remained unknown. Here, we report a crystal structure of hTDG (catalytic domain, hTDGcat) in complex with abasic DNA, at 2.8 Å resolution. Surprisingly, the enzyme crystallized in a 2:1 complex with DNA, one subunit bound at the abasic site, as anticipated, and the other at an undamaged (nonspecific) site. Isothermal titration calorimetry and electrophoretic mobility-shift experiments indicate that hTDG and hTDGcat can bind abasic DNA with 1:1 or 2:1 stoichiometry. Kinetics experiments show that the 1:1 complex is sufficient for full catalytic (base excision) activity, suggesting that the 2:1 complex, if adopted in vivo, might be important for some other activity of hTDG, perhaps binding interactions with other proteins. Our structure reveals interactions that promote the stringent specificity for guanine versus adenine as the pairing partner of the target base and interactions that likely confer CpG sequence specificity. We find striking differences between hTDG and its prokaryotic ortholog (MUG), despite the relatively high (32%) sequence identity.
Polyorganophosphazenes are water-soluble macromolecules with immunoadjuvant activity that self-assemble with proteins to enable biological functionality. Direct imaging by cryogenic electron ...microscopy uncovers the coil structure of those highly charged macromolecules. The successful visualization of individual polymer chains within the vitrified state is achieved in the absence of additives for contrast enhancement and is attributed to the high mass contrast of the inorganic backbone. Upon assembly with proteins, multiple protein copies bind at the single polymer chain level resulting in structures reminiscent of compact spherical complexes or stiffened coils. The outcome depends on protein characteristics and cannot be deduced by commonly used characterization techniques, such as light scattering, thus revealing direct morphological insights crucial for understanding biological activity. Atomic force microscopy supports the morphology outcomes while advanced analytical techniques confirm protein-polymer binding. The chain visualization methodology provides tools for gaining insights into the processes of supramolecular assembly and mechanistic aspects of polymer enabled vaccine delivery.
DNA base excision repair is essential for maintaining genomic integrity and for active DNA demethylation, a central element of epigenetic regulation. A key player is thymine DNA glycosylase (TDG), ...which excises thymine from mutagenic G·T mispairs that arise by deamination of 5-methylcytosine (mC). TDG also removes 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC produced by Tet enzymes. Recent studies show that the glycosylase activity of TDG is essential for active DNA demethylation and for embryonic development. Our understanding of how repair enzymes excise modified bases without acting on undamaged DNA remains incomplete, particularly for mismatch glycosylases such as TDG. We solved a crystal structure of TDG (catalytic domain) bound to a substrate analog and characterized active-site residues by mutagenesis, kinetics, and molecular dynamics simulations. The studies reveal how TDG binds and positions the nucleophile (water) and uncover a previously unrecognized catalytic residue (Thr197). Remarkably, mutation of two active-site residues (Ala145 and His151) causes a dramatic enhancement in G·T glycosylase activity but confers even greater increases in the aberrant removal of thymine from normal A·T base pairs. The strict conservation of these residues may reflect a mechanism used to strike a tolerable balance between the requirement for efficient repair of G·T lesions and the need to minimize aberrant action on undamaged DNA, which can be mutagenic and cytotoxic. Such a compromise in G·T activity can account in part for the relatively weak G·T activity of TDG, a trait that could potentially contribute to the hypermutability of CpG sites in cancer and genetic disease.
Cathelicidins form a family of small host defense peptides distinct from another class of cationic antimicrobial peptides, the defensins. They are expressed as large precursor molecules with a highly ...conserved pro-domain known as the cathelin-like domain (CLD). CLDs have high degrees of sequence homology to cathelin, a protein isolated from pig leukocytes and belonging to the cystatin family of cysteine protease inhibitors. In this report, we describe for the first time the X-ray crystal structure of the human CLD (hCLD) of the sole human cathelicidin, LL-37. The structure of the hCLD, determined at 1.93 Å resolution, shows the cystatin-like fold and is highly similar to the structure of the CLD of the pig cathelicidin, protegrin-3. We assayed the in vitro antibacterial activities of the hCLD, LL-37, and the precursor form, pro-cathelicidin (also known as hCAP18), and we found that the unprocessed protein inhibited the growth of Gram-negative bacteria with efficiencies comparable to that of the mature peptide, LL-37. In addition, the antibacterial activity of LL-37 was not inhibited by the hCLD intermolecularly, because exogenously added hCLD had no effect on the bactericidal activity of the mature peptide. The hCLD itself lacked antimicrobial function and did not inhibit the cysteine protease, cathepsin L. Our results contrast with previous reports of hCLD activity. A comparative structural analysis between the hCLD and the cysteine protease inhibitor stefin A showed why the hCLD is unable to function as an inhibitor of cysteine proteases. In this respect, the cystatin scaffold represents an ancestral structural platform from which proteins evolved divergently, with some losing inhibitory functions.
Lipoplex preparations are heterogeneous mixtures of lipoplex particles of different structure. Because these structures determine the efficiency of genetic material delivery, it is important to ...characterize the distribution of particles of different types in lipoplex preparations with good statistics. We describe the application of flow fluorometry which allow producing such distributions (in terms of lipoplex particle size and composition) within minutes using basic flow cytometer (Pozharski and MacDonald, Anal Biochem 341:230-240, 2005).
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