Search

The Protein Data Bank (PDB) includes a carefully curated treasury of experimentally derived structural data on biological macromolecules and their various complexes. Such information is fundamental for a multitude of projects that involve large‐scale data mining and/or detailed evaluation of individual structures of importance to chemistry, biology and, most of all, to medicine, where it provides the foundation for structure‐based drug discovery. However, despite extensive validation mechanisms, it is almost inevitable that among the ∼215 000 entries there will occasionally be suboptimal or incorrect structure models. It is thus vital to apply careful verification procedures to those segments of the PDB that are of direct medicinal interest. Here, such an analysis was carried out for crystallographic models of l‐asparaginases, enzymes that include approved drugs for the treatment of certain types of leukemia. The focus was on the adherence of the atomic coordinates to the rules of stereochemistry and their agreement with the experimental electron‐density maps. Whereas the current clinical application of l‐asparaginases is limited to two bacterial proteins and their chemical modifications, the field of investigations of such enzymes has expanded tremendously in recent years with the discovery of three entirely different structural classes and with numerous reports, not always quite reliable, of the anticancer properties of l‐asparaginases of different origins. [ABSTRACT FROM AUTHOR]

l‐asparaginases from mesophilic bacteria (ASNases), including two enzymes very successfully used in the treatment of leukaemia, have been consistently described as homotetramers. On the contrary, structural studies show that homodimers of these enzymes should be sufficient to carry out the catalytic reaction. In this report, we investigated whether the type I Yersinia pestis asparaginase (YpAI) is active in a dimeric form or whether the tetrameric quaternary structure is critical for its activity. Using multiple biophysical techniques that investigate enzymatic properties and quaternary structure at either high or low protein concentration, we found that dimeric YpAI is fully active, suggesting that the tetrameric form of this subfamily of enzymes does not bear significant enzymatic relevance. In this process, we extensively characterized YpAI, showing that it is a cooperative enzyme, although the mechanism of allostery is still not definitely established. We showed that, like most type I ASNases, the substrate affinity of YpAI is low and this enzyme is very similar in terms of both the structure and enzymatic properties to homologous type I ASNase from Escherichia coli (EcAI). We extended these studies to more medically relevant type II ASNases, used as anti‐leukaemia drugs. We confirmed that type II ASNases are not allosteric, and that they might also be functional in a dimeric form. However, the determination of the accurate tetramer⇆dimer dissociation constants of these enzymes that most likely lie in the picomolar range is not possible with currently available biophysical techniques. [ABSTRACT FROM AUTHOR]

Triggering receptor expressed on myeloid cells--like (TREM-like) transcript-1 (TLT-1), a type 1 single Ig domain orphan receptor specific to platelet and megakaryocyte [alpha]-granules, relocates to the platelet surface upon platelet stimulation. We found here that patients diagnosed with sepsis, in contrast to healthy individuals, had substantial levels of soluble TLT-1 (sTLT-1) in their plasma that correlated with the presence of disseminated intravascular coagulation. sTLT-1 bound to fibrinogen and augmented platelet aggregation in vitro. Furthermore, the cytoplasmic domain of TLT-1 could also bind ezrin/radixin/moesin family proteins, suggesting its ability to link fibrinogen to the platelet cytoskeleton. Accordingly, platelets of Treml1-/- mice failed to aggregate efficiently, extending tail-bleeding times. Lipopolysaccharide-treated Treml1-/- mice developed higher plasma levels of TNF and D-dimers than wild-type mice and were more likely to succumb during challenge. Finally, Treml1-/- mice were predisposed to hemorrhage associated with localized inflammatory lesions. Taken together, our findings suggest that TLT-1 plays a protective role during inflammation by dampening the inflammatory response and facilitating platelet aggregation at sites of vascular injury. Therefore, therapeutic modulation of TLT-1-mediated effects may provide clinical benefit to patients with hypercoagulatory conditions, including those associated with inflammation., Introduction Septic shock claims over 200,000 people a year in the United States and is a leading cause of morbidity and mortality. Death from sepsis occurs when the deposition of [...]

Sexually transmitted human papillomaviruses (HPVs) are the primary cause of cervical cancer. Recent advances in techniques for production of papillomaviral vectors [known as pseudoviruses (PsVs)] have made it possible to perform high-throughput screens for compounds that might block the initial stages of papillomavirus infection. We have used PsVs to screen a variety of compounds that might function as inhibitors of HPV infection, with emphasis on human peptides previously implicated in innate antimicrobial immunity. Little is known about the possible activity of these peptides against nonenveloped viruses, such as HPVs. Our screen revealed that human [alpha]-defensins 1-3 [known as human neutrophil peptides (HNPs) 1-3] and human [alpha]-defensin 5 (HD-5) are potent antagonists of infection by both cutaneous and mucosal papillomavirus types. In contrast, human [beta]-defensins 1 and 2 displayed little or no anti-HPV activity. HD-5 was particularly active against sexually transmitted HPV types, with 50% inhibitory doses in the high ng/ml range. Microscopic studies of PsV inhibition by the [alpha]-defensins revealed that they block virion escape from endocytic vesicles but not virion binding or internalization. Consistent with this finding, PsVs remained susceptible to inhibition by [alpha]-defensins for many hours after initial binding to cells. HNPs 1-3 and HD-5 have been reported to be present in the female genital tract at levels that overlap those that inhibit HPVs in vitro, suggesting that they could present a natural barrier to the sexual transmission of HPV and could serve as the basis of a broad-spectrum topical microbicide. cathelicidin | hCAP18 | lactoferrin | LL-37 | microbicide

Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into [alpha] and [beta] families. Structures of several [beta]-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., [Cys.sup.1]-[Cys.sup.5], [Cys.sup.2]-[Cys.sup.4], and [Cys.sup.3]-[Cys.sup.6]. We found that human [beta]-defensin 3 (hBD3), a recently described member of the growing [beta] family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of [Cys.sup.1]-[Cys.sup.5] and of [Cys.sup.1]-[Cys.sup.6], we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native [beta] pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coli, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by [alpha]-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human [beta]-defensins and the design of novel peptide antibiotics.

High-resolution crystal structures of Erwinia chrysanthemi L-asparaginase complexes with L-glutamic acid, L-succinic acid, and D-aspartic acid are described. Data reveal that structurally these complexes are similar to other bacterial L-asparaginases and their ligand complexes and ligands bind to all four active sites on the enzyme.

Molecular dynamics simulations of the crystal complex of feline immunodeficiency virus (FIV) were utilized to investigate their effect on the ligand/enzyme interactions. Results have shown that the carbonyl oxygen of the scissile bond from the substrate was not hydrated. It was indicated that the carboxyl group at the C-terminus of the N-terminal product interacted with aspartate.

The 2 angstroms crystallographic structure and amino acid sequence of Pseudomonas 7A glutaminase-asparaginase (PGA) are similar to those of other tetrameric bacterial amidohydrolases. The active site loop is present in one monomer of PGA in an open position. This open position is different for other amidohydrolases and is in a disordered state. The active site loop presence is indicated by the low occupancy of products of enzymatic interactions.

In-depth biochemical, crystallographic and computational studies are performed to demonstrate the origin of the observed affinity enhancement in a class of small molecules, called ARM-Ps (antibody-recruiting molecule targeting prostate cancer) towards antibody-mediated immune recognition of prostate cancer cells. The results provide critical insights into the design of prostate specific membrane antigen (PSMA)-targeted small molecules for prostate cancer diagnosis and treatment and the presence of similar arene-binding sites throughout the proteome which could enable the optimization of small molecule-protein interactions.

A mutant of human GCPII [GCPII(E424A)], in which Glu424, a putative proton shuttle residue, is substituted with alanine is constructed to study in detail peptide hydrolysis by Glutamate carboxypeptidase II (GCPII). The results provided a detailed and consistent picture of the reaction mechanism of GCPII enzyme at the atomic level which is a zinc-dependent exopeptidase and an important therapeutic target for neurodegeneration and prostate cancer.

CGL is a 150 amino-acid residue lectin that was originally isolated from the sea mussel Crenomytilus grayanus. It is specific for binding GalNAc/Gal-containing carbohydrate moieties and in general does not share sequence homology with other known galectins or lectins. Since CGL displays antibacterial, antifungal and antiviral activities, and interacts with high affinity with mucin-type receptors, which are abundant on some cancer cells, knowledge of its structure is of significant interest. Conditions have been established for the expression, purification and crystallization of a recombinant variant of CGL. The crystal structure of recombinant CGL was determined and refined at a resolution of 2.12 Å. The amino-acid sequence of CGL contains three homologous regions (73% similarity) and the folded protein has a β-trefoil topology. Structural comparison of CGL with the closely related lectin MytiLec allowed description of the glycan-binding pockets. [ABSTRACT FROM AUTHOR]