Peptidoglycan is a giant molecule that forms the cell wall that surrounds bacterial cells. It is composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues connected by ß-(1,4)-glycosidic bonds and cross-linked with short polypeptide chains. Owing to the increasing antibiotic resistance against drugs targeting peptidoglycan synthesis, studies of enzymes involved in the degradation of peptidoglycan, such as N-acetylglucosaminidases, may expose new, valuable drug targets. The scientific challenge addressed here is how lysozymes, muramidases which are likely to be the most studied enzymes ever, and bacterial N-acetylglucosaminidases discriminate between two glycosidic bonds that are different in sequence yet chemically equivalent in the same NAG-NAM polymers. In spite of more than fifty years of structural studies of lysozyme, it is still not known how the enzyme selects the bond to be cleaved. Using macromolecular crystallography, chemical synthesis and molecular modelling, this study explains how these two groups of enzymes based on an equivalent structural core exhibit a difference in selectivity. The crystal structures of Staphylococcus aureus N-acetylglucosaminidase autolysin E (AtlE) alone and in complex with fragments of peptidoglycan revealed that N-acetylglucosaminidases and muramidases approach the substrate at alternate glycosidic bond positions from opposite sides. The recognition pocket for NAM residues in the active site of N-acetylglucosaminidases may make them a suitable drug target.
COBISS.SI-ID: 30287911
Bacterial cell wall proteins play crucial roles in cell survival, growth, and environmental interactions. In Gram-positive bacteria, cell wall proteins include several types that are non-covalently attached via cell wall binding domains. Of the two conserved surface-layer (S-layer)-anchoring modules composed of three tandem SLH or CWB2 domains, the latter have so far eluded structural insight. The crystal structures of Cwp8 and Cwp6 reveal multi-domain proteins, each containing an embedded CWB2 module. It consists of a triangular trimer of Rossmann-fold CWB2 domains, a feature common to 29 cell wall proteins in Clostridium difficile 630. The structural basis of the intact module fold necessary for its binding to the cell wall is revealed. A comparison with previously reported atomic force microscopy data of S-layers suggests that C. difficile S-layers are complex oligomeric structures, likely composed of several different proteins.
COBISS.SI-ID: 30263847
Cathepsin L is a ubiquitously expressed papain-like cysteine protease involved in the endosomal degradation of proteins and has numerous roles in physiological and pathological processes, such as arthritis, osteoporosis, and cancer. Insight into the specificity of cathepsin L is important for elucidating its physiological roles and drug discovery. To study interactions with synthetic ligands, we prepared a presumably inactive mutant and crystallized it. Unexpectedly, the crystal structure determined at 1.4 A revealed that the cathepsin L molecule is cleaved, Cathepsin L is a ubiquitously expressed papain-like cysteine protease involved in the endosomal degradation of proteins and has numerous roles in physiological and pathological processes, such as arthritis, osteoporosis, and cancer. Insight into the specificity of cathepsin L is important for elucidating its physiological roles and drug discovery. To study interactions with synthetic ligands, we prepared a presumably inactive mutant and crystallized it. Unexpectedly, the crystal structure determined at 1.4 A revealed that the cathepsin L molecule is cleaved, with the cleaved region trapped in the active site cleft of the neighboring molecule. Hence, the catalytic mutant demonstrated low levels of catalytic activity.
COBISS.SI-ID: 29921831
MAIN is software designed to interactively perform the complex tasks of macromolecular crystal structure determination and validation. With MAIN, it is possible to perform density modifications, manual and semiautomated or automated model building and rebuilding, real and reciprocal space structure optimization and refinement, map calculations, and various types of molecular structure validation. The prompt availability of various analytical tools and immediate visualization of molecular and map objects allow a user to efficiently progress towards the completed, refined structure. The extraordinary depth perception of molecular objects in 3D that is provided by MAIN is achieved by the clarity and contrast of colours and smooth rotation of the displayed objects. MAIN allows simultaneous work on several molecular models and various crystal forms. The strength of MAIN lies in its manipulation of averaged density maps and molecular models when noncrystallographic symmetry (NCS) is present. With MAIN, it is possible to optimize NCS parameters and envelopes and to refine the structure in single or multiple crystal forms.
COBISS.SI-ID: 26802727
Comparison of structures indicated that the selectivity of interactions between cysteine cathepsins and the p41 fragment is far from being understood and requires further investigation. The p41 fragment has now been shown also to inhibit human cathepsins V, K and F (also, presumably, O) and mouse cathepsin L with Ki values in the low nM range and surprisingly cathepsin S too. These findings suggest that regulation of the proteolytic activity of most of the cysteine cathepsins by the p41 fragment is an important and widespread control mechanism of antigen presentation.
COBISS.SI-ID: 21555495
Protease research has undergone a major expansion in the last decade, largely due to the extremely rapid development of new technologies, such as quantitative proteomics and in-vivo imaging, as well as an extensive use of in-vivo models. These have led to identification of physiological substrates and resulted in a paradigm shift from the concept of proteases as protein-degrading enzymes to proteases as key signalling molecules. However, we are still at the beginning of an understanding of protease signalling pathways. We have only identified a minor subset of true physiological substrates for a limited number of proteases, and their physiological regulation is still not well understood. Similarly, links with other signalling systems are not well established. Herein, we will highlight current challenges in protease research.
COBISS.SI-ID: 25737767
Use of reliable density maps is crucial for rapid and successful crystal structure determination. Here, the averaged kick (AK) map approach is investigated, its application is generalized and it is compared with other map-calculation methods. The conclusion is that AK maps can be useful throughout the entire progress of crystal structure determination, offering the possibility of improved map interpretation.
COBISS.SI-ID: 22793511
Mycocypins are cysteine protease inhibitors isolated from the mushrooms Clytocibe nebularis and Macrolepiota procera. The crystal structures of the complex of clitocypin with the papain-like cysteine protease cathepsin V and macrocypin and clitocypin alone have revealed yet another motif of binding to papain-like cysteine proteases. The binding loops present a versatile surface with the potential to bind to additional classes of proteases. When appropriately engineered, they could provide the basis for possible exploitation in crop protection.
COBISS.SI-ID: 23263527
It is more than 50 years since the lysosome was discovered. Since then its hydrolytic machinery, including proteases and other hydrolases, has been fairly well identified and characterized. Among these are the cysteine cathepsins, members of the family of papain-like cysteine proteases. They have unique reactive site properties and an uneven tissue specific expression pattern. In living organisms their activity is a delicate balance of expression, targeting, zymogen activation, inhibition by protein inhibitors and degradation. The specificity of their substrate binding sites, small molecule inhibitor repertoire and crystal structures are providing new tools for research and development. Their unique reactive site properties have made it possible to confine the targets simply by the use of appropriate reactive groups. The epoxysuccinyls still dominate the field, but now nitriles seem to be the most appropriate “warhead”. The view of cysteine cathepsins as lysosomal proteases is changing as there is now clear evidence of their localization in other cellular compartments. Besides being involved in protein turnover, they build an important part of the endosomal antigen presentation machinery. Together with the growing number of nonendosomal roles of cysteine cathepsins is growing also the knowledge of their involvement in diseases, such as cancer and rheumatoid arthritis, among others. Finally, cysteine cathepsins are important regulators and signalling molecules of an unimaginable number of biological processes. The current challenge is to identify their endogenous substrates, in order to gain an insight into the mechanisms of substrate degradation and processing. In this review, some of the remarkable advances that have taken place in the past decade are presented. The article (as well as the whole issue BBA Proteins and proteomics vol. 1824/1, 2012) was dedicated to Nobel laureate Christian de Duve.
COBISS.SI-ID: 25347623
We presented the first experimental characterisation of a prokaryotic caspase homologue that we named 'orthocaspase'. The putative caspase-like gene MaOC1 from the toxic bloom-forming cyanobacterium Microcystis aeruginosa PCC 7806 was expressed in Escherichia coli. We elucidated the mode of proenzyme activation and determined amino acid preference at the substrate cleavage site. In contrast to plant or metazoan caspase-like proteins, whose activity is calcium-dependent or requires dimerization for activation, MaOC1 is activated by autoproteolytic cleavage of the precursor.
COBISS.SI-ID: 1536383427