Validation of three-dimensional structures is at the core of structural determination methods. The local validation criteria, such as deviations from ideal bond length and bonding angles, ...Ramachandran plot outliers and clashing contacts, are a standard part of structure analysis before structure deposition, whereas the global and regional packing may not yet have been addressed. In the last two decades, three-dimensional models of macromolecules such as proteins have been successfully described by a network of nodes and edges. Amino acid residues as nodes and close contact between the residues as edges have been used to explore basic network properties, to study protein folding and stability and to predict catalytic sites. Using complex network analysis, we introduced common network parameters to distinguish between correct and incorrect three-dimensional protein structures. The analysis showed that correct structures have a higher average node degree, higher graph energy, and lower shortest path length than their incorrect counterparts. Thus, correct protein models are more densely intra-connected, and in turn, the transfer of information between nodes/amino acids is more efficient. Moreover, protein graph spectra were used to investigate model bias in protein structure.
MAIN is software that has been designed to interactively perform the complex tasks of macromolecular crystal structure determination and validation. Using MAIN, it is possible to perform density ...modification, manual and semi‐automated 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 the 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 three dimensions that is provided by MAIN is achieved by the clarity and contrast of colours and the 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. Using MAIN, it is possible to optimize NCS parameters and envelopes and to refine the structure in single or multiple crystal forms.
The Future of Cysteine Cathepsins in Disease Management Kramer, Lovro; Turk, Dušan; Turk, Boris
Trends in pharmacological sciences (Regular ed.),
October 2017, 2017-10-00, 20171001, Letnik:
38, Številka:
10
Journal Article
Recenzirano
Since the discovery of the key role of cathepsin K in bone resorption, cysteine cathepsins have been investigated by pharmaceutical companies as drug targets. The first clinical results from ...targeting cathepsins by activity-based probes and substrates are paving the way for the next generation of molecular diagnostic imaging, whereas the majority of antibody–drug conjugates currently in clinical trials depend on activation by cathepsins. Finally, cathepsins have emerged as suitable vehicles for targeted drug delivery. It is therefore timely to review the future of cathepsins in drug discovery. We focus here on inflammation-associated diseases because dysregulation of the immune system accompanied by elevated cathepsin activity is a common feature of these conditions.
The newly discovered roles of secreted cysteine cathepsins are dominating preclinical research, with major developments in pain regulation and cancer chemotherapy resistance.
Cathepsin K inhibitors, including odanacatib, have all failed in osteoporosis clinical trials, but novel strategies to target cathepsin K are emerging.
The antibody–drug conjugate field is dominated by cathepsin-cleavable linkers.
First cathepsin-targeting imaging probes have entered clinical trials with favorable initial results.
A new concept for targeted drug delivery based on cathepsin-targeting is emerging.
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.
The identification of physiological protease substrates has resulted in a shift of paradigm from proteases as protein‐degrading enzymes to establishing their role as key signalling molecules.
SecA protein is a major component of the general bacterial secretory system. It is an ATPase that couples nucleotide hydrolysis to protein translocation. In some Gram-positive pathogens, a second ...paralogue, SecA2, exports a different set of substrates, usually virulence factors. To identify SecA2 features different from SecA(1)s, we determined the crystal structure of SecA2 from
, an important nosocomial pathogen, in apo and ATP-γ-S-bound form. The structure reveals a closed monomer lacking the C-terminal tail (CTT) with an otherwise similar multidomain organization to its SecA(1) homologues and conserved binding of ATP-γ-S. The average in vitro ATPase activity rate of
SecA2 was 2.6 ± 0.1 µmolPi/min/µmol. Template-based modeling combined with evolutionary conservation analysis supports a model where
SecA2 in open conformation binds the target protein, ensures its movement through the SecY channel, and enables dimerization through PPXD/HWD cross-interaction of monomers during the process. Both approaches exposed regions with differences between SecA(1) and SecA2 homologues, which are in agreement with the unique adaptation of SecA2 proteins for a specific type of substrate, a role that can be addressed in further studies.
Autolysin E (AtlE), from Staphylococcus aureus, is a cell-wall-degrading enzyme that is a potential drug target. It is a member of the glycoside hydrolase (GH) class, enzymes that commonly have ...either two catalytic residues and hydrolyze their substrates by inverting or retaining mechanisms or one catalytic residue and undergo retaining, substrate-assisted catalysis. Here, we address the catalytic mechanism of AtlE. Site-directed mutagenesis studies identified Glu138 as the only catalytic residue. Quantum mechanics/molecular mechanics (QM/MM) simulations of the possible reaction pathways suggest that hydrolysis proceeds via a retaining, water-assisted mechanism and an oxocarbenium ion like transition state. These results, on the basis of data from a member of the hydrolase GH73 family, support the hypothesis of the presence of an alternative catalytic mechanism in glycoside hydrolases, which can be considered in the design of future AtlE inhibitors.
Bacterial DNA gyrase is a well-established and validated target for the development of novel antibacterials. Starting from the available structural information about the binding of the natural ...product inhibitor, clorobiocin, we identified a novel series of 4′-methyl-N 2-phenyl-4,5′-bithiazole-2,2′-diamine inhibitors of gyrase B with a low micromolar inhibitory activity by implementing a two-step structure-based design procedure. This novel class of DNA gyrase inhibitors was extensively investigated by various techniques (differential scanning fluorimetry, surface plasmon resonance, and microscale thermophoresis). The binding mode of the potent inhibitor 18 was revealed by X-ray crystallography, confirming our initial in silico binding model. Furthermore, the high resolution of the complex structure allowed for the placement of the Gly97–Ser108 flexible loop, thus revealing its role in binding of this class of compounds. The crystal structure of the complex protein G24 and inhibitor 18 provides valuable information for further optimization of this novel class of DNA gyrase B inhibitors.
Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed ...the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the "lock and key" mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors.
Macromolecular crystallography and electron microscopy (single-particle and in situ tomography) are merging into a single approach used by the two coalescing scientific communities. The merger is a ...consequence of technical developments that enabled determination of atomic structures of macromolecules by electron microscopy. Technological progress in experimental methods of macromolecular structure determination, computer hardware, and software changed and continues to change the nature of model building and visualization of molecular structures. However, the increase in automation and availability of structure validation are reducing interactive manual model building to fiddling with details. On the other hand, interactive modeling tools increasingly rely on search and complex energy calculation procedures, which make manually driven changes in geometry increasingly powerful and at the same time less demanding. Thus, the need for accurate manual positioning of a model is decreasing. The user's push only needs to be sufficient to bring the model within the increasing convergence radius of the computing tools. It seems that we can now better than ever determine an average single structure. The tools work better, requirements for engagement of human brain are lowered, and the frontier of intellectual and scientific challenges has moved on. The quest for resolution of new challenges requires out-of-the-box thinking. A few issues such as model bias and correctness of structure, ongoing developments in parameters defining geometric restraints, limitations of the ideal average single structure, and limitations of Bragg spot data are discussed here, together with the challenges that lie ahead.
Determination of protease specificity is of crucial importance for understanding protease function. We have developed the first gel‐based label‐free proteomic approach (DIPPS—direct in‐gel profiling ...of protease specificity) that enables quick and reliable determination of protease cleavage specificities under large variety of experimental conditions. The methodology is based on in‐gel digestion of the gel‐separated proteome with the studied protease, enrichment of cleaved peptides by gel extraction, and subsequent mass spectrometry analysis combined with a length‐limited unspecific database search. We applied the methodology to profile ten proteases ranging from highly specific (trypsin, endoproteinase GluC, caspase‐7, and legumain) to broadly specific (matrix‐metalloproteinase‐3, thermolysin, and cathepsins K, L, S, and V). Using DIPPS, we were able to perform specificity profiling of thermolysin at its optimal temperature of 75°C, which confirmed the applicability of the method to extreme experimental conditions. Moreover, DIPPS enabled the first global specificity profiling of legumain at pH as low as 4.0, which revealed a pH‐dependent change in the specificity of this protease, further supporting its broad applicability.
Synopsis
Direct In‐gel Profiling of Protease Specificity (DIPPS) enables quick and reliable determination of protease cleavage specificities under a large variety of experimental conditions, offering an excellent starting point for protease characterization and substrate design.
Gel‐based, label‐free whole‐proteome degradomics provides an easy‐to‐use approach applicable under a broad range of assay conditions.
DIPPS validation on 10 distinct proteases from three major mechanistic classes shows an excellent correlation with published protease specificities, without observable experimental biases.
DIPPS profiling of legumain reveals a pH‐dependent specificity switch between Asn and Asn/Asp cleavage specificity.
Direct In‐gel Profiling of Protease Specificity (DIPPS) under a large variety of experimental conditions offers a quick and reliable starting point for protease functional characterization and substrate design.
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