Lysine 156 promotes the anomalous proenzyme activity of tPA: X-ray crystal structure of single-chain human tPA.
TL;DR: Comparisons with the structures of other serine proteinases that also possess Lys156, such as trypsin, factor Xa and human urokinase plasminogen activator (uPA), identify a set of secondary interactions which are required for Lys156 to fulfil this activating role.
Abstract: Tissue type plasminogen activator (tPA) is the physiological initiator of fibrinolysis, activating plasminogen via highly specific proteolysis; plasmin then degrades fibrin with relatively broad specificity. Unlike other chymotrypsin family serine proteinases, tPA is proteolytically active in a single-chain form. This form is also preferred for therapeutic administration of tPA in cases of acute myocardial infarction. The proteolytic cleavage which activates most other chymotrypsin family serine proteinases increases the catalytic efficiency of tPA only 5- to 10-fold. The X-ray crystal structure of the catalytic domain of recombinant human single-chain tPA shows that Lys156 forms a salt bridge with Asp194, promoting an active conformation in the single-chain form. Comparisons with the structures of other serine proteinases that also possess Lys156, such as trypsin, factor Xa and human urokinase plasminogen activator (uPA), identify a set of secondary interactions which are required for Lys156 to fulfil this activating role. These findings help explain the anomalous single-chain activity of tPA and may suggest strategies for design of new therapeutic plasminogen activators.
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TL;DR: This paper presents a meta-review of the literature on Vinyl Sulfones, Michael Acceptors, and Heterocyclic Inhibitors dating back to the 1970s, which revealed a wide diversity of opinions about the properties of these substances and their role in the human immune system.
Abstract: F. Vinyl Sulfones and Other Michael Acceptors 4683 G. Azodicarboxamides 4695 IV. Acylating Agents 4695 A. Aza-peptides 4695 B. Carbamates 4699 C. Peptidyl Acyl Hydroxamates 4700 D. â-Lactams and Related Inhibitors 4704 E. Heterocyclic Inhibitors 4714 1. Isocoumarins 4715 2. Benzoxazinones 4722 3. Saccharins 4725 4. Miscellaneous Heterocyclic Inhibitors 4728 V. Phosphonylation Agents 4728 A. Peptide Phosphonates 4728 B. Phosphonyl Fluorides 4734 VI. Sulfonylating Agents 4735 A. Sulfonyl Fluorides 4735 VII. Miscellaneous Inhibitors 4736 VIII. Summary and Perspectives 4737 IX. Acknowledgments 4740 X. Note Added in Proof 4740 XI. References 4740
961 citations
TL;DR: A comprehensive review of the caspases, their regulators and inhibitors from a structural and mechanistic point of view, and with an aim to consolidate the many threads that define the rapid growth of this field.
Abstract: The death morphology commonly known as apoptosis results from a post-translational pathway driven largely by specific limited proteolysis. In the last decade the structural basis for apoptosis regulation has moved from nothing to 'quite good', and we now know the fundamental structures of examples from the initiator phase, the pre-mitochondrial regulator phase, the executioner phase, inhibitors and their antagonists, and even the structures of some substrates. The field is as well advanced as the best known of proteolytic pathways, the coagulation cascade. Fundamentally new mechanisms in protease regulation have been disclosed. Structural evidence suggests that caspases have an unusual catalytic mechanism, and that they are activated by apparently unrelated events, depending on which position in the apoptotic pathway they occupy. Some naturally occurring caspase inhibitors have adopted classic inhibition strategies, but other have revealed completely novel mechanisms. All of the structural and mechanistic information can, and is, being applied to drive therapeutic strategies to combat overactivation of apoptosis in degenerative disease, and underactivation in neoplasia. We present a comprehensive review of the caspases, their regulators and inhibitors from a structural and mechanistic point of view, and with an aim to consolidate the many threads that define the rapid growth of this field.
864 citations
TL;DR: The structural basis for inhibition by activation segments is focused upon, as well as the molecular events that lead to the conversion of zymogens to active enzymes.
Abstract: Proteolytic enzymes are synthesized as inactive precursors, or "zymogens," to prevent unwanted protein degradation, and to enable spatial and temporal regulation of proteolytic activity. Upon sorting or appropriate compartmentalization, zymogen conversion to the active enzyme typically involves limited proteolysis and removal of an "activation segment." The sizes of activation segments range from dipeptide units to independently folding domains comprising more than 100 residues. A common form of the activation segment is an N-terminal extension of the mature enzyme, or "prosegment," that sterically blocks the active site, and thereby prevents binding of substrates. In addition to their inhibitory role, prosegments are frequently important for the folding, stability, and/or intracellular sorting of the zymogen. The mechanisms of conversion to active enzymes are diverse in nature, ranging from enzymatic or nonenzymatic cofactors that trigger activation, to a simple change in pH that results in conversion by an autocatalytic mechanism. Recent X-ray crystallographic studies of zymogens and comparisons with their active counterparts have identified the structural changes that accompany conversion. This review will focus upon the structural basis for inhibition by activation segments, as well as the molecular events that lead to the conversion of zymogens to active enzymes.
453 citations
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TL;DR: In this paper, a new paradigm emerges where two forms of the protease, E* and E, are in allosteric equilibrium and determine biological activity and specificity, and the success of this expansion resides in a highly efficient fold that couples catalysis and regulatory interactions.
Abstract: Over one third of all known proteolytic enzymes are serine proteases. Among these, the trypsins underwent the most predominant genetic expansion yielding the enzymes responsible for digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis, and immunity. The success of this expansion resides in a highly efficient fold that couples catalysis and regulatory interactions. Added complexity comes from the recent observation of a significant conformational plasticity of the trypsin fold. A new paradigm emerges where two forms of the protease, E* and E, are in allosteric equilibrium and determine biological activity and specificity. © 2009 IUBMB IUBMB Life 61(5): 510–515, 2009
296 citations
TL;DR: The crystal structure of streptokinase complexed with the catalytic unit of human plasmin was solved and the amino-terminal domain of strePTokinase in the complex is hypothesized to enhance the substrate recognition.
Abstract: Streptokinase is a plasminogen activator widely used in treating blood-clotting disorders. Complexes of streptokinase with human plasminogen can hydrolytically activate other plasminogen molecules to plasmin, which then dissolves blood clots. A similar binding activation mechanism also occurs in some key steps of blood coagulation. The crystal structure of streptokinase complexed with the catalytic unit of human plasmin was solved at 2.9 angstroms. The amino-terminal domain of streptokinase in the complex is hypothesized to enhance the substrate recognition. The carboxyl-terminal domain of streptokinase, which binds near the activation loop of plasminogen, is likely responsible for the contact activation of plasminogen in the complex.
245 citations
References
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TL;DR: The PROCHECK suite of programs as mentioned in this paper provides a detailed check on the stereochemistry of a protein structure and provides an assessment of the overall quality of the structure as compared with well refined structures of the same resolution.
Abstract: The PROCHECK suite of programs provides a detailed check on the stereochemistry of a protein structure Its outputs comprise a number of plots in PostScript format and a comprehensive residue-by-residue listing These give an assessment of the overall quality of the structure as compared with well refined structures of the same resolution and also highlight regions that may need further investigation The PROCHECK programs are useful for assessing the quality not only of protein structures in the process of being solved but also of existing structures and of those being modelled on known structures
22,829 citations
TL;DR: The CCP4 (Collaborative Computational Project, number 4) program suite is a collection of programs and associated data and subroutine libraries which can be used for macromolecular structure determination by X-ray crystallography.
Abstract: The CCP4 (Collaborative Computational Project, number 4) program suite is a collection of programs and associated data and subroutine libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims and so there may be more than one program to cover each function. The programs are written mainly in standard Fortran77. They are from a wide variety of sources but are connected by standard data file formats. The package has been ported to all the major platforms under both Unix and VMS. The suite is distributed by anonymous ftp from Daresbury Laboratory and is widely used throughout the world.
17,220 citations
TL;DR: In this paper, a new molecular-replacement package is presented, which is an improvement on conventional methods, based on more powerful algorithms and a new conception that enables automation and rapid solution.
Abstract: A new molecular-replacement package is presented. It is an improvement on conventional methods, based on more powerful algorithms and a new conception that enables automation and rapid solution.
5,166 citations
"Lysine 156 promotes the anomalous p..." refers methods in this paper
...Navaza,J. (1994) AMoRe: an automated package for molecular replacement....
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...The structure was solved by Pattersonplasminogenolytic activity by a factor of 550 (Bringmann search techniques with the program AMoRe (Navaza, 1994), using anet al., 1995) to 3800 (Tachias and Madison, 1997)....
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TL;DR: In this article, a statistical survey of X-ray structures of small compounds from the Cambridge Structural Database is used for the refinement of protein structures determined by X-Ray crystallography.
Abstract: Bond-length and bond-angle parameters are derived from a statistical survey of X-ray structures of small compounds from the Cambridge Structural Database. The side chains of the common amino acids and the polypeptide backbone were represented by appropriate chemical fragments taken from the Database. Average bond lengths and bond angles are determined from the resulting samples and the sample standard deviations provide information regarding the expected variability of the average values which can be parametrized as force constants. These parameters are ideally suited for the refinement of protein structures determined by X-ray crystallography since they are derived from X-ray structures, are accurate to within the deviations from target values suggested for X-ray structure refinement and use force constants which directly reflect the variability or uncertainty of the average values. Tests of refinement of the structures of BPTI and phycocyanin demonstrate the integrity of the parameters and comparisons of equivalent refinements with XPLOR parameters show improvement in R-factors and geometry statistics.
2,512 citations
"Lysine 156 promotes the anomalous p..." refers methods in this paper
...Conventional crystallographic refinement (rigid-body, positional and temperature factor) was carried out using the parameters o...
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TL;DR: An automatic algorithm STRIDE for protein secondary structure assignment from atomic coordinates based on the combined use of hydrogen bond energy and statistically derived backbone torsional angle information is developed.
Abstract: We have developed an automatic algorithm STRIDE for protein secondary structure assignment from atomic coordinates based on the combined use of hydrogen bond energy and statistically derived backbone torsional angle information. Parameters of the pattern recognition procedure were optimized using designations provided by the crystallographers as a standard-of-truth. Comparison to the currently most widely used technique DSSP by Kabsch and Sander (Biopolymers 22:2577-2637, 1983) shows that STRIDE and DSSP assign secondary structural states in 58 and 31% of 226 protein chains in our data sample, respectively, in greater agreement with the specific residue-by-residue definitions provided by the discoverers of the structures while in 11% of the chains, the assignments are the same. STRIDE delineates every 11th helix and every 32nd strand more in accord with published assignments.
2,390 citations