Trypsin Fold Research Articles

Origin and Expansion of the Serine Protease Repertoire in the Myelomonocyte Lineage.

The deepest evolutionary branches of the trypsin/chymotrypsin family of serine proteases are represented by the digestive enzymes of the gastrointestinal tract and the multi-domain proteases of the blood coagulation and complement system. Similar to the very old digestive system, highly diverse cleavage specificities emerged in various cell lineages of the immune defense system during vertebrate evolution. The four neutrophil serine proteases (NSPs) expressed in the myelomonocyte lineage, neutrophil elastase, proteinase 3, cathepsin G, and neutrophil serine protease 4, collectively display a broad repertoire of (S1) specificities. The origin of NSPs can be traced back to a circulating liver-derived trypsin-like protease, the complement factor D ancestor, whose activity is tightly controlled by substrate-induced activation and TNFα-induced locally upregulated protein secretion. However, the present-day descendants are produced and converted to mature enzymes in precursor cells of the bone marrow and are safely sequestered in granules of circulating neutrophils. The potential site and duration of action of these cell-associated serine proteases are tightly controlled by the recruitment and activation of neutrophils, by stimulus-dependent regulated secretion of the granules, and by various soluble inhibitors in plasma, interstitial fluids, and in the inflammatory exudate. An extraordinary dynamic range and acceleration of immediate defense responses have been achieved by exploiting the high structural plasticity of the trypsin fold.

Papain-like cysteine proteinase zone (PCP-zone) and PCP structural catalytic core (PCP-SCC) of enzymes with cysteine proteinase fold

There are several families of cysteine proteinases with different folds – for example the (chymo)trypsin fold family and papain-like fold family – but in both families the hydrolase activity of cysteine proteinases requires a cysteine residue as the catalytic nucleophile. In this work, we have analyzed the topology of the active site regions in 146 three-dimensional structures of proteins belonging to the Papain-like Cysteine Proteinase (PCP) superfamily, which includes papain as a typical representative of this protein superfamily. All analyzed enzymes contain a unique structurally closed conformation – a “PCP-Zone” – which can be divided into two groups, Class A and Class B. Eight structurally conserved amino acids of the PCP-Zone form a common Structural Core. The Structural Core, catalytic nucleophile, catalytic base and residue Xaa – which stabilizes the side-chain conformation of the catalytic base – make up a PCP Structural Catalytic Core (PCP-SCC). The PCP-SCC of Class A and Class B are divided into 5 and 2 types, respectively. Seven variants of the mutual arrangement of the amino-acid side chains of the catalytic triad – nucleophile, base and residue Xaa – within the same fold clearly demonstrate how enzymes with the papain-like fold adapt to the need to perform diverse functions in spite of their limited structural diversity. The roles of both the PCP-Zone of SARS-CoV-2-PLpro described in this study and the NBCZone of SARS-CoV-2-3CLpro presented in our earlier article (Denesyuk AI, Johnson MS, Salo-Ahen OMH, Uversky VN, Denessiouk K. Int J Biol Macromol. 2020;153:399-411) that are in contacts with inhibitors are discussed.

Role of the I16-D194 ionic interaction in the trypsin fold

Activity in trypsin-like proteases is the result of proteolytic cleavage at R15 followed by an ionic interaction that ensues between the new N terminus of I16 and the side chain of the highly conserved D194. This mechanism of activation, first proposed by Huber and Bode, organizes the oxyanion hole and primary specificity pocket for substrate binding and catalysis. Using the clotting protease thrombin as a relevant model, we unravel contributions of the I16-D194 ionic interaction to Na+ binding, stability of the transition state and the allosteric E*-E equilibrium of the trypsin fold. The I16T mutation abolishes the I16-D194 interaction and compromises the architecture of the oxyanion hole. The D194A mutation also abrogates the I16-D194 interaction but, surprisingly, has no effect on the architecture of the oxyanion hole that remains intact through a new H-bond established between G43 and G193. In both mutants, loss of the I16-D194 ionic interaction compromises Na+ binding, reduces stability of the transition state, collapses the 215–217 segment into the primary specific pocket and abrogates the allosteric E*-E equilibrium in favor of a rigid conformation that binds ligand at the active site according to a simple lock-and-key mechanism. These findings refine the structural role of the I16-D194 ionic interaction in the Huber-Bode mechanism of activation and reveal a functional linkage with the allosteric properties of the trypsin fold like Na+ binding and the E*-E equilibrium.

Residues W215, E217 and E192 control the allosteric E*-E equilibrium of thrombin

A pre-existing, allosteric equilibrium between closed (E*) and open (E) conformations of the active site influences the level of activity in the trypsin fold and defines ligand binding according to the mechanism of conformational selection. Using the clotting protease thrombin as a model system, we investigate the molecular determinants of the E*-E equilibrium through rapid kinetics and X-ray structural biology. The equilibrium is controlled by three residues positioned around the active site. W215 on the 215–217 segment defining the west wall of the active site controls the rate of transition from E to E* through hydrophobic interaction with F227. E192 on the opposite 190–193 segment defining the east wall of the active site controls the rate of transition from E* to E through electrostatic repulsion of E217. The side chain of E217 acts as a lever that moves the entire 215–217 segment in the E*-E equilibrium. Removal of this side chain converts binding to the active site to a simple lock-and-key mechanism and freezes the conformation in a state intermediate between E* and E. These findings reveal a simple framework to understand the molecular basis of a key allosteric property of the trypsin fold.

Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin.

Thrombin exists as an ensemble of active (E) and inactive (E*) conformations that differ in their accessibility to the active site. Here we show that redistribution of the E*-E equilibrium can be achieved by perturbing the electrostatic properties of the enzyme. Removal of the negative charge of the catalytic Asp102 or Asp189 in the primary specificity site destabilizes the E form and causes a shift in the 215-217 segment that compromises substrate entrance. Solution studies and existing structures of D102N document stabilization of the E* form. A new high-resolution structure of D189A also reveals the mutant in the collapsed E* form. These findings establish a new paradigm for the control of the E*-E equilibrium in the trypsin fold.

Kinetic Dissection of the Pre-Existing Conformational Equilibrium in the Trypsin Fold

Structural biology has recently documented the conformational plasticity of the trypsin fold for both the protease and zymogen in terms of a pre-existing equilibrium between closed (E∗) and open (E) forms of the active site region. How such plasticity is manifested in solution and affects ligand recognition by the protease and zymogen is poorly understood in quantitative terms. Here we dissect the E∗-E equilibrium with stopped-flow kinetics in the presence of excess ligand or macromolecule. Using the clotting protease thrombin and its zymogen precursor prethrombin-2 as relevant models we resolve the relative distribution of the E∗ and E forms and the underlying kinetic rates for their interconversion. In the case of thrombin, the E∗ and E forms are distributed in a 1:4 ratio and interconvert on a time scale of 45 ms. In the case of prethrombin-2, the equilibrium is shifted strongly (10:1 ratio) in favor of the closed E∗ form and unfolds over a faster time scale of 4.5 ms. The distribution of E∗ and E forms observed for thrombin and prethrombin-2 indicates that zymogen activation is linked to a significant shift in the pre-existing equilibrium between closed and open conformations that facilitates ligand binding to the active site. These findings broaden our mechanistic understanding of how conformational transitions control ligand recognition by thrombin and its zymogen precursor prethrombin-2 and have direct relevance to other members of the trypsin fold.

Kinetic Dissection of the Pre-existing Conformational Equilibrium in the Trypsin Fold

Structural biology has recently documented the conformational plasticity of the trypsin fold for both the protease and zymogen in terms of a pre-existing equilibrium between closed (E*) and open (E) forms of the active site region. How such plasticity is manifested in solution and affects ligand recognition by the protease and zymogen is poorly understood in quantitative terms. Here we dissect the E*-E equilibrium with stopped-flow kinetics in the presence of excess ligand or macromolecule. Using the clotting protease thrombin and its zymogen precursor prethrombin-2 as relevant models we resolve the relative distribution of the E* and E forms and the underlying kinetic rates for their interconversion. In the case of thrombin, the E* and E forms are distributed in a 1:4 ratio and interconvert on a time scale of 45 ms. In the case of prethrombin-2, the equilibrium is shifted strongly (10:1 ratio) in favor of the closed E* form and unfolds over a faster time scale of 4.5 ms. The distribution of E* and E forms observed for thrombin and prethrombin-2 indicates that zymogen activation is linked to a significant shift in the pre-existing equilibrium between closed and open conformations that facilitates ligand binding to the active site. These findings broaden our mechanistic understanding of how conformational transitions control ligand recognition by thrombin and its zymogen precursor prethrombin-2 and have direct relevance to other members of the trypsin fold.

Why Ser and not Thr brokers catalysis in the trypsin fold.

Although Thr is equally represented as Ser in the human genome and as a nucleophile is as good as Ser, it is never found in the active site of the large family of trypsin-like proteases that utilize the Asp/His/Ser triad. The molecular basis of the preference of Ser over Thr in the trypsin fold was investigated with X-ray structures of the thrombin mutant S195T free and bound to an irreversible active site inhibitor. In the free form, the methyl group of T195 is oriented toward the incoming substrate in a conformation seemingly incompatible with productive binding. In the bound form, the side chain of T195 is reoriented for efficient substrate acylation without causing steric clash within the active site. Rapid kinetics prove that this change is due to selection of an active conformation from a preexisting ensemble of reactive and unreactive rotamers whose relative distribution determines the level of activity of the protease. Consistent with these observations, the S195T substitution is associated with a weak yet finite activity that allows identification of an unanticipated important role for S195 as the end point of allosteric transduction in the trypsin fold. The S195T mutation abrogates the Na(+)-dependent enhancement of catalytic activity in thrombin, activated protein C, and factor Xa and significantly weakens the physiologically important allosteric effects of thrombomodulin on thrombin and of cofactor Va on factor Xa. The evolutionary selection of Ser over Thr in trypsin-like proteases was therefore driven by the need for high catalytic activity and efficient allosteric regulation.

Conformational selection in trypsin-like proteases

For over four decades, two competing mechanisms of ligand recognition--conformational selection and induced-fit--have dominated our interpretation of protein allostery. Defining the mechanism broadens our understanding of the system and impacts our ability to design effective drugs and new therapeutics. Recent kinetics studies demonstrate that trypsin-like proteases exist in equilibrium between two forms: one fully accessible to substrate (E) and the other with the active site occluded (E*). Analysis of the structural database confirms existence of the E* and E forms and vouches for the allosteric nature of the trypsin fold. Allostery in terms of conformational selection establishes an important paradigm in the protease field and enables protein engineers to expand the repertoire of proteases as therapeutics.

Modeling and structural analysis of PA clan serine proteases

BackgroundSerine proteases account for over a third of all known proteolytic enzymes; they are involved in a variety of physiological processes and are classified into clans sharing structural homology. The PA clan of endopeptidases is the most abundant and over two thirds of this clan is comprised of the S1 family of serine proteases, which bear the archetypal trypsin fold and have a catalytic triad in the order Histidine, Aspartate, Serine. These proteases have been studied in depth and many three dimensional structures have been experimentally determined. However, these structures mostly consist of bacterial and animal proteases, with a small number of plant and fungal proteases and as yet no structures have been determined for protozoa or archaea. The core structure and active site geometry of these proteases is of interest for many applications. This study investigated the structural properties of different S1 family serine proteases from a diverse range of taxa using molecular modeling techniques.ResultsOur predicted models from protozoa, archaea, fungi and plants were combined with the experimentally determined structures of 16 S1 family members and used for analysis of the catalytic core. Amino acid sequences were submitted to SWISS-MODEL for homology-based structure prediction or the LOOPP server for threading-based structure prediction. Predicted models were refined using INSIGHT II and SCRWL and validated against experimental structures. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. The structural geometry of the catalytic core shows clear deviations between taxa, but the relative positions of the catalytic triad residues were conserved. Some highly conserved residues potentially contributing to the stability of the structural core were identified. Evolutionary divergence was also exhibited by large variation in secondary structure features outside the core, differences in overall amino acid distribution, and unique surface electrostatic potential patterns between species.ConclusionsEncompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on S1 family serine proteases. Focusing on the common core containing the catalytic site of the enzyme, this analysis is beneficial for future molecular modeling strategies and structural analysis of serine protease models.

Thermodynamic activation and structural analysis of trypsin I from Monterey sardine (Sardinops sagax caerulea)

Abstract In this work, we report the molecular characterisation of trypsin I (Try I) from Monterey sardine (Sardinops sagax caerulea). Aspects such as thermodynamic activation parameters, molecular model and cDNA-deduced amino acid sequence allow a more in depth understanding of its activity at low temperatures. The analysis of the thermodynamic activation parameters suggests that this molecule is a cold-adapted protease. From the molecular cloning, we deduced the amino acid sequence and predicted a theoretical structural model of sardine Try I with a classical trypsin fold. Cold-adaptation of this enzyme probably comes from amino acid replacement of key residues to improve flexibility at low temperature, thus increasing kcat. The cold-adaptation of sardine Try I opens a wide range of biotechnological applications for this protease and also it is interesting from the structure function relationship point of view of serine protease proteins.

Crystallographic and Kinetic Evidence of Allostery in a Trypsin-like Protease

Protein allostery is based on the existence of multiple conformations in equilibrium linked to distinct functional properties. Although evidence of allosteric transitions is relatively easy to identify by functional studies, structural detection of a pre-existing equilibrium between alternative conformations remains challenging even for textbook examples of allosteric proteins. Kinetic studies show that the trypsin-like protease thrombin exists in equilibrium between two conformations where the active site is either collapsed (E*) or accessible to substrate (E). However, structural demonstration that the two conformations exist in the same enzyme construct free of ligands has remained elusive. Here we report the crystal structure of the thrombin mutant N143P in the E form, which complements the recently reported structure in the E* form, and both the E and E* forms of the thrombin mutant Y225P. The side chain of W215 moves 10.9 Å between the two forms, causing a displacement of 6.6 Å of the entire 215-217 segment into the active site that in turn opens or closes access to the primary specificity pocket. Rapid kinetic measurements of p-aminobenzamidine binding to the active site confirm the existence of the E*-E equilibrium in solution for wild-type and the mutants N143P and Y225P. These findings provide unequivocal proof of the allosteric nature of thrombin and lend strong support to the recent proposal that the E*-E equilibrium is a key property of the trypsin fold.

Evidence of the E*−E Equilibrium from Rapid Kinetics of Na+ Binding to Activated Protein C and Factor Xa*

Na(+) binding to thrombin enhances the procoagulant and prothrombotic functions of the enzyme and obeys a mechanism that produces two kinetic phases: one fast (in the microsecond time scale) due to Na(+) binding to the low activity form E to produce the high activity form E:Na(+) and another considerably slower (in the millisecond time scale) that reflects a pre-equilibrium between E and the inactive form E*. In this study, we demonstrate that this mechanism also exists in other Na(+)-activated clotting proteases like factor Xa and activated protein C. These findings, along with recent structural data, suggest that the E*-E equilibrium is a general feature of the trypsin fold.

Combinatorial Enzyme Design Probes Allostery and Cooperativity in the Trypsin Fold

Converting one enzyme into another is challenging due to the uneven distribution of important amino acids for function in both protein sequence and structure. We report a strategy for protein engineering allowing an organized mixing and matching of genetic material that leverages lower throughput with increased quality of screens. Our approach successfully tested the contribution of each surface-exposed loop in the trypsin fold alone and the cooperativity of their combinations towards building the substrate selectivity and Na+-dependent allosteric activation of the protease domain of human coagulation factor Xa into a bacterial trypsin. As the created proteases lack additional protein domains and protein co-factor activation mechanism requisite for the complexity of blood coagulation, they are stepping-stones towards further understanding and engineering of artificial clotting factors.

Engineering Ecotin for Identifying Proteins with a Trypsin Fold

Ecotin is a bidentate, fold-specific inhibitor of mammalian serine-proteases produced by Escherichia coli. This molecule may be engineered to increase and/or change its affinity and specificity providing significant biotechnological potential. Since ecotin binds tightly to serine proteases of the trypsin fold, it may help to identify the role of these enzymes in different biological processes. In this work, we tested ecotin variants as an affinity purification reagent for identifying enzymes in samples of tumor progression and mammary gland involution. Initially, we used a commercial source of urokinase-type plasminogen activator (u-PA) that remained fully active after elution from an affinity column of the ecotin variant (M84R, M85R). We then successfully identified u-PA from more complex mixtures including lysates from a prostate cancer cell line and involuting mouse mammary glands. Interestingly, a membrane-type serine protease 1 was isolated from the Triton X-100-solubilized PC-3 cell lysates, and surprisingly, haptoglobin, a serine-protease homolog protein, was also identified in mammary gland lysates and in blood. Haptoglobin does not prevent ecotin inhibition of u-PA, but it may act as a carrier within blood when ecotin is used in vivo. Finally, this affinity purification matrix was also able to identify a thrombin-like enzyme from snake venom using an ecotin variant directed against thrombin. Overall, the ecotin variants acted as robust tools for the isolation and characterization of proteins with a trypsin fold. Thus, they may assist in the understanding of the role of these serine proteases and homologous proteins in different biological processes.

Serine proteases

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.

Crystal structure of the serine protease domain of Sesbania mosaic virus polyprotein and mutational analysis of residues forming the S1-binding pocket

Sesbania mosaic virus (SeMV) polyprotein is processed by its N-terminal serine protease domain. The crystal structure of the protease domain was determined to a resolution of 2.4 Å using multiple isomorphous replacement and anomalous scattering. The SeMV protease domain exhibited the characteristic trypsin fold and was found to be closer to cellular serine proteases than to other viral proteases. The residues of the S1-binding pocket, H298, T279 and N308 were mutated to alanine in the ΔN70-Protease–VPg polyprotein, and the cis-cleavage activity was examined. The H298A and T279A mutants were inactive, while the N308A mutant was partially active, suggesting that the interactions of H298 and T279 with P1-glutamate are crucial for the E–T/S cleavage. A region of exposed aromatic amino acids, probably essential for interaction with VPg, was identified on the protease domain, and this interaction could play a major role in modulating the function of the protease.