Insight Derived from Molecular Dynamics Simulation into Substrate-Induced Changes in Protein Motions of Proteinase K

Abstract

Because of the significant industrial, agricultural and biotechnological importance of serine protease proteinase K, it has been extensively investigated using experimental approaches such as X-ray crystallography, site-directed mutagenesis and kinetic measurement. However, detailed aspects of enzymatic mechanism such as substrate binding, release and relevant regulation remain unstudied. Molecular dynamics (MD) simulations of the proteinase K alone and in complex with the peptide substrate AAPA were performed to investigate the effect of substrate binding on the dynamics/molecular motions of proteinase K. The results indicate that during simulations the substrate-complexed proteinase K adopt a more compact and stable conformation than the substrate-free form. Further essential dynamics (ED) analysis reveals that the major internal motions are confined within a subspace of very small dimension. Upon substrate binding, the overall flexibility of the protease is reduced; and the noticeable displacements are observed not only in substrate-binding regions but also in regions opposite the substrate-binding groove/pockets. The dynamic pockets caused by the large concerted motions are proposed to be linked to the substrate recognition, binding, orientation and product release; and the significant displacements in regions opposite the binding groove/pockets are considered to play a role in modulating the dynamics of enzyme-substrate interaction. Our simulation results complement the biochemical and structural studies, highlighting the dynamic mechanism of the functional properties of proteinase K.     

Insights derived from molecular dynamics simulation into the molecular motions of serine protease proteinase K

Serine protease proteinase K, a member of the subtilisin family of enzymes, is of significant industrial, agricultural and biotechnological importance. Despite the wealth of structural information about proteinase K provided by static X-ray structures, a full understanding of the enzymatic mechanism requires further insight into the dynamic properties of this enzyme. Molecular dynamics simulations and essential dynamics (ED) analysis were performed to investigate the molecular motions in proteinase K. The results indicate that the internal core of proteinase K is relatively rigid, whereas the surface-exposed loops, most notably the substrate-binding regions, exhibit considerable conformational fluctuations. Further ED analysis reveals that the large concerted motions in the substrate-binding regions cause opening/closing of the substrate-binding pockets, thus supporting the proposed induced-fit mechanism of substrate binding. The distinct electrostatic/hydrogen-bonding interactions between Asp39 and His69 and between His69 and Ser224 within the catalytic triad lead to different thermal motions and orientations of these three catalytic residues, which can be related to their different functional roles in the catalytic process. Statistical analyses of the geometrical/functional properties as well as evolutionary conservation of the glycines in proteinase K-like proteins reveal that glycines may play an important role in determining the folding architecture and structural flexibility of this class of enzymes. Our simulation study complements the biochemical and structural studies and provides new insights into the dynamic structural basis of the functional properties of this class of enzymes.     

A molecular dynamics analysis of protein structural elements

The relation between protein secondary structure and internal motions was examined by using molecular dynamics to calculate positional fluctuations of individual helix, beta-sheet, and loop structural elements in free and substrate-bound hen egg-white lysozyme. The time development of the fluctuations revealed a general correspondence between structure and dynamics; the fluctuations of the helices and beta-sheets converged within the 101 psec period of the simulation and were lower than average in magnitude, while the fluctuations of the loop regions were not converged and were mostly larger than average in magnitude. Notable exceptions to this pattern occurred in the substrate-bound simulation. A loop region (residues 101-107) of the active site cleft had significantly reduced motion due to interactions with the substrate. Moreover, part of a loop and a 3(10) helix (residues of 67-88) not in contact with the substrate showed a marked increase in fluctuations. That these differences in dynamics of free and substrate-bound lysozyme did not result simply from sampling errors was established by an analysis of the variations in the fluctuations of the two halves of the 101 psec simulation of free lysozyme. Concerted transitions of four to five mainchain phi and psi angles between dihedral wells were shown to be responsible for large coordinate shifts in the loops. These transitions displaced six or fewer residues and took place either abruptly, in 1 psec or less, or with a diffusive character over 5-10 psec. Displacements of rigid secondary structures involved longer timescale motions in bound lysozyme; a 0.5 A rms change in the position of a helix occurred over the 55 psec simulation period. This helix reorientation within the protein appears to be a response to substrate binding. There was little correlation between the solvent accessible surface area and the dynamics of the different structural elements.     

Isolation and Characterization of a Serine Protease from the Nematophagous Fungus, <Emphasis Type="Italic">Lecanicillium psalliotae</Emphasis>, Displaying Nematicidal Activity

Lecanicillium psalliotae produced an extracellular protease (Ver112) which was purified to apparent homogeneity giving a single band on SDS-PAGE with a molecular mass of 32 kDa. The optimum activity of Ver112 was at pH 10 and 70 °C (over 5 min). The purified protease degraded a broad range of substrates including casein, gelatin, and nematode cuticle with 81% of a nematode (Panagrellus redivivus) being degraded after treating with Ver112 for 12 h. The protease was highly sensitive to PMSF (1 mM) indicating it to be a serine protease. The N-terminal amino acid residues of Ver112 shared a high degree of similarity with other cuticle-degrading proteases from nematophagous fungi which suggests a role in nematode infection.     

Investigation of the induced-fit mechanism and catalytic activity of the human cytomegalovirus protease homodimer via molecular dynamics simulations

Human cytomegalovirus (HCMV) is a highly species-specific DNA virus infecting up to 80% of the general population. The viral genome contains the open reading frame UL80, which encodes the full-length 80 kDa HCMV serine protease and its substrate. Full-length HCMV protease is composed of an N-terminal 256-amino-acid proteolytic domain, called assemblin, a linker region, and a C-terminal structural domain, the assembly protein precursor. Biochemical studies have shown that dimerization activates assemblin because of an induced stabilization of the oxyanion hole (Arg166). Thus, we performed here molecular dynamics (MD) simulations on HCMV protease models to study the induced-fit mechanism of the enzyme upon the binding of substrates and peptidyl inhibitors, and structural and energetic factors that are responsible for the catalytic activity of the enzyme dimer. Long and stable trajectories were obtained for the models of the monomeric and dimeric states, free in solution and bound to a peptidyl-activated carbonyl inhibitor, with very good agreement between theoretical and experimental results. Our results suggest that HCMV protease is indeed a novel example of serine protease that operates by an induced-fit mechanism. Also, in agreement with mutagenesis studies, our MD simulations suggest that the dimeric form is necessary to activate the enzyme because of an induced stabilization of the oxyanion hole.     

Homology modeling and molecular dynamics study of West Nile virus NS3 protease: a molecular basis for the catalytic activity increased by the NS2B cofactor

The West Nile virus (WNV) NS3 serine protease, which plays an important role in assembly of infective virion, is an attractive target for anti-WNV drug development. Cofactors NS2B and NS4A increase the catalytic activity of NS3 in dengue virus and Hepatitis C virus, respectively. Recent studies on the WNV-NS3 characterize the catalytically active form of NS3 by tethering the 40-residue cofactor NS2B. It is suggested that NS2B is essential for the NS3 activity in WNV, while there is no information of the WNV-NS3-related crystal structure. To understand the role of NS2B/substrate in the NS3 catalytic activity, we built a series of models: WNV-NS3 and WNV-NS3-NS2B and WNV-NS3-NS2B-substrate using homology modeling and molecular modeling techniques. Molecular dynamics (MD) simulations were performed for 2.75 ns on each model, to investigate the structural stabilization and catalytic triad motion of the WNV NS3 protease with and without NS2B/substrate. The simulations show that the NS3 rearrangement occurs upon the NS2B binding, resulting in the stable D75-OD1...H51-NH hydrogen bonding. After the substrate binds to the NS3-NS2B active site, the NS3 protease becomes more stable, and the catalytic triad is formed. These results provide a structural basis for the activation and stabilization of the enzyme by its cofactor and substrate.     

Influence of modulated structural dynamics on the kinetics of alpha-chymotrypsin catalysis. Insights through chemical glycosylation, molecular dynamics and domain motion analysis

Although the chemical nature of the catalytic mechanism of the serine protease alpha-chymotrypsin (alpha-CT) is largely understood, the influence of the enzyme's structural dynamics on its catalysis remains uncertain. Here we investigate whether alpha-CT's structural dynamics directly influence the kinetics of enzyme catalysis. Chemical glycosylation [Solá RJ & Griebenow K (2006) FEBS Lett 580, 1685-1690] was used to generate a series of glycosylated alpha-CT conjugates with reduced structural dynamics, as determined from amide hydrogen/deuterium exchange kinetics (k(HX)). Determination of their catalytic behavior (K(S), k(2), and k(3)) for the hydrolysis of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA) revealed decreased kinetics for the catalytic steps (k(2) and k(3)) without affecting substrate binding (K(S)) at increasing glycosylation levels. Statistical correlation analysis between the catalytic (DeltaG( not equal)k(i)) and structurally dynamic (DeltaG(HX)) parameters determined revealed that the enzyme acylation and deacylation steps are directly influenced by the changes in protein structural dynamics. Molecular modelling of the alpha-CT glycoconjugates coupled with molecular dynamics simulations and domain motion analysis employing the Gaussian network model revealed structural insights into the relation between the protein's surface glycosylation, the resulting structural dynamic changes, and the influence of these on the enzyme's collective dynamics and catalytic residues. The experimental and theoretical results presented here not only provide fundamental insights concerning the influence of glycosylation on the protein biophysical properties but also support the hypothesis that for alpha-CT the global structural dynamics directly influence the kinetics of enzyme catalysis via mechanochemical coupling between domain motions and active site chemical groups.     

Evaluation of extensional and torsional stiffness of single actin filaments by molecular dynamics analysis

It is essential to investigate the mechanical behaviour of cytoskeletal actin filaments in order to understand their critical role as mechanical components in various cellular functional activities. These actin filaments consisting of monomeric molecules function in the thermal fluctuations. Hence, it is important to understand their mechanical behaviour on the microscopic scale by comparing the stiffness based on thermal fluctuations with the one experimentally measured on the macroscopic scale. In this study, we perform a large-scale molecular dynamics (MD) simulation for a half-turn structure of an actin filament. We analyse its longitudinal and twisting Brownian motions in equilibrium and evaluated its apparent extensional and torsional stiffness on the nanosecond scale. Upon increasing the sampling-window durations for analysis, the apparent stiffness gradually decreases and exhibits a trend to converge to a value that is close to the experimental value. This suggests that by extrapolating the data obtained in the MD analysis, we can estimate the experimentally determined stiffness on the microsecond to millisecond scales. For shorter temporal scales, the apparent stiffness is larger than experimental values, indicating that fast, local motions of the molecular structure are dominant. To quantify the local structural changes within the filament on the nanosecond scale and investigate the molecular mechanisms, such as the binding of the actin-regulatory proteins to the filaments, it is preferable to analyse the mechanical behaviour on the nanometre and nanosecond scales using MD simulation.     

Long-term molecular dynamics simulation of copper azurin: structure, dynamics and functionality

A long-term molecular dynamics simulation (1.1 ns), at 300 K, of fully hydrated azurin has been performed to put into relationship the protein dynamics to functional properties with particular attention to those structural elements involved in the electron transfer process. A detailed analysis of the root mean square deviations and fluctuations and of the intraprotein H-bonding pattern has allowed us to demonstrate that a rigid arrangement of the beta-stranded protein skeleton is maintained during the simulation run, while a large mobility is registered in the solvent-exposed connecting regions (turns) and in the alpha-helix. Moreover, the structural elements, likely involved in the electron transfer path, show a stable H-bonding arrangement and low fluctuations. Analysis of the dynamical cross-correlation map has revealed the existence of correlated motions among residues connected by hydrogen bonds and of correlated and anti-correlated motions between regions which are supposed to be involved in the functional process, namely the hydrophobic patch and the regions close to the copper reaction center. The results are briefly discussed also in connection to the current through-bond tunneling model for the electron transfer process. Finally, a comparison with the structural and the dynamical behaviour of plastocyanin, whose structure and functional role are very similar to those of azurin, has been performed.     

Collective dynamics of EcoRI-DNA complex by elastic network model and molecular dynamics simulations

Anisotropic network model (ANM) is used to analyze the collective motions of restriction enzyme EcoRI in free form and in complex with DNA. For comparison, three independent molecular dynamics (MD) simulations, each of 1.5 ns duration, are also performed for the EcoRI-DNA complex in explicit water. Although high mobility (equilibrium fluctuations) of inner and outer loops that surround the DNA is consistent in both methods and experiments, MD runs sample different conformational subspaces from which reliable collective dynamics cannot be extracted. However, ANM employed on different conformations from MD simulations indicates very similar collective motions. The stems of the inner loops are quite immobile even in the free enzyme and form a large, almost fixed, pocket for DNA binding. As a result, the residues that make specific and non-specific interactions with the DNA exhibit very low fluctuations in the free enzyme. The vibrational entropy difference between the EcoRI complex and free protein + unkinked DNA is positive (favorable), which may partially counteract the unfavorable enthalpy difference of DNA kink formation. Dynamic domains in EcoRI complex and cross-correlations between residue fluctuations indicate possible means of communication between the distal active sites.     

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure–function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca²⁺ removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca²⁺ removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein–ligand binding, including the concept of the free energy landscape (FEL) of the protein–solvent system, how the ruggedness and variability of FEL determine protein’s dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth.     

Regulatory phosphorylation of cyclin-dependent kinase 2: insights from molecular dynamics simulations

The structures of fully active cyclin-dependent kinase-2 (CDK2) complexed with ATP and peptide substrate, CDK2 after the catalytic reaction, and CDK2 inhibited by phosphorylation at Thr14/Tyr15 were studied using molecular dynamics (MD) simulations. The structural details of the CDK2 catalytic site and CDK2 substrate binding box were described. Comparison of MD simulations of inhibited complexes of CDK2 was used to help understand the role of inhibitory phosphorylation at Thr14/Tyr15. Phosphorylation at Thr14/Tyr15 causes ATP misalignment for the phosphate-group transfer, changes in the Mg²⁺ coordination sphere, and changes in the H-bond network formed by CDK2 catalytic residues (Asp127, Lys129, Asn132). The inhibitory phosphorylation causes the G-loop to shift from the ATP binding site, which leads to opening of the CDK2 substrate binding box, thus probably weakening substrate binding. All these effects explain the decrease in kinase activity observed after inhibitory phosphorylation at Thr14/Tyr15 in the G-loop. Interaction of the peptide substrate, and the phosphorylated peptide product, with CDK2 was also studied and compared. These results broaden hypotheses drawn from our previous MD studies as to why a basic residue (Arg/Lys) is preferred at the P₊₂ substrate position. Figure View of the substrate binding site of the fully active cyclin-dependent kinase-2 (CDK2) (pT160-CDK2/cyclin A/ATP). The pThr160 activation site is located in the T-loop (yellow secondary structure). The G-loop, which partly forms the ATP binding site, is shown in blue. The Thr14 and Tyr15 inhibitory phosphorylation sites located in the G-loop are shown in licorice representation     

Conformational regulation of the EGFR kinase core by the juxtamembrane and C-terminal tail: a molecular dynamics study

The catalytic domain of epidermal growth factor receptor (EGFR) is activated by dimerization, which requires allosteric coupling between distal dimerization and catalytic sites. Although crystal structures of EGFR kinases, solved in various conformational states, have provided important insights into EGFR activation by dimerization, the atomic details of how dimerization signals are dynamically coupled to catalytic regions of the kinase core are not fully understood. In this study, we have performed unrestrained and targeted molecular dynamics simulations on the active and inactive states of EGFR, followed by principal component analysis on the simulated trajectories, to identify correlated motions in the EGFR kinase domain upon dimerization. Our analysis reveals that the conformational changes associated with the catalytic functions of the kinase core are highly correlated with motions in the juxtamembrane (JM) and C-terminal tail, two flexible structural elements that play an active role in EGFR kinase activation and dimerization. In particular, the opening and closing of the ATP binding lobe relative to the substrate binding lobe is highly correlated with motions in the JM and C-terminal tail, suggesting that ATP and substrate binding can be coordinated with dimerization through conformational changes in the JM and C-terminal tail. Our study pinpoints key residues involved in this conformational coupling, and provides new insights into the role of the JM and C-terminal tail segments in EGFR kinase functions.     

Relating molecular flexibility to function: a case study of tubulin

Microtubules (MT), along with a variety of associated motor proteins, are involved in a range of cellular functions including vesicle movement, chromosome segregation, and cell motility. MTs are assemblies of heterodimeric proteins, alpha beta-tubulins, the structure of which has been determined by electron crystallography of zinc-induced, pacilitaxel-stabilized tubulin sheets. These data provide a basis for examining relationships between structural features and protein function. Here, we study the fluctuation dynamics of the tubulin dimer with the aim of elucidating its functional motions relevant to substrate binding, polymerization/depolymerization and MT assembly. A coarse-grained model, harmonically constrained according to the crystal structure, is used to explore the global dynamics of the dimer. Our results identify six regions of collective motion, comprised of structurally close but discontinuous sequence fragments, observed only in the dimeric form, dimerization being a prerequisite for domain identification. Boundaries between regions of collective motions appear to act as linkages, found primarily within secondary-structure elements that lack sequence conservation, but are located at minima in the fluctuation curve, at positions of hydrophobic residues. Residue fluctuations within these domains identify the most mobile regions as loops involved in recognition of the adjacent regions. The least mobile regions are associated with nucleotide binding sites where lethal mutations occur. The functional coupling of motions between and within regions identifies three global motions: torsional and wobbling movements, en bloc, between the alpha- and beta-tubulin monomers, and stretching longitudinally. Further analysis finds the antitumor drug pacilitaxel (TaxotereR) to reduce flexibility in the M loop of the beta-tubulin monomer; an effect that may contribute to tightening lateral interactions between protofilaments assembled into MTs. Our analysis provides insights into relationships between intramolecular tubulin movements of MT organization and function.     

Cold-active enzymes studied by comparative molecular dynamics simulation

Enzymes from cold-adapted species are significantly more active at low temperatures, even those close to zero Celsius, but the rationale of this adaptation is complex and relatively poorly understood. It is commonly stated that there is a relationship between the flexibility of an enzyme and its catalytic activity at low temperature. This paper gives the results of a study using molecular dynamics simulations performed for five pairs of enzymes, each pair comprising a cold-active enzyme plus its mesophilic or thermophilic counterpart. The enzyme pairs included α-amylase, citrate synthase, malate dehydrogenase, alkaline protease and xylanase. Numerous sites with elevated flexibility were observed in all enzymes; however, differences in flexibilities were not striking. Nevertheless, amino acid residues common in both enzymes of a pair (not present in insertions of a structure alignment) are generally more flexible in the cold-active enzymes. The further application of principle component analysis to the protein dynamics revealed that there are differences in the rate and/or extent of opening and closing of the active sites. The results indicate that protein dynamics play an important role in catalytic processes where structural rearrangements, such as those required for active site access by substrate, are involved. They also support the notion that cold adaptation may have evolved by selective changes in regions of enzyme structure rather than in global change to the whole protein. Figure Collective motions in C^α atoms of the active site of cold-active xylanase Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Flexibility of prolyl oligopeptidase: molecular dynamics and molecular framework analysis of the potential substrate pathways

The flexibility of prolyl oligopeptidase has been investigated using molecular dynamics (MD) and molecular framework approaches to delineate the route of the substrate to the active site. The selectivity of the enzyme is mediated by a seven-bladed beta-propeller that in the crystal structure does not indicate the possible passage for the substrate to the catalytic center. Its open topology however, could allow the blades to move apart and let the substrate into the large central cavity. Flexibility analysis of prolyl oligopeptidase structure using the FIRST (Floppy Inclusion and Rigid Substructure Topology) approach and the atomic fluctuations derived from MD simulations demonstrated the rigidity of the propeller domain, which does not permit the substrate to approach the active site through this domain. Instead, a smaller tunnel at the inter-domain region comprising the highly flexible N-terminal segment of the peptidase domain and a facing hydrophilic loop from the propeller (residues 192-205) was identified by cross-correlation analysis and essential dynamics as the only potential pathway for the substrate. The functional importance of the flexible loop has been also verified by kinetic analysis of the enzyme with a split loop. Catalytic effect of engineered disulfide bridges was rationalized by characterizing the concerted motions of the two domains.     

Activation and inhibition of cyclin-dependent kinase-2 by phosphorylation; a molecular dynamics study reveals the functional importance of the glycine-rich loop

Nanoseconds long molecular dynamics (MD) trajectories of differently active complexes of human cyclin-dependent kinase 2 (inactive CDK2/ATP, semiactive CDK2/Cyclin A/ATP, fully active pT160-CDK2/Cyclin A/ATP, inhibited pT14-; pY15-; and pT14,pY15,pT160-CDK2/Cyclin A/ATP) were compared. The MD simulations results of CDK2 inhibition by phosphorylation at T14 and/or Y15 sites provide insight into the structural aspects of CDK2 deactivation. The inhibitory sites are localized in the glycine-rich loop (G-loop) positioned opposite the activation T-loop. Phosphorylation of T14 and both inhibitory sites T14 and Y15 together causes ATP misalignment for phosphorylation and G-loop conformational change. This conformational change leads to the opening of the CDK2 substrate binding box. The phosphorylated Y15 residue negatively affects substrate binding or its correct alignment for ATP terminal phospho-group transfer to the CDK2 substrate. The MD simulations of the CDK2 activation process provide results in agreement with previous X-ray data.     

Role of conformational fluctuations in the enzymatic reaction of HIV-1 protease

The emergence of compensatory drug-resistant mutations in HIV-1 protease challenges the common view of the reaction mechanism of this enzyme. Here, we address this issue by performing classical and ab initio molecular dynamics simulations (MD) on a complex between the enzyme and a peptide substrate. The classical MD calculation reveals large-scale protein motions involving the flaps and the cantilever. These motions modulate the conformational properties of the substrate at the cleavage site. The ab initio calculations show in turn that substrate motion modulates the activation free energy barrier of the enzymatic reaction dramatically. Thus, the catalytic power of the enzyme does not arise from the presence of a pre-organized active site but from the protein mechanical fluctuations. The implications of this finding for the emergence of drug-resistance are discussed.     

Functional stability and structural transitions of Kallikrein: spectroscopic and molecular dynamics studies

Kallikrein, a physiologically vital serine protease, was investigated for its functional and conformational transitions during chemical (organic solvents, Gdn-HCl), thermal, and pH induced denaturation using biochemical and biophysical techniques and molecular dynamics (MD) simulations approach. The enzyme was exceptionally stable in isopropanol and ethanol showing 110% and 75% activity, respectively, after 96 h, showed moderate tolerance in acetonitrile (45% activity after 72 h) and much lower stability in methanol (40% activity after 24 h) (all the solvents [90% v/v]). Far UV CD and fluorescence spectra indicated apparent reduction in compactness of KLKp structure in isopropanol system. MD simulation studies of the enzyme in isopropanol revealed (1) minimal deviation of the structure from native state (2) marginal increase in radius of gyration and solvent accessible surface area (SASA) of the protein and the active site, and (3) loss of density barrier at the active site possibly leading to increased accessibility of substrate to catalytic triad as compared to methanol and acetonitrile. Although kallikrein was structurally stable up to 90 °C as indicated by secondary structure monitoring, it was functionally stable only up to 45 °C, implicating thermolabile active site geometry. In GdnHCl [1.0 M], 75% of the activity of KLKp was retained after incubation for 4 h, indicating its denaturant tolerance. A molten globule-like structure of KLKp formed at pH 1.0 was more thermostable and exhibited interesting structural transitions in organic solvents. The above results provide deeper understanding of functional and structural stability of the serine proteases at molecular level.