Dissecting substrate recognition by thrombin using the inactive mutant S195A

The catalytically inactive mutant S195A was used to study the interaction of thrombin with substrates under equilibrium conditions. By monitoring changes in intrinsic fluorescence, we measured dissociation constants for a variety of synthetic substrates, PAR peptides and the inhibitor PPACK. The S195A mutant retains the Na⁺-binding properties of the wild type, and substrate binding to the mutant is enhanced by the presence of Na⁺. Temperature dependence studies allowed calculation of the thermodynamic parameters of substrate binding at the active site and showed a negligible ΔC_p. Titration of synthetic substrates carrying substitutions at the P1–P3 positions revealed energetics consistent with the specificity hierarchy identified in hydrolysis by the wild type. Titration with PAR peptides, which interact with both the active site and exosite I of thrombin, also showed consistency with the results obtained with the wild type at steady state. These findings demonstrate that inactive mutants of enzymes make it possible to dissect the equilibrium components linked to substrate binding and complement information on the kinetic properties of the wild type.     

Rigidification of a flexible protease inhibitor variant upon binding to trypsin

The Tyr35-->Gly replacement in bovine pancreatic trypsin inhibitor (BPTI) has previously been shown to dramatically enhance the flexibility of the trypsin-binding region of the free inhibitor and to destabilize the interaction with the protease by about 3 kcal/mol. The effects of this replacement on the enzyme-inhibitor interaction were further studied here by X-ray crystallography and isothermal titration calorimetry (ITC). The co-crystal structure of Y35G BPTI bound to trypsin was determined using 1.65 A resolution X-ray diffraction data collected from cryopreserved crystals, and a new structure of the complex with wild-type BPTI under the same conditions was determined using 1.62 A data. These structures reveal that, in contrast to the free protein, Y35G BPTI adopts a conformation nearly identical with that of the wild-type protein, with a water-filled cavity in place of the missing Tyr side-chain. The crystallographic temperature factors for the two complexes indicate that the mutant inhibitor is nearly as rigid as the wild-type protein when bound to trypsin. Calorimetric measurements show that the change in enthalpy upon dissociation of the complex is 2.5 kcal/mol less favorable for the complex containing Y35G BPTI than for the complex with the wild-type inhibitor. Thus, the destabilization of the complex resulting from the Y35G replacement is due to a more favorable change in entropy upon dissociation. The heat capacity changes for dissociation of the mutant and wild-type complexes were very similar, suggesting that the entropic effects probably do not arise from solvation effects, but are more likely due to an increase in protein conformational entropy upon dissociation of the mutant inhibitor. These results define the biophysical role of a highly conserved core residue located outside of a protein-binding interface, demonstrating that Tyr35 has little impact on the trypsin-bound BPTI structure and acts primarily to define the structure of the free protein so as to maximize binding affinity.     

Partially folded bovine pancreatic trypsin inhibitor analogues attain fully native structures when co-crystallized with S195A rat trypsin

Crystal structures, at 1.7 Å resolution, were solved for complexes between each of two chemically synthesized partially folded analogues of bovine pancreatic trypsin inhibitor (BPTI) with the proteolytically inactive rat trypsin mutant S195A. The BPTI analogue termed [14-38]_Abu retains only the disulfide bond between Cys14 and Cys38, while Cys5, Cys30, Cys51, and Cys55 are replaced by isosteric α-amino-n-butyric acid residues. The analogue K26P,A27D[14-38]_Abu contains two further replacements, by statistically favored residues, in the type I β-turn that has been suggested to be a main site for initiation of BPTI folding. As a control, the structure of the complex between S195A trypsin and wild-type BPTI was also solved. Despite significant differences in the degree of structure detected among these three BPTIs in solution by several biophysical techniques, their tertiary folds once bound to S195A trypsin in a crystalline lattice are essentially superimposable.     

A structure of potentially active and inactive genes of chicken erythrocyte chromatin upon decondensation.

In the presence of 3 mM MgCl2 DNase I cleavage of bulk, globin and ovalbumin gene chromatin in chicken erythrocyte nuclei generates fragments which are multiples of a double-nucleosome repeat. However, in addition to the dinucleosomal periodicity beta-globin gene chromatin was fragmented into multiples of a 100 b.p. interval which is characteristic for partially unfolded chromatin. This distinction correlates with higher sensitivity of beta-globin domain to DNase I and DNase II as compared to the inactive ovalbumin gene. At 0.7 mM MgCl2 where these DNases fragment bulk chromatin into series of fragments with a 100 b.p. interval, the difference in digestibility of the investigated genes is dramatically decreased. When chromatin has been decondensed by incubation of nuclei in 10 mM Tris-buffer, DNase II generates a typical nucleosomal repeat, and the differential nuclease sensitivity of the analyzed genes is not observed. The data suggest that higher nuclease sensitivity of potentially active genes is due to irregularities in higher order chromatin structure.     

Conformational dynamics of threonine 195 and the S1 subsite in functional trypsin variants

Replacing the catalytic serine in trypsin with threonine (S195T variant) leads to a nearly complete loss of catalytic activity, which can be partially restored by eliminating the C42-C58 disulfide bond. The 0.69 μs of combined explicit solvent molecular dynamics (MD) simulations revealed continuous rearrangement of T195 with different conformational preferences between five trypsin variants tested. Among three conformational families observed for the T195 residue, one showed the T195 hydroxyl in a conformation analogous to that of the serine residue in wild-type trypsin, positioning the hydroxyl oxygen atom for attack on the carbonyl carbon of the peptide substrate. MD simulations demonstrated that this conformation was more populated for the C42A/C58V/S195T and C42A/C58A/S195T triple variants than for the catalytically inactive S195T variant and correlated with restored enzymatic activities for triple variants. In addition, observation of the increased motion of the S214-G219 segment in the S195T substituted variants suggested an existence of open and closed conformations for the substrate binding pocket. The closed conformation precludes access to the S1 binding site and could further reduce enzymatic activities for triple variants. Double variants with intact serine residues (C42A/C58A/S195 and C42A/C58V/S195) also showed interchange between closed and open conformations for the S214-G219 segment, but to a lesser extent than the triple variants. The increased conformational flexibility of the S1 subsite, which was not observed for the wild-type, correlated with reduced enzymatic activities and suggested a possible mode of substrate regulation for the trypsin variants tested.     

A theoretical study of the active sites of papain and S195C rat trypsin: implications for the low reactivity of mutant serine proteinases.

The serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residues, Asp...His...Ser. The hydroxyl group of serine functions as a nucleophile and the imidazole ring of histidine functions as a general acid/general base during catalysis. Similarly, the active site of the cysteine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to exist as a zwitterion (Asn...His+...Cys-) with the thiolate anion of the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilisin and thiol trypsin, which have the hybrid Asp...His...Cys triad, are almost catalytically inert. In this study, ab initio Hartree-Fock calculations have been performed on the active sites of papain and the mutant serine proteinase S195C rat trypsin. These calculations predict that the active site of papain exists predominately as a zwitterion (Cys-...His+...Asn). However, similar calculations on S195C rat trypsin demonstrate that the thiol mutant is unable to form a reactive thiolate anion prior to catalysis. Furthermore, structural comparisons between native papain and S195C rat trypsin have demonstrated that the spatial juxtapositions of the triad residues have been inverted in the serine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site-directed mutagenesis.     

Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

Metabotropic glutamate receptors (mGluRs) are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects--enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%), the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%), the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86%) and 8/9 (89%) for ArgusLab and 10/14 (71%) and 7/9 (78%) for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by photochemical isomerization of the retinal ligand--despite the extensive differences in sequences between mGluRs and rhodopsin.     

Protection of prostaglandin H synthase from trypsin upon binding of heme

The sensitivity of the apo- and holoenzyme forms of prostaglandin H synthase to trypsin have been investigated. Both the cyclooxygenase and peroxidase activities associated with the synthase were rapidly lost from the apoenzyme when incubated with trypsin. However, both activities were resistant to trypsin in the holoenzyme, suggesting that some structural change accompanies heme binding. Inactive protein present in some holoenzyme preparations, although indistinguishable from the synthase subunit by polyacrylamide gel electrophoresis, was also hydrolyzed by trypsin.     

Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin

Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.     

Subsites of trypsin active site favor catalysis or substrate binding.

Enzymes enhance chemical reaction rates by lowering the activation energy, the energy barrier of the reaction leading to products. This occurs because enzymes bind the high-energy intermediate of the reaction (the transition state) more strongly than the substrate. We studied details of this process by determining the substrate binding energy (DeltaG(s), calculated from K(m) values) and the activation energy (DeltaG(T), determined from k(cat)/K(m) values) for the trypsin-catalyzed hydrolysis of oligopeptides. Plots of DeltaG(T) versus DeltaG(s) for oligopeptides with 15 amino acid replacements at each of the positions P(1)', P(1), and P(2) were straight lines, as predicted by a derived equation that relates DeltaG(T) and DeltaG(s). The data led to the conclusion that the trypsin active site has subsites that bind moieties of substrate and of transition state in characteristic ratios, whichever substrate is used. This was unexpected and means that each subsite characteristically favors substrate binding or catalysis.     

Use of psoralen monoadducts to compare the structure of 16S rRNA in active and inactive 30S ribosomal subunits

E. coli 30S ribosomes in the inactive conformation were irradiated at 390 nm in the presence of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT). This produces monoadducts in which AMT is attached to only one strand of an RNA duplex region. After unbound AMT was removed, some ribosomes were activated and then subjected to 360 nm irradiation; others were reirradiated without activation. Electron microscopic examination of 16S rRNA extracted from these two samples showed covalent rRNA loops indicative of rRNA crosslinks. The general pattern of loops closely matched that seen previously after direct psoralen crosslinking of 30S particles. However, the frequency of occurrence of one major class of loops formed by crosslinks between residues near position 500 and the 3' end was substantially lower for the activated samples, implying that the structure of the 16S rRNA in active and inactive 30S particles is different.     

Inhibitor binding to active and inactive CDK2: the crystal structure of CDK2-cyclin A/indirubin-5-sulphonate

BACKGROUND: Cyclin-dependent kinase 2 (CDK2) is an important target for structure-based design of antitumor agents. Monomeric CDK2 is inactive. Activation requires rearrangements to key structural elements of the enzyme's active site, which accompany cyclin binding and phosphorylation. To assess the validity of using monomeric CDK2 as a model for the active kinase in structure-based drug design, we have solved the structure of the inhibitor indirubin-5-sulphonate (E226) complexed with phospho-CDK2-cyclin A and compared it with the structure of E226 bound to inactive, monomeric CDK2. RESULTS: Activation of monomeric CDK2 leads to a rotation of its N-terminal domain relative to the C-terminal lobe. The accompanying change in position of E226 follows that of the N-terminal domain, and its interactions with residues forming part of the adenine binding pocket are conserved. The environment of the ATP-ribose site, not explored by E226, is significantly different in the binary complex compared to the monomeric complex due to movement of the glycine loop. Conformational changes also result in subtle differences in hydrogen bonding and electrostatic interactions between E226's sulphonate and CDK2's phosphate binding site. Affinities calculated by LUDI for the interaction of E226 with active or inactive CDK2 differ by a factor of approximately ten. CONCLUSIONS: The accuracy of monomeric CDK2 as an inhibitor design template is restricted to the adenine binding site. The general flexibility observed for the glycine loop and subtle changes to the phosphate binding site suggest a need to study interactions between inhibitors and active CDK2 in structure-based drug design programs.     

Induced resistance of trypsin to sodium dodecylsulfate upon complex formation with trypsin inhibitor

The stabilities of trypsin and soybean trypsin inhibitor in sodium dodecylsulfate (SDS) were examined by SDS-polyacrylamide gel electrophoresis (PAGE). Both samples contained several bands, all of which migrated to positions corresponding to the appropriate molecular weight or less, even when the samples were unheated, suggesting that both the trypsin and trypsin inhibitor are susceptible to SDS-induced denaturation. When they were mixed together prior to addition of SDS-PAGE sample buffer (1% SDS), a new smearing band appeared which corresponded to a molecular weight of around 46,000, suggesting that these proteins form a stable complex in SDS. This was confirmed by electroblotting and sequence analysis, which indicated that this band contains both the trypsin and inhibitor sequences. At a fixed concentration of the inhibitor, increasing concentrations of the trypsin resulted in an increase in the intensity of the complex band. When the mixture was heated for 10 min in 1% SDS, the complex band disappeared in a temperature-dependent manner. The melting temperature determined under the experimental conditions used was about 35|MoC. Similar results were obtained with Bowman-Birk trypsin inhibitor, except that the complex with the above inhibitor had a higher melting temperature, around 41|MoC, suggesting that the Bowman-Birk inhibitor/trypsin complex is more stable than the soybean inhibitor/trypsin complex.     

A method of active conformation search based on active and inactive analogues and its application to allylamine antimycotics

A new program ACSBAIA (Active Conformation Search Based on Active and Inactive Analogues) for determination of the active conformations was developed based on the rationales that specific functional groups of active analogues could reach and interact with the active site of target receptor by means of the change of conformations, but that of inactive analogues could not interact with the active site owing to conformational restriction. The program consisted of 4 sub-programs: conformation sampling system, active conformation constraint system, inactive conformation exclusion system, and activity prediction system. Pharmacophoric conformation of allylamine antimycotics was studied by this method. Activities of 2 analogues were predicted and tested. The results suggested that the method was scientific and practical. The application of this method was not restricted by the three-dimensional structural knowledge of target receptor. In the absence of structural information about the receptor, the method was     

A method of active conformation search based on active and inactive analogues and its application to allylamine antimycotics

A new program ACSBAIA (Active Conformation Search Based on Active and Inactive Analogues) for determination of the active conformations was developed based on the rationales that specific functional groups of active analogues could reach and interact with the active site of target receptor by means of the change of conformations, but that of inactive analogues could not interact with the active site owing to conformational restriction. The program consisted of 4 sub-programs: conformation sampling system, active conformation constraint system, inactive conformation exclusion system, and activity prediction system. Pharmacophoric conformation of allylamine antimycotics was studied by this method. Activities of 2 analogues were predicted and tested. The results suggested that the method was scientific and practical. The application of this method was not restricted by the three-dimensional structural knowledge of target receptor. In the absence of structural information about the receptor, the method was particularly applicable.     

Free energy calculations show that acidic P1 variants undergo large pKa shifts upon binding to trypsin

Serine proteinases and their protein inhibitors belong to one of the most comprehensively studied models of protein-protein interactions. It is well established that the narrow trypsin specificity is caused by the presence of a negatively charged aspartate at the specificity pocket. X-ray crystallography as well as association measurements revealed, surprisingly, that BPTI with glutamatic acid as the primary binding (P1) residue was able to bind to trypsin. Previous free energy calculations showed that there was a substantially unfavorable binding free energy associated with accommodation of ionized P1 Glu at the S1-site of trypsin. In this study, the binding of P1 Glu to trypsin has been systematically investigated in terms of the protonation states of P1 Glu and Asp189, the orientation of Gln192, as well as the possible presence of counterions using the linear interaction energy (LIE) approach and the free energy perturbation (FEP) method. Twenty-four conceivable binding arrangements were evaluated and quantitative agreement with experiments is obtained when the P1 Glu binds in its protonated from. The results suggest that P1 Glu is one of the variants of BPTI that inhibit trypsin strongest at low pH, contrary to the specificity profile of trypsin, suggesting a new regulation mechanism of trypsin-like enzymes.