Conformational stability of serine proteinase inhibitor from the sea anemone Heteractis crispa

The influence of different environmental values of the pH and temperature on the spatial organization of serine proteinase inhibitor from the sea anemone Heteractis crispa (=Radianthus macrodactylus) on the level of tertiary and secondary structure was studied by CD spectroscopy. The molecule InhVJ was shown to possess a high conformational thermo- and pH-stability. We determined the point of conformational thermotransition of polypeptide (70 degrees C) after which the molecule gets denaturational stable state with conservation of 80% proteinase inhibitory activity. The significant partial reversible changes of molecule spatial organization were established to occur at the level of tertiary structure in the process of acid-base titration in the range of pH 11.0-13.0. This can be explained by of ionization of tyrosine residues. The molecule InhVJ is conformationally stable at the low pH values (2.0). The quenching of tyrosine residues by acrylamide showed that two of these residues are accessible to the quencher in full, while the third part is available.     

From determination of amino acid residue conformation to reconstruction of the spatial structure of proteins (from the data of nuclear Overhauser enhancement spectroscopy

A new approach to the calculation of the spatial protein structure based on the joint utilization of the theoretical conformational analysis method and nuclear Overhauser enhancement (NOE) spectroscopy data is proposed and verified. The quality in determining various molecule structural parameters is estimated in terms of the expected NOE spectral parameters derived from the X-ray analysis data of the avian pancreatic polypeptide. The proposed approach is shown to correctly determine such structural parameters of protein molecules as local amino acid residue conformations, reciprocal spatial orientation of the C alpha atoms neighbouring along amino acid sequence and reapproached segments of the polypeptide chain. Spatially remote molecule fragments are mainly responsible for the error in determining structural parameters.     

Global and Local Structural Properties of the Principal Neutralizing Determinant of the HIV-1 Envelope Protein gp120

Abstract

The model of spatial structure for the principal neutralizing determinant (PND) of the HIV-1 envelope protein gpl20 is proposed in terms of two-dimensional nuclear Overhauser effect (NOE) spectroscopy data. To build the model, the NMR-based theoretical conformational analysis of synthetic PND peptides of length 40, 24, and 12 residues is carried out. The modeling of the molecular spatial structures is performed by a new approach to research of conformationally mobile peptides using the algorithms of the restrained molecular mechanics method developed earlier. The following major conclusions are made based on the analysis of the simulated peptide conformations: i) there is not unique PND structure in solution, ii) there are seven different PND structures each of which agrees with the experimental data and stereochemical criteria used in computing its spatial model, iii) the PND is characterized by irregular conformation containing a number of reverse turns, iv) all of the selected conformations are conserved in the Gly-Pro-Gly-Arg-Ala-Phe stretch, the most provable viral immunodominant epitope. These data allow to suppose that binding properties of this site are determined by the structural motif which forms the conformation of a double β-turn and appears common for all hexapeptide structures.     

Global and local structural properties of the principal neutralizing determinant of the HIV-1 envelope protein gp120

The model of spatial structure for the principal neutralizing determinant (PND) of the HIV-1 envelope protein gp120 is proposed in terms of two-dimensional nuclear Overhauser effect (NOE) spectroscopy data. To build the model, the NMR-based theoretical conformational analysis of synthetic PND peptides of length 40, 24, and 12 residues is carried out. The modeling of the molecular spatial structures is performed by a new approach to research of conformationally mobile peptides using the algorithms of the restrained molecular mechanics method developed earlier. The following major conclusions are made based on the analysis of the simulated peptide conformations: i) there is not unique PND structure in solution, ii) there are seven different PND structures each of which agrees with the experimental data and stereochemical criteria used in computing its spatial model, iii) the PND is characterized by irregular conformation containing a number of reverse turns, iv) all of the selected conformations are conserved in the Gly-Pro-Gly-Arg-Ala-Phe stretch, the most probable viral immunodominant epitope. These data allow to suppose that binding properties of this site are determined by the structural motif which forms the conformation of a double beta-turn and appears common for all hexapeptide structures.     

Conformational-functional relationships of tetragastrin analogs]

Sets of low-energy backbone conformations of the active tetragastrin analogue Boc-Trp-Leu-Asp-Phe-NH2 and two competitive antagonists Boc-Trp-Leu psi (CH2NH)-Asp-Phe-NH2 and Boc-Trp-Leu-Asp-O-CH2-CH2-C6H5 were obtained using theoretical conformational analysis methods. Groups of the conformations were selected for the three analogues, allowing a spatial matching of Trp, Asp and Phe residues responsible for the gastrin receptor binding. Three conformations possessing the lowest energies among the geometrically similar structures of these three peptides are suggested as a model for the "receptor-bound" conformations of these analogues. Backbone spatial folding resembling an alpha-helix turn is characteristic of these conformations. The correspondence of the proposed model to the available data on structure--activity relationships for tetragastrin analogues is discussed. Orientations of the putative receptor-bound conformations in a "water--lypophylic medium" two-phase system were investigated.     

Method of determining protein conformations by the two-dimensional nuclear Overhauser enhancement spectroscopy data

A method is suggested to determine the most probable values of the angles phi, psi of the protein backbone by the data on the availability and absence of d connectivities in the two-dimensional nuclear Overhauser enhancement spectra. In view of this, the dependences of the proton-proton distances in dipeptide units of L-amino acid residues on the dihedral angles phi, psi, chi1 are considered and the conformational states of amino acid residues of the proteins with the known spatial structure are analysed statistically. The potentialities of the method are assessed with the aid of model spectral nuclear magnetic resonance (NMR) parameters obtained from the X-ray data for the bovine pancreatic trypsin inhibitor and avian pancreatic polypeptide. It is shown that the developed procedure of structural interpretation of the NMR data allows one to correctly reproduce the local conformation of the protein backbone. The obtained backbone conformation may serve as a starting point to build and refine molecular three-dimensional structure.     

Mechanism of action of aspartyl proteinases. VI. Nonvalent enzyme-inhibitory and enzyme-substrate complexes of the aspartyl proteinase rhizopus pepsin

The structure of a complex of rhizopuspepsin, a fungal aspartyl protease, with Pro1-Phe2-His3-Phe4-psi[CH2-NH]-Phe5-Val6, its substrate-like inhibitor, was calculated by theoretical conformational analysis. The search for energetically favorable conformational variants of the ligand structure was based on the fragmental approach using the dynamic library of peptide fragments, which were successively extended in the potential field of the protein. The root-mean-square deviation of atom positions in the calculated and experimental inhibitor conformations was 0.56 A. A similar approach was used to model a noncovalent complex of rhizopuspepsin with Pro1-Phe2-His3-Lys4-Phe5-Val6, its specific substrate. As a result, two isoenergetic structures of the complex with different arrangements of the cleavable peptide group and a nucleophilic water molecule were calculated. The possibility of the achieving each of these conformations during the catalytic act is considered. It is shown that there are no structural prerequisites for the distortion of the cleavable bond in the active site of the enzyme. On the basis of the resulting structural data, the assumption was made that Asp35 may be protonated at a late stage of formation of the tetrahedral intermediate rather than at the basic state of the complex.     

A concerted structural transition in the plasminogen activator inhibitor-1 mechanism of inhibition

The inhibition mechanism of serpins requires a change in structure to entrap the target proteinase as a stable acyl-enzyme complex. Although it has generally been assumed that reactive center loop insertion and associated conformational change proceeds in a concerted manner, this has not been demonstrated directly. Through the substitution of tryptophan with 7-azatryptophan and an analysis of transient reaction kinetics, we have described the formation of an inhibited serpin-proteinase complex as a single concerted transition of the serpin structure. Replacement of the four tryptophans of plasminogen activator inhibitor type-1 (PAI-1) with the spectrally unique analogue 7-azatryptophan permitted observations of conformational changes in the serpin but not those of the proteinase. Formation of covalent acyl-enzyme complexes, but not noncovalent Michaelis complexes, with tissue-type plasminogen activator (t-PA) or urokinase (u-PA) resulted in rapid decreases of fluorescence coinciding with insertion of the reactive center loop and expansion of beta-sheet A. Insertion of an octapeptide consisting of the P14-P7 residues of the reactive center loop into beta-sheet A produced the same conformational change in serpin structure measured by 7-azatryptophan fluorescence, suggesting that introduction of the proximal loop residues induces the structural rearrangement of the serpin molecule. The atom specific modification of the tryptophan indole rings through analogue substitution produced a proteinase specific effect on function. The reduced inhibitory activity of PAI-1 against t-PA but not u-PA suggested that the mechanism of loop insertion is sensitive to the intramolecular interactions of one or more tryptophan residues.     

Latent S49P neuroserpin forms polymers in the dementia familial encephalopathy with neuroserpin inclusion bodies

The serpinopathies result from conformational transitions in members of the serine proteinase inhibitor superfamily with aberrant tissue deposition or loss of function. They are typified by mutants of neuroserpin that are retained within the endoplasmic reticulum of neurons as ordered polymers in association with dementia. We show here that the S49P mutant of neuroserpin that causes the dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) forms a latent species in vitro and in vivo in addition to the formation of polymers. Latent neuroserpin is thermostable and inactive as a proteinase inhibitor, but activity can be restored by refolding. Strikingly, latent S49P neuroserpin is unlike any other latent serine proteinase inhibitor (serpin) in that it spontaneously forms polymers under physiological conditions. These data provide an alternative method for the inactivation of mutant neuroserpin as a proteinase inhibitor in FENIB and demonstrate a second pathway for the formation of intracellular polymers in association with disease.     

Solution Structure of the Squash Aspartic Acid Proteinase Inhibitor (SQAPI) and Mutational Analysis of Pepsin Inhibition

The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel β-sheet gripping an α-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting β-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S′ side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp³²–Asp²¹⁵ diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin.     

Crystal and molecular structure of the serine proteinase inhibitor CI-2 from barley seeds

Chymotrypsin inhibitor 2 (CI-2), a serine proteinase inhibitor from barley seeds, has been crystallized and its three-dimensional structure determined at 2.0-A resolution by the molecular replacement method. The structure has been refined by restrained-parameter least-squares methods to a crystallographic R factor (= sigma parallel Fo magnitude of-Fo parallel/sigma magnitude of Fo) o of 0.198. CI-2 is a member of the potato inhibitor 1 family. It lacks the characteristic stabilizing disulfide bonds of most other members of serine proteinase inhibitor families. The body of CI-2 shows few conformational changes between the free inhibitor and the previously reported structure of CI-2 in complex with subtilisin Novo [McPhalen, C.A., Svendsen, I., Jonassen, I., & James, M.N.G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7242-7246]. However, the reactive site loop has some significant conformational differences between the free inhibitor and its complexed form. The residues in this segment of polypeptide exhibit relatively large thermal motion parameters and some disorder in the uncomplexed form of the inhibitor. The reactive site bond is between Met-59I and Glu-60I in the consecutive sequential numbering of CI-2 (Met-60-Glu-61 according to the alignment of Svendsen et al. [Svendsen, I., Hejgaard, J., & Chavan, J.K. (1984) Carlsberg Res. Commun. 49, 493-502]). The network of hydrogen bonds and electrostatic interactions stabilizing the conformation of the reactive site loop is much less extensive in the free than in the complexed inhibitor.     

An approach to the problem of the structuro-functional organization of natural peptides

Theory and computational scheme of three-dimensional structure and dynamic conformational properties of naturally occurring peptides are proposed basing on a known amino acid sequence. The diverse biological activity of a low-molecular peptide is shown to arise from a restricted number of preferable spatial structures which may occur under physiological conditions. Each particular function of an oligopeptide is connected to a definite spatial structure, belonging to the set of low-energy conformations from one biological activity of a peptide shift of the conformational equilibrium caused by a change of environmental conditions. This shift is provided for by specific intramolecular interactions, alternative in their nature, which stabilize a particular structure. An approach is suggested which enables to construct a synthetic analog with the predetermined physiologically active conformation, prior to all chemical and biological tests.     

Partitioning of serpin-proteinase reactions between stable inhibition and substrate cleavage is regulated by the rate of serpin reactive center loop insertion into beta-sheet A

The serpin family of serine proteinase inhibitors is a mechanistically unique class of naturally occurring proteinase inhibitors that trap target enzymes as stable covalent acyl-enzyme complexes. This mechanism appears to require both cleavage of the serpin reactive center loop (RCL) by the proteinase and a significant conformational change in the serpin structure involving rapid insertion of the RCL into the center of an existing beta-sheet, serpin beta-sheet A. The present study demonstrates that partitioning between inhibitor and substrate modes of reaction can be altered by varying either the rates of RCL insertion or deacylation using a library of serpin RCL mutants substituted in the critical P(14) hinge residue and three different proteinases. We further correlate the changes in partitioning with the actual rates of RCL insertion for several of the variants upon reaction with the different proteinases as determined by fluorescence spectroscopy of specific RCL-labeled inhibitor mutants. These data demonstrate that the serpin mechanism follows a branched pathway, and that the formation of a stable inhibited complex is dependent upon both the rate of the RCL conformational change and the rate of enzyme deacylation.     

Method of Determining Protein Conformations by the Two-Dimensional Nuclear Overhauser Enhancement Spectroscopy Data

Abstract

A method is suggested to determine the most probable values of the angles Φ, Ψ of the protein backbone by the data on the availability and absence of d connectivities in the two-dimensional nuclear Overhauser enhancement spectra. In view of this, the dependences of the protonproton distances in dipeptide units of L-amino acid residues on the dihedral angles Φ, Ψ, χ, are considered and the conformational states of amino acid residues of the proteins with the known spatial structure are analysed statistically. The potentialities of the method are assessed with the aid of model spectral nuclear magnetic resonance (NMR) parameters obtained from the X-ray data for the bovine pancreatic trypsin inhibitor and avian pancreatic polypeptide.

It is shown that the developed procedure of structural interpretation of the NMR data allows one to correctly reproduce the local conformation of the protein backbone. The obtained backbone conformation may serve as a starting point to build and refine molecular three- dimensional structure.     

Conformational changes of calpain from human erythrocytes in the presence of Ca2+

Small angle x-ray scattering has been used to monitor calpain structural transitions during the activation process triggered by Ca(2+) binding. The scattering pattern of the unliganded enzyme in solution does not display any significant difference with that calculated from the crystal structure. The addition of Ca(2+) promotes the formation of large aggregates, indicating the exposure of hydrophobic patches on the surface of the protease. In contrast, Ca(2+) addition in the presence of the thiol proteinase inhibitor E64 or of the inhibitor leupeptin causes a small conformational change with no dissociation of the heterodimer. The resulting conformation appears to be slightly more extended than the unliganded form. From the comparison between ab initio models derived from our data with the crystal structure, the major observable conformational change appears to be localized at level of the L-subunit and in particular seems to confirm the mutual movement already observed by the crystallographic analysis of the dII (dIIb) and the dI (dIIa) domains creating a functional active site. This work not only provides another piece of supporting evidence for the calpain conformational change in the presence of Ca(2+), but actually constitutes the first experimental observation of this change for intact heterodimeric calpain in solution.     

Isolation and characterization of an alpha-macroglobulin from the gastropod mollusc Helix pomatia with tetrameric structure and preserved activity after methylamine treatment

A proteinase inhibitor with M(r) 697000 and 20.3% (w/w) carbohydrate was isolated from the haemolymph of the snail Helix pomatia and characterized. It was shown to have a tetrameric structure with subunits disulphide linked by two. It inhibited the activity of several types of proteinases against large substrates but not that of trypsin against N-alpha-benzoyl-DL-arginine-4-nitroanilide. This indicated a nonspecific and steric hindrance mode of inhibition. The ratio of trypsin molecules inactivated per inhibitor amounted to 1.5. This interaction led to a cleavage of the subunits into two equal fragments and to a slow to fast conformational change of the whole molecules. Experiments with 125I-labelled trypsin indicated that the proteinase had become covalently linked to one of the fragments. Heating of the inhibitor led to autolytic cleavage products but not when methylamine treated. Thiol titration after trypsin or methylamine treatment indicated the presence of one thiol ester bond per subunit. These facts are all indicative of an alpha-macroglobulin type of inhibitor. However, unlike for most of them the methylamine treatment did not induce a conformational change nor suppress its proteinase inhibitory activity. Moreover, invertebrate alpha-macroglobulins are mostly dimeric in structure but tetramers likewise do occur in Biomphalaria glabrata.     

Oxidized caprine alpha-2-macroglobulin: damaged but not completely dysfunctional

Caprine alpha-2-macroglobulin (alpha2M) is a broad-spectrum, homotetrameric proteinase inhibitor that can maximally bind a single molecule of proteinase. Inhibition of proteinases by caprine alpha2M results from a series of conformational changes that are initiated by the proteinase and results in physical sequestration of the proteinase within the closed cage-like structure of conformationally altered alpha2M. In a previous study, uric acid-generated superoxide anion was identified as one of the physiologically relevant inactivators of alpha2M S.A. Khan, F.H. Khan [Free. Radic. Res. 34 (2001) 113]. We now demonstrate that hypochlorous acid (HOCl) and, to lesser extent, hydrogen peroxide (H2O2) destroy the antiproteolytic potential of caprine alpha2M. At physiologically attainable concentration, we found that HOCl significantly compromised functional integrity of the inhibitor. High concentrations of H2O2 also partially diminished proteinase inhibitory capacity of alpha2M by a mechanism not involving formation of hydroxyl radicals. For hydrogen peroxide, catalase completely protected alpha2M activity while the ability to protect the inhibitor from HOCl-induced inactivation was limited by availability of albumin. Structure function analysis demonstrated that oxidized caprine inhibitor, unlike its human counterpart, retained its tetrameric configuration as well as its characteristic ability to undergo major conformational change upon trypsinization. It is proposed that inhibition of alpha2M activity may be due to oxidation of essential residues of the inhibitor and/or structural rearrangement of the subunits.     

Structure of a serpin-enzyme complex probed by cysteine substitutions and fluorescence spectroscopy

The x-ray crystal structure of the serpin-proteinase complex is yet to be determined. In this study we have investigated the conformational changes that take place within antitrypsin during complex formation with catalytically inactive (thrombin(S195A)) and active thrombin. Three variants of antitrypsin Pittsburgh (an effective thrombin inhibitor), each containing a unique cysteine residue (Cys(232), Cys(P3'), and Cys(313)) were covalently modified with the fluorescence probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine. The presence of the fluorescent label did not affect the structure or inhibitory activity of the serpin. We monitored the changes in the fluorescence emission spectra of each labeled serpin in the native and cleaved state, and in complex with active and inactive thrombin. These data show that the serpin undergoes conformational change upon forming a complex with either active or inactive proteinase. Steady-state fluorescence quenching measurements using potassium iodide were used to further probe the nature and extent of this conformational change. A pronounced conformational change is observed upon locking with an active proteinase; however, our data reveal that docking with the inactive proteinase thrombin(S195A) is also able to induce a conformational change in the serpin.     

pH-dependent stability of sperm whale myoglobin in water-guanidine hydrochloride solutions

An experimental-theoretical approach for the elucidation of protein stability is proposed. The theoretical prediction of pH-dependent protein stability is based on the macroscopic electrostatic model for calculation of the pH-dependent electrostatic free energy of proteins. As a test of the method we have considered the pH-dependent stability of sperm whale metmyoglobin. Two theoretical methods for evaluation of the electrostatic free energy and p K values are applied: the finite-difference Poisson-Boltzmann method and the semiempirical approach based on the modified Tanford-Kirkwood theory. The theoretical results for electrostatic free energy of unfolding are compared with the experimental data for guanidine hydrochloride unfolding under equilibrium conditions over a wide pH range. Using the optical parameters of the Soret absorbance to monitor conformational equilibrium and Tanford's method to estimate the resulting data, it was found that the conformational free energy of unfolding of metmyoglobin is 16.3 kcal mol(-1) at neutral pH values. The total unfolding free energies were calculated on the basis of the theoretically predicted electrostatic unfolding free energies and the experimentally measured midpoints (pH(1/2)) of acidic and alkaline denaturation transitions. Experimental data for alkaline denaturation were used for the first time in theoretical analysis of the pH-dependent unfolding of myoglobin. The present results demonstrate that the simultaneous application of appropriate theoretical and experimental methods permits a more complete analysis of the pH-dependent and pH-independent properties and stability of globular proteins.     

Intrinsic fluorescence changes and rapid kinetics of proteinase deformation during serpin inhibition

The X-ray crystal structure of the serpin-proteinase complex suggested that the serpin deformed the proteinase thereby inactivating the molecule. Using a variant of alpha(1)-antitrypsin in which both tryptophan residues have been replaced by phenylalanine, we have shown that the proteinase becomes partially unfolded during serpin inhibition. The tryptophan free variant, alpha(1)-antitrypsin((FF)), is fully active as an inhibitor of thrombin. Thrombin has a fluorescence emission maximum of 340 nm which blue shifts to 346 nm, concomitant with a 40% increase in intensity, upon formation of the serpin-proteinase complex indicative of substantial conformational change within the proteinase. Stopped-flow analysis of the fluorescence changes within the proteinase indicated a two-step mechanism. A fast bimolecular reaction with a rate constant of 2.8x10(6) M(-1) s(-1) is followed by a slow unimolecular process with a rate of 0.26 s(-1) that is independent of concentration. We propose that the first rate is formation of an initial complex which is then followed by a slower process involving the partial unfolding of the proteinase during its translocation to the opposite pole of the serpin.