Predicting protein interaction interfaces from protein sequences: case studies of subtilisin and phycocyanin

Identification of protein interaction interfaces is very important for understanding the molecular mechanisms underlying biological phenomena. Here, we present a novel method for predicting protein interaction interfaces from sequences by using PAM matrix (PIFPAM). Sequence alignments for interacting proteins were constructed and parsed into segments using sliding windows. By calculating distance matrix for each segment, the correlation coefficients between segments were estimated. The interaction interfaces were predicted by extracting highly correlated segment pairs from the correlation map. The predictions achieved an accuracy 0.41-0.71 for eight intraprotein interaction examples, and 0.07-0.60 for four interprotein interaction examples. Compared with three previously published methods, PIFPAM predicted more contacting site pairs for 11 out of the 12 example proteins, and predicted at least 34% more contacting site pairs for eight proteins of them. The factors affecting the predictions were also analyzed. Since PIFPAM uses only the alignments of the two interacting proteins as input, it is especially useful when no three-dimensional protein structure data are available.     

Characterization of a bifunctional wheat inhibitor of endogenous α-amylase and subtilisin

A bifunctional α-amylase/serine protease inhibitor which inhibits germination-specific cereal α-amylases of the Graminae subfamily Festucoideae as well as bacterial subtilisins has been isolated from wheat grains. This protein has M_r ≈20500 and pI ≈7.2. The amino acid composition and N-teminal sequence (45 residues) show that the inhibitor is homologous with cereal and leguminous inhibitors of the soybean trypsin inhibitor (Kunitz) family.     

Structural and mutational analyses of the interaction between the barley alpha-amylase/subtilisin inhibitor and the subtilisin savinase reveal a novel mode of inhibition

Subtilisins represent a large class of microbial serine proteases. To date, there are three-dimensional structures of proteinaceous inhibitors from three families in complex with subtilisins in the Protein Data Bank. All interact with subtilisin via an exposed loop covering six interacting residues. Here we present the crystal structure of the complex between the Bacillus lentus subtilisin Savinase and the barley α-amylase/subtilisin inhibitor (BASI). This is the first reported structure of a cereal Kunitz-P family inhibitor in complex with a subtilisin. Structural analysis revealed that BASI inhibits Savinase in a novel way, as the interacting loop is shorter than loops previously reported. Mutational analysis showed that Thr88 is crucial for the inhibition, as it stabilises the interacting loop through intramolecular interactions with the BASI backbone.     

Urea-induced conformational changes in cold- and heat-denatured states of a protein, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor (SSI) is known to exist in at least two distinct denatured states, cold-denatured (D') and heat-denatured (D) under acidic conditions. In the present work, we investigated the manner how increasing urea concentration from 0 to 8 M changes the polypeptide chain conformation of SSI that exists initially in the D' and D states as well as in the native state (N), in terms of the secondary structure, the tertiary structure, and the chain form, based on the results of the experiments using circular dichroism (CD), small-angle X-ray scattering (SAXS) and 1H-NMR spectroscopy. Our results indicate that the urea-induced conformational transitions of SSI under typical conditions of D' (pH 1.8, 3 degrees C) occur at least in two steps. In the urea concentration range of 0-2 M (step 1), a cooperative destruction of the tertiary structure occurs, resulting in a mildly denatured state (DU), which may still contain a little amount of secondary structures. In the concentration range of 2-4 M urea (step 2), the DU state gradually loses its residual secondary structure, and increases the radius of gyration nearly to a maximum value. At 4 M urea, the polypeptide chain is highly disordered with highly mobile side chains. Increasing the urea concentration up to 8 M probably results in the more highly denatured or alternatively the stiffer chain conformations. The conformational transition starting from the N state proceeds essentially the same way as in the above scheme in which D' is replaced with N. The conformational transition starting from the D state lacks step 1 because the D state contains no tertiary structures and is similar to the DU state. The fact that similar conformations are reached at urea concentrations above 2 M from different conformations of D', D, and N indicates that the effect of urea dominates in determining the polypeptide conformation of SSI in the denatured states rather than the pH and temperature.     

Structural determinants of ligand imprinting: a molecular dynamics simulation study of subtilisin in aqueous and apolar solvents

The phenomenon known as "ligand imprinting" or "ligand-induced enzyme memory" was first reported in 1988, when Russell and Klibanov observed that lyophilizing subtilisin in the presence of competitive inhibitors (that were subsequently removed) could significantly enhance its activity in an apolar solvent. (Russell and Klibanov, J Biol Chem 1988;263:11624-11626). They further observed that this enhancement did not occur when similar assays were carried out in water. Herein, we shed light on the molecular determinants of ligand imprinting using a molecular dynamics (MD) approach. To simulate the effect of placing an enzyme in the presence of a ligand before its lyophilization, an inhibitor was docked in the active site of subtilisin and 20 ns MD simulations in water were performed. The ligand was then removed and the resulting structure was used for subsequent MD runs using hexane and water as solvents. As a control, the same simulation setup was applied using the structure of subtilisin in the absence of the inhibitor. We observed that the ligand maintains the active site in an open conformation and that this configuration is retained after the removal of the inhibitor, when the simulations are carried out in hexane. In agreement with experimental findings, the structural configuration induced by the ligand is lost when the simulations take place in water. Our analysis of fluctuations indicates that this behavior is a result of the decreased flexibility displayed by enzymes in an apolar solvent, relatively to the aqueous situation.     

Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

Purification and properties of a protease inhibitor from crayfish hemolymph

A protease inhibitor from the hemolymph of crayfish, Astacus astacus, has been purified by differential centrifugation, acid precipitation and preparative isoelectric focusing. The inhibitor was apparent homogenous in SDS-electrophoresis and had a molecular weight of 23,000. pI was determined to be 4.7 by isoelectric focusing. No inhibitory activity was lost when the inhibitor was incubated in a pH range of 1–11.5. The purified inhibitor was heat stable. Urea (6 m) had no effect upon the inhibitor. The inhibitor was active against subtilisin and a partly purified protease from the fungus Aphanomyces astaci. Pronase was slightly inhibited whereas trypsin, chymotrypsin, papain, Arthrobacter protease, and extracellular proteases from the fungi Aphanomyces stellatus and A. laevis were unaffected. The importance of protease inhibitors in pathogenesis between the parasitic fungus, A. astaci, and its crayfish host, A. astacus is discussed.     

The evaluation of Cr-curcumin-DNA complexation by experimental and theoretical approaches

Chromium(III) chloride mediates DNA-DNA cross-linking. Some chromium complexes promote programmed cell death in specific ligand environment through binding to DNA. One strategy that can be supposed for reduction of Cr³⁺ binding affinity to DNA is using curcumin as a chelator. In the current study, the [Cr(Curcumin)(EtOH)₂](NO₃)₂ (CCC) was synthesized and characterized by UV/Vis, FT-IR, CHN and spectrophotometric titration techniques. The mole ratio plot revealed a 1:1 complex between Cr³⁺ and curcumin in solution. Binding interaction of this complex with calf thymus-DNA (CT-DNA) was investigated using UV/Vis, circular dichroism (CD), FT-IR and cyclic voltammetry. The intrinsic binding constants of CCC with DNA, measured by UV/Vis and cyclic voltammetry, were 1.60 × 10⁵ and 1.13 × 10⁵, respectively. The thermodynamic studies showed that the reaction is enthalpy and entropy favoured. CD analysis revealed that only Λ-CCC interacts with DNA and Δ-CCC form has no tendency towards DNA. Based on FT-IR studies, it was understood that CCC interacts with DNA via minor groove binding. The docking simulation was carried out for finding the binding mode of CCC to DNA, too. All of data demonstrated that the curcumin significantly reduced the affinity of Cr³⁺ to the DNA and the form of Δ-CCC has no interaction with DNA.     

Nucleophilic cleavage of the complex between human alpha-1-antitrypsin and porcine pancreatic elastase

Several nucleophilic amines were examined to determine their ability to split the alpha-1-antitrypsin-elastase complex. Hydrazine and hydroxylamine, their methyl derivatives, and methylamine were tested in the pH range of 8 to 10.6. Only hydrazine and methylamine produced complete and clean cleavage of the complex into its inhibitor and enzyme components. When [¹⁴C]-methylamine at pH 10.6 was used about 0.3 mol of the nucleophile was specifically bound per mol of the inhibitor component. An interfering reaction between methylamine and the native inhibitor was controlled by preincubation with unlabeled methylamine.     

Coordination topology and stability for the native and binding conformers of chymotrypsin inhibitor 2

We demonstrate that the stabilization of the binding region is accomplished at the expense of a loss in the stability of the rest of the protein. A novel molecular mechanics (MM) approach is introduced to distinguish residue stabilities of proteins in a given conformation. As an example, the relative stabilities of folded chymotrypsin inhibitor 2 (CI2) in unbound form, and CI2 in complex with subtilisin novo is investigated. The conformation of the molecule in the two states is almost identical, with an approximately 0.6-A root-mean-square deviation (RMSD) of the Calpha atoms. On binding, the packing density changes only at the binding loop. However, residue fluctuations in the rest of the protein are greatly altered solely due to those contacts, indicating the effective propagation of perturbation and the presence of remotely controlling residues. To quantify the interplay between packing density, packing order, residue fluctuations, and residue stability, we adopt an MM approach whereby small displacements are inserted at selected residues, followed by energy minimization; the displacement of each residue in response to such perturbations are organized in a perturbation-response matrix L. We define residue stability lambda(i) = summation operator((j)L(ij))/ summation operator((j) L(ji)) as the ratio of the amount of change to which the residue is amenable, to the ability of a given residue to induce change. We then define the free energy associated with residue stability, DeltaG(lambda) = -RT ln lambda. DeltaG(lambda) intrinsically selects the residues that are in the folding core. Upon complexation, the binding loop becomes more resistant to perturbation, in contrast to the alpha-helix that favors change. Although the two forms of CI2 are structurally similar, residue fluctuations differ vastly, and the stability of many residues is altered upon binding. The decrease in entropy introduced by binding is thus compensated by these changes.     

The transglutaminase activating metalloprotease inhibitor from Streptomyces mobaraensis is a glutamine and lysine donor substrate of the intrinsic transglutaminase

Transglutaminase (TGase) from Streptomyces mobaraensis is an extra-cellular enzyme that cross-links proteins to high molecular weight aggregates. Screening for intrinsic substrates now revealed the dual Streptomyces subtilisin inhibitor-like inhibitor Streptomyces subtilisin and transglutaminase activating metalloprotease (TAMEP) inhibitor (SSTI), equally directed against subtilisin and the TGase activating metalloprotease TAMEP, is both a glutamine and a lysine donor protein. Reactivity of glutamines is lost during culture, most likely by TGase mediated deamidation, and, accordingly, cross-linking only occurred if SSTI from early cultures was used. Interestingly, release of buried endo-glutamines by the lipoamino acid N-lauroylsarcosine could restore SSTI reactivity. Formation of lipoamino acids by Streptomycetes suggests such compounds could also modulate in vivo TGase mediated SSTI cross-linking.     

Molecular identification of wheat endoxylanase inhibitor TAXI-II and the determinants of its inhibition specificity

Wheat grains contain Triticum aestivum xylanase inhibitor (TAXI) proteins which inhibit microbial xylanases, some of which are used in cereal based food industries. These inhibitors may play a role in plant defence. Among the TAXI isoforms described so far, TAXI-II displays a deviating inhibition specificity pattern. Here, we report on the molecular identity of TAXI-II and the basis of its inhibition specificity. Three candidate TAXI-II encoding sequences were isolated and recombinantly expressed in Pichia pastoris. To identify TAXI-II, the resulting proteins were tested against glycoside hydrolase family (GHF) 11 xylanases of Aspergillus niger (ANX) and Bacillus subtilis (BSX). One of these proteins (rTAXI-IB) inhibited both enzymes, like natural TAXI-I. The other candidates (rTAXI-IIA and rTAXI-IIB) showed an inhibition pattern typical for natural TAXI-II, only clearly inhibiting BSX. Comparative analysis of these highly similar sequences with distinct inhibition activity patterns, combined with information on the structural basis for ANX inhibition by TAXI-I [S. Sansen, C.J. De Ranter, K. Gebruers, K. Brijs, C.M. Courtin, J.A. Delcour, A. Rabijns, Structural basis for inhibition of Aspergillus niger xylanase by Triticum aestivum xylanase inhibitor-I, J. Biol. Chem. 279 (2004) 36022-36028], indicated a crucial role for Pro294 of TAXI-IIA and Gln376 of TAXI-IIB in determining the reduced inhibition activity towards ANX. Consequently, single point mutants rTAXI-IIA[P294L] and rTAXI-IIB[Q376H], both displaying the Leu/His combination corresponding to TAXI-I, were able to inhibit ANX. These results show that TAXI-II inhibition specificity bears on the identity of two key residues at positions 294 and 376, which are involved in the interaction at the -2 glycon subsite and the active site of GHF 11, respectively.     

Specificity of covalently stabilized complexes of 125I-labeled human somatotropin and components of the lactogenic binding sites of rat liver

¹²⁵I-labeled human somatotropin specifically bound to the lactogenic sites of microsomal membranes from pregnant rat liver, originated a radioactive covalent complex of M_r 63,000 upon reaction with dimethyl suberimidate, disuccinimidyl suberate (DSS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The amoun of this species was closely parallel with the preexisting amount of the ligand-receptor complex. Photoactivation of a hormone derivative bound to the receptor also gave rise to the 63 K species. A ternary complex of receptor, hormone and Triton X-100 cross-linked with DSS yielded the 63 K species and a new one of 96 K. The data indicate that the 63 K complex involves the radioactive hormone and a constituent of the binding site. The 96 K species could comprise a second component of the receptor.     

Netrin, Slit and Wnt receptors allow axons to choose the axis of migration

One of the challenges to understanding nervous system development has been to establish how a fairly limited number of axon guidance cues can set up the patterning of very complex nervous systems. Studies on organisms with relatively simple nervous systems such as Drosophila melanogaster and C. elegans have provided many insights into axon guidance mechanisms. The axons of many neurons migrate along both the dorsal-ventral (DV) and the anterior-posterior (AP) axes at different phases of development, and in addition they may also cross the midline. Axon migration in the dorsal-ventral (DV) direction is mainly controlled by Netrins with their receptors; UNC-40/DCC and UNC-5, and the Slits with their receptors; Robo/SAX-3. Axon guidance in the anterior-posterior (AP) axis is mainly controlled by Wnts with their receptors; the Frizzleds/Fz. An individual axon may be subjected to opposing attractive and repulsive forces coming from opposite sides in the same axis but there may also be opposing cues in the other axis of migration. All the information from the cues has to be integrated within the growth cone at the leading edge of the migrating axon to elicit a response. Recent studies have provided insight into how this is achieved.Evidence suggests that the axis of axon migration is determined by the manner in which Netrin, Slit and Wnt receptors are polarized (localized) within the neuron prior to axon outgrowth. The same molecules are involved in both axon outgrowth and axon guidance, for at least some neurons in C. elegans, whether the cue is the attractive cue UNC-6/Netrin working though UNC-40/DCC or the repulsive cue SLT-1/Slit working though the receptor SAX-3/Robo (Adler et al., 2006, Chang et al., 2006, Quinn et al., 2006, 2008). The molecules involved in cell signaling in this case are polarized within the cell body of the neuron before process outgrowth and direct the axon outgrowth. Expression of the Netrin receptor UNC-40/DCC or the Slit receptor SAX-3/Robo in axons that normally migrate in the AP direction causes neuronal polarity reversal in a Netrin and Slit independent manner (Levy-Strumpf and Culotti 2007, Watari-Goshima et al., 2007). Localization of the receptors in this case is caused by the kinesin-related VAB-8L which appears to govern the site of axon outgrowth in these neurons by causing receptor localization. Therefore, asymmetric localization of axon guidance receptors is followed by axon outgrowth in vivo using the receptor's normal cue, either attractive, repulsive or unknown cues.     

Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery

The epithelial and endothelial barriers of the human body are major obstacles for drug delivery to the systemic circulation and to organs with unique environment and homeostasis, like the central nervous system. Several transport routes exist in these barriers, which potentially can be exploited for enhancing drug permeability. Beside the transcellular pathways via transporters, adsorptive and receptor-mediated transcytosis, the paracellular flux for cells and molecules is very limited. While lipophilic molecules can diffuse across the cellular plasma membranes, the junctional complexes restrict or completely block the free passage of hydrophilic molecules through the paracellular clefts. Absorption or permeability enhancers developed in the last 40 years for modifying intercellular junctions and paracellular permeability have unspecific mode of action and the effective and toxic doses are very close. Recent advances in barrier research led to the discovery of an increasing number of integral membrane, adaptor, regulator and signalling proteins in tight and adherens junctions. New tight junction modulators are under development, which can directly target tight or adherens junction proteins, the signalling pathways regulating junctional function, or tight junction associated lipid raft microdomains. Modulators acting directly on tight junctions include peptides derived from zonula occludens toxin, or Clostridium perfringens enterotoxin, peptides selected by phage display that bind to integral membrane tight junction proteins, and lipid modulators. They can reversibly increase paracellular transport and drug delivery with less toxicity than previous absorption enhancers, and have a potential to be used as pharmaceutical excipients to improve drug delivery across epithelial barriers and the blood-brain barrier.