Studies on simultaneous inhibition of trypsin and chymotrypsin by horsegram Bowman-Birk inhibitor

Bowman-Birk inhibitors (BBI) isolated from plant seeds are small proteins active against trypsin and/or chymotrypsin. These inhibitors have been extensively studied in terms of their structure, interactions, function and evolution. Examination of the known three-dimensional structures of BBIs revealed similarities and subtle differences. The hydrophobic core, deduced from surface accessibility and hydrophobicity plots, corresponding to the two tandem structural domains of the double headed BBI are related by an almost exact two-fold, in contrast to the reactive site loops which depart appreciably from the two-fold symmetry. Also, the orientations of inhibitory loops in soybean and peanut inhibitors were different with respect to the rigid core. Based on the structure of Adzuki bean BBI-trypsin complex, models of trypsin and chymotryspin bound to the monomeric soybean BBI (SBI) were constructed. There were minor short contacts between the two enzymes bound to the inhibitor suggesting near independence of binding. Binding studies revealed that the inhibition of one enzyme in the presence of the other is associated with a minor negative cooperativity. In order to assess the functional significance of the reported oligomeric forms of BBI, binding of proteases to the crystallographic and non-crystallographic dimers as found in the crystal structure of peanut inhibitor were examined. It was found that all the active sites in these oligomers cannot simultaneously participate in inhibition.     

Unusual evolution of a catalytic core element in CCA-adding enzymes

CCA-adding enzymes are polymerases existing in two distinct enzyme classes that both synthesize the C-C-A triplet at tRNA 3′-ends. Class II enzymes (found in bacteria and eukaryotes) carry a flexible loop in their catalytic core required for switching the specificity of the nucleotide binding pocket from CTP- to ATP-recognition. Despite this important function, the loop sequence varies strongly between individual class II CCA-adding enzymes. To investigate whether this loop operates as a discrete functional entity or whether it depends on the sequence context of the enzyme, we introduced reciprocal loop replacements in several enzymes. Surprisingly, many of these replacements are incompatible with enzymatic activity and inhibit ATP-incorporation. A phylogenetic analysis revealed the existence of conserved loop families. Loop replacements within families did not interfere with enzymatic activity, indicating that the loop function depends on a sequence context specific for individual enzyme families. Accordingly, modeling experiments suggest specific interactions of loop positions with important elements of the protein, forming a lever-like structure. Hence, although being part of the enzyme’s catalytic core, the loop region follows an extraordinary evolutionary path, independent of other highly conserved catalytic core elements, but depending on specific sequence features in the context of the individual enzymes.     

Studies on molecular evolution and structural features of double-headed inhibitors of α-amylase and trypsin in plants

Plant seeds usually have high concentrations of proteinase and amylase inhibitors. These inhibitors exhibit a wide range of specificity, stability and oligomeric structure. In this communication, we report analysis of sequences that show statistically significant similarity to the double-headed α-amylase/trypsin inhibitor of ragi (Eleusine coracana). Our aim is to understand their evolutionary and structural features. The 14 sequences of this family that are available in the SWISSPROT database form three evolutionarily distinct branches. The branches relate to enzyme specificities and also probably to the oligomeric state of the proteins and not to the botanical class of the plant from which the enzymes are derived. This suggests that the enzyme specificities of the inhibitors evolved before the divergence of commercially cultivated cereals. The inhibitor sequences have three regions that display periodicity in hydrophobicity. It is likely that this feature reflects extended secondary structure in these segments. One of the most variable regions of the polypeptide corresponds to a loop, which is most probably exposed in the native structure of the inhibitors and is responsible for the inhibitory property.     

Steered molecular dynamics simulations reveal important mechanisms in reversible monoamine oxidase B inhibition

The monotopic membrane protein monoamine oxidase B (MAO B) is an important drug target for Parkinson's disease. In order to design more specific, and thereby more effective, inhibitors for this enzyme, it is necessary to determine what factors govern inhibitor specificity and the inhibitor binding process, including the roles of the lipid bilayer, the active site loop, and several key residues within the binding pocket. Atomistic molecular dynamics simulations of MAO B either embedded in a lipid bilayer or free in solution have been performed. The simulations suggest that the bilayer controls the availability of the active site cavity by regulating the degree of fluctuation in two key loops that form the greater part of the active site entrance (residues 85-110 and 155-165). In turn, the enzyme itself causes local thinning and a decrease in area per lipid of the surrounding bilayer environment. Additional MD simulations of MAO B in complex with seven different reversible inhibitors followed by nonequilibrium steered MD simulations of the inhibitor unbinding have also been performed. The simulations demonstrate that the average energy of interaction between inhibitor and MAO B residues during inhibitor egress is an effective indicator of inhibitor strength and is also useful for identifying key residues that govern inhibitor specificity. These data provide researchers with valuable tools for designing effective MAO B inhibitors as well as outline a method that can be translated to the study of other enzyme-inhibitor complexes.     

The mechanism of inhibition of antibody-based inhibitors of membrane-type serine protease 1 (MT-SP1)

The mechanisms of inhibition of two novel scFv antibody inhibitors of the serine protease MT-SP1/matriptase reveal the basis of their potency and specificity. Kinetic experiments characterize the inhibitors as extremely potent inhibitors with K(I) values in the low picomolar range that compete with substrate binding in the S1 site. Alanine scanning of the loops surrounding the protease active site provides a rationale for inhibitor specificity. Each antibody binds to a number of residues flanking the active site, forming a unique three-dimensional binding epitope. Interestingly, one inhibitor binds in the active site cleft in a substrate-like manner, can be processed by MT-SP1 at low pH, and is a standard mechanism inhibitor of the protease. The mechanisms of inhibition provide a rationale for the effectiveness of these inhibitors, and suggest that the development of specific antibody-based inhibitors against individual members of closely related enzyme families is feasible, and an effective way to develop tools to tease apart complex biological processes.     

Prediction of residues involved in inhibitor specificity in the dihydrofolate reductase family

Dihydrofolate reductase (DHFR) is of significant recent interest as a target for drugs against parasitic and opportunistic infections. Understanding factors which influence DHFR homolog inhibitor specificity is critical for the design of compounds that selectively target DHFRs from pathogenic organisms over the human homolog. This paper presents a novel approach for predicting residues involved in ligand discrimination in a protein family using DHFR as a model system. In this approach, the relationship between inhibitor specificity and amino acid composition for sets of protein homolog pairs is examined. Similar inhibitor specificity profiles correlate with increased sequence homology at specific alignment positions. Residue positions that exhibit the strongest correlations are predicted as specificity determinants. Correlation analysis requires a quantitative measure of similarity in inhibitor specificity (S(lig)) for a pair of homologs. To this end, a method of calculating S(lig) values using K(I) values for the two homologs against a set of inhibitors as input was developed. Correlation analysis of S(lig) values to amino acid sequence similarity scores - obtained via multiple sequence alignments - was performed for individual residue alignment positions and sets of residues on 13 DHFRs. Eighteen alignment positions were identified with a strong correlation of S(lig) to sequence similarity. Of these, three lie in the active site; four are located proximal to the active site, four are clustered together in the adenosine binding domain and five on the βFβG loop. The validity of the method is supported by agreement between experimental findings and current predictions involving active site residues.     

Navigating within thiamine diphosphate-dependent decarboxylases: Sequences,structures, functional positions,and binding sites

Thiamine diphosphate-dependent decarboxylases catalyze both cleavage and formation of C C bonds in various reactions, which have been assigned to different homologous sequence families. This work compares 53 ThDP-dependent decarboxylases with known crystal structures. Both sequence and structural information were analyzed synergistically and data were analyzed for global and local properties by means of statistical approaches (principle component analysis and principal coordinate analysis) enabling complexity reduction. The different results obtained both locally and globally, that is, individual positions compared with the overall protein sequence or structure, revealed challenges in the assignment of separated homologous families. The methods applied herein support the comparison of enzyme families and the identification of functionally relevant positions. The findings for the family of ThDP-dependent decarboxylases underline that global sequence identity alone is not sufficient to distinguish enzyme function. Instead, local sequence similarity, defined by comparisons of structurally equivalent positions, allows for a better navigation within several groups of homologous enzymes. The differentiation between homologous sequences is further enhanced by taking structural information into account, such as BioGPS analysis of the active site properties or pairwise structural superimpositions. The methods applied herein are expected to be transferrable to other enzyme families, to facilitate family assignments for homologous protein sequences.     

Identification of Serpin Determinants of Specificity and Selectivity for Furin Inhibition through Studies of α1PDX (α1-Protease Inhibitor Portland)-Serpin B8 and Furin Active-site Loop Chimeras

α₁-Protease inhibitor Portland (α₁PDX) is an engineered serpin family inhibitor of the proprotein convertase (PC), furin, that exhibits high specificity but limited selectivity for inhibiting furin over other PC family proteases. Here, we characterize serpin B8, a natural inhibitor of furin, together with α₁PDX-serpin B8 and furin-PC chimeras to identify determinants of serpin specificity and selectivity for furin inhibition. Replacing reactive center loop (RCL) sequences of α₁PDX with those of serpin B8 demonstrated that both the P4–P1 RXXR recognition sequence as well as the P1′–P5′ sequence are critical determinants of serpin specificity for furin. Alignments of PC catalytic domains revealed four variable active-site loops whose role in furin reactivity with serpin B8 was tested by engineering furin-PC loop chimeras. The furin(298–300) loop but not the other loops differentially affected furin reactivity with serpin B8 and α₁PDX in a manner that depended on the serpin RCL-primed sequence. Modeling of the serpin B8-furin Michaelis complex identified serpin exosites in strand 3C close to the 298–300 loop whose substitution in α₁PDX differentially affected furin reactivity depending on the furin loop and serpin RCL-primed sequences. These studies demonstrate that RCL-primed residues, strand 3C exosites, and the furin(298–300) loop are critical determinants of serpin reactivity with furin, which may be exploited in the design of specific and selective α₁PDX inhibitors of PCs.     

Regulation of cellular signals by G-proteins

Extracellular signals are transduced across the cell by the cell surface receptors, with the aid of G-proteins, which act at a critical point of signal transduction and cellular regulation. Structurally, G-proteins are heterotrimeric consisting α, β and γ subunits but in functionally active state they dissociate into α subunit coupled to GTP and as βγ dimer. G-proteins can be broadly divided into two classes based on their sensitivity to pertussis toxin and cholera toxin. Existence of various forms of each of the subunit allows molecular diversity in the subunit species of G-proteins. These subunits interact with a wide range of receptors and effectors, facilitated by post translational modification of their subunits. Different types of G-proteins mediate several signalling events in different parts of the body. This review summarizes the features of (i) structural and functional heterogenity among different subunits of G-proteins, (ii) interaction of G-proteins and their subunits with effectors with specific cases of G-protein mediated signalling in olfaction, phototransduction in the retina, ras andras related transduction and (iii) disease conditions associated with malfunctioning of G-proteins.     

Protein inhibitors of serine proteinases

Serine proteinases and their natural protein inhibitors belong to the most intensively studied models of protein-protein recognition. Protein inhibitors do not form a single group but can be divided into about 20 different families. Global structures of proteins representing different inhibitor families are completely different and comprise alpha-helical proteins, beta-sheet proteins, alpha/beta-proteins and different folds of small disulfide-rich proteins. Three different types of inhibitors can be distinguished: canonical (standard mechanism) inhibitors, non-canonical inhibitors, and serpins. The canonical inhibitor binds to the enzyme through the exposed and convex binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is consistently very similar and resembles that of an ideal substrate. Non-canonical inhibitors, originating from blood sucking organisms, specifically block enzymes of the blood clotting cascade. The interaction is mediated through inhibitor N-terminus which binds to the proteinase forming a parallel beta-sheet. There are also extensive secondary interactions which provide an additional buried area and contribute significantly to the strength and specificity of recognition. Serpins are major proteinase inhibitors occurring in plasma. Similarly to canonical inhibitors, serpins interact with their target proteinases in a substrate-like manner. However, in the case of serpins, cleavage of a single peptide bond in a flexible and exposed binding loop leads to dramatic structural changes.     

Activity of wheat alpha-amylase inhibitors towards bruchid alpha-amylases and structural explanation of observed specificities.

Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Five alpha-amylase inhibitors of the structural 0.19 family were isolated from wheat kernels, and assayed against three insect alpha-amylases and porcine pancreatic alpha-amylase, revealing several intriguing differences in inhibition profiles, even between proteins sharing sequence identity of up to 98%. Inhibition of the enzyme from a commercially important pest, the bean weevil Acanthoscelides obtectus, is observed for the first time. Using the crystal structure of an insect alpha-amylase in complex with a structurally related inhibitor, models were constructed and refined of insect and human alpha-amylases bound to 0.19 inhibitor. Four key questions posed by the differences in biochemical behaviour between the five inhibitors were successfully explained using these models. Residue size and charge, loop lengths, and the conformational effects of a Cys to Pro mutation, were among the factors responsible for observed differences in specificity. The improved structural understanding of the bases for the 0.19 structural family inhibitor specificity reported here may prove useful in the future for the rational design of inhibitors possessing altered inhibition characteristics.     

Association of HSP90 with the heme-regulated eukaryotic initiation factor 2α kinase—A collaboration for regulating protein synthesis

Among the various heat shock proteins (HSPs), members of the HSP70 and HSP90 families have drawn particular attention due to their heat shock-unrelated functions. HSP90, an ubiquitous and abundant member of the HSP90 family has been shown to be associated with a large array of protein factors. These proteins reside in the nucleus as well as in the cytoplasm and are involved in various physiological processes, such as, regulation of chromatin structure, cell cycle, cytoskelelal architecture, protein trafficking and protein synthesis. In this article, we focus our interest on the role of HSP90 in protein synthesis. Recent data obtained from a few laboratories strongly suggest that HSP90 interacts with the heme-regulated eukaryotic initiation factor 2α (elF-2α) kinase, also called the heme-regulated inhibitor, and causes its activation which leads to inhibition of protein synthesis. On the basis of data reported from various laboratories, including our own, we propose a possible model on the mechanism of HSP90-mediated activation of heme-regulated inhibitor and regulation of protein synthesis.     

Use of phage display to probe the evolution of binding specificity and affinity in integrins

The specific binding of RGD-containing proteins to integrin is a function of both the conformation of and the local sequence surrounding the RGD motif. To study the effect of these factors on integrin binding affinity and specificity, we obtained RGD-containing ligands specific for different integrins presented on the same protein scaffold. The beta-turn region between two anti-parallel beta-strands on the loop I of tendamistat, an inhibitor of alpha-amylase, was extended by two residues and randomized in a phagemid library. This library and two subsequently constructed RGD-containing loop I libraries were biopanned with purified integrins alphaIIbbeta3, alphaVbeta3 and alphaVbeta5 individually. The sequence analysis of selected tendamistat variants and characterization by phage ELISA revealed that phage adhesion is mediated exclusively by an RGD motif located at only two out of four possible positions on loop I. Further, sequences flanking the RGD motif were specific for different integrin targets. Interestingly, selected tendamistat variants mimic natural integrin ligands, both in sequence similarity and in integrin binding specificity, indicating that various ligand specificity patterns can be generated by driving towards maximum affinity in the integrin-ligand complexes.     

Endothelin-1 induced sustained contractions of tracheal smooth muscle involve an activation of protein kinase C

Endothelin-1, a potent vasoconstrictor peptide produces concentration dependent contractions in lamb tracheal smooth muscle. These contractions are not inhibited by low doses (up to 20 μM) of trifluoroperazine and W-7, the calmodulin/myosin light chain kinase (MLCK) inhibitors. At higher concentrations (200 μM), a delayed and poor reversal of isometric tensions results. These relaxations are coupled with a partial dephosphorylation of regulatory myosin light chain (MLC). Preincubation of fiber strips in MLCK inhibitors (200 μM) results in a delayed and attenuated contractile response but without a dephosphorylation of MLC. H-7, a putative protein kinase C antagonist (25–100 μM) abolishes endothelin-1 induced contractile effects rapidly (50% relaxation within 1–3 min). Moreover, such relaxations are accompanied by complete dephosphorylation of MLC. Phorbol 12, 13-dibutyrate, an exogenous activator of protein kinase C potentiates the endothelin induced contractions. Inactive phorbol ester, 4α-phorbol ester does not elicit any contractile response in the muscle. The down regulation of protein kinase C, on the other hand suppresses such potentiated contractile responses. These results suggest that endothelin-1 induced contractile tensions in tracheal smooth muscle are mediated by a mechanism that involves an activation of enzyme protein kinase C.     

The phylogeny of trypsin-related serine proteases and their zymogens. New methods for the investigation of distant evolutionary relationships.

The sequence of all presently known trypsin-related serine proteases and their zymogens of animal and bacterial origin were optimally aligned on the basis of three different scoring schemes for amino acid comparisons. Sequence homology was found to extend into the activation peptides. The gaps resulting from the alignment of the sequences of the active enzymes formed the basis for a new procedure based on position and number of gaps, which allowed the correct topology of the evolutionary relationship of thrombin and the pancreatic enzymes trypsin, chymotrypsin and elastase to be determined. The procedure was applied in an analogous manner to changes in disulfide bridges as well as to a selected set of amino acid positions.Evolutionary distances between proteins were estimated by minimum, base differences as well as according to the stochastic model of evolution. These distances were used successfully to find the best topology of evolutionary relationships. The fact that the branch lengths in evolutionary trees were less affected by the number of sequences considered when evolutionary distances between contemporary sequences were measured in minimum base differences than when measured according to the stochastic model of evolution, suggested in our specific case, that minimum base differences yielded estimates of evolutionary distance closer to reality than the stochastic model of evolution.All these techniques combined yielded the following picture for the evolution of the four protease families. Prothrombin and the zymogens of the pancreatic serine proteases had a common ancestor with tryptic specificity. After the initial divergence, the gene for trypsinogen duplicated. Evidence was found that the duplicated gene underwent drastic changes for a short period of time to become eventually the common ancestor of chymotrypsin and elastase. The phylogenetic tree elaborated for these enzyme families and the methods introduced to determine its topology, should readily allow determination of the attachment site of branches leading to newly sequenced serine proteases, provided their amino acid sequence can be aligned fairly unambiguously. In addition, the consequences of the alignment of the different serine proteases for the relationship of zymogen to enzyme are discussed.     

Inhibition of human beta-tryptase by Bowman-Birk inhibitor derived peptides: creation of a new tri-functional inhibitor

Bowman-Birk inhibitor proteins (BBIs), which are potent inhibitors of chymotrypsin-like proteases, do not inhibit human beta-tryptase despite this protein having a chymotrypsin-like fold. We have reported previously that, in contrast, BBI-derived peptides (whose sequences incorporate the solvent exposed reactive site loop motif) are able to inhibit human beta-tryptase. This is due to their small size, which allows them to access the restricted active site(s) of tryptase, which has an unusual tetrameric arrangement with four active sites flanking a central pore. In this paper, we have examined the possibility of creating additional interactions within this pore by adding extensions to the BBI-peptide motif. We have taken the core disulfide-bridged sequence SCTKSIPPQCY and examined a series of extensions, at both the C- and N-termini, that bear a second positively charged Lys residue at their end. The aim was to construct inhibitors that could make additional interactions in tryptase by spanning the gap between adjacent active sites in the enzyme, producing a double-headed inhibitor; a positively charged group was used as the dominant specificity of this enzyme is for a positively charged P1 residue. Both N- and C-terminal extensions are found to produce inhibitors of much increased potency, with a strong dependence of potency on chain length. Moreover, it was found that the C- and N-terminal extensions were able to synergise, with their combination on the same peptide producing an even better inhibitor with a potency 10(4)-fold greater than the original sequence. We suggest that the C- and N-terminal extensions are picking up interactions with separate additional sites on the tryptase, making the doubly extended BBI peptide a tri-functional tryptase inhibitor.     

Roles of ubiquitin-mediated proteolysis in cell cycle control

Selective degradation of cyclins, inhibitors of cyclin-dependent kinases and anaphase inhibitors is responsible for several major cell cycle transitions. The degradation of these cell cycle regulators is controlled by the action of ubiquitin—protein-ligase complexes, which target the regulators for degradation by the 26S proteasome. Recent results indicate that two types of multisubunit ubiquitin ligase complexes, which are connected to the protein kinase regulatory network of the cell cycle in different ways, are responsible for the specific and programmed degradation of many cell cycle regulators.     

Kazal型蛋白酶抑制剂结构与功能研究进展

蛋白酶抑制剂广泛存在于生物体内,在许多生命活动过程中发挥必不可少的作用,特别是对蛋白酶活性进行精确调控。其中Kazal型蛋白酶抑制剂是最重要的、研究最为广泛的酶抑制剂之一,该类抑制剂一般由一个或几个结构域组成,每一个结构域具有保守的序列和分子构象,同时发现该类抑制剂与蛋白酶作用的结合部位高度易变,它们大多数暴露于与溶剂接触的环上,其中P1部位是抑制作用的关键部位,抑制剂的专一性由P1部位氨基酸残基的性质决定,其它残基取代结合部位残基对抑制剂-酶的结合常数有显著的影响。Laskowski算法可直接从Kazal型丝氨酸蛋白酶抑制剂的序列推测其与6种丝氨酸蛋白酶之间的抑制常数(Ki)。目前在生物体内发现大量的Kazal型蛋白酶抑制剂,并证实其有重要的生物学功能。     

Within- and among-population variation in oviposition preference for urea-supplemented food inDrosophila melanogaster

Oviposition preference for ureasupplemented food was assayed by simultaneous choice trials on five pairs of closely related laboratory populations of Drosophila melanogaster.Each pair of populations had been derived from a separate ancestral population about 85 generations prior to this study. One population in each pair had been subjected to selection for larval tolerance to the toxic effects of urea; the other population served as a control. Considerable variation in oviposition preference was seen both within and among populations, with four of the ten populations showing a significant mean preference for ureasupplemented food. The degree of specificity shown by individual females was surprisingly high, leading to a bimodal distribution of oviposition preference in some populations. Overall, selection for larval tolerance to urea did not significantly affect oviposition preference. However, the data indicated that pairwise comparisons between randomly selected populations from the two larval selection regimes would lead to a range of possible outcomes, suggesting, in several cases, that selection for larval urea tolerance had led to significant differentiation of adult oviposition preference for urea in one or the other direction. The results, therefore, highlight the importance of population level replication and caution against the practice, common in ecological studies, of assaying oviposition preference in two populations that utilize different hosts in nature, and then drawing broad evolutionary inferences from the results.