Protein and Peptide Letters - Volume 12, Issue 5, 2005

Interaction of proteinase inhibitors with proteinases is one of the best understood protein-protein interaction systems. The past decade has seen explosive growth in our understanding of the structure and function of proteinase inhibitors and their molecular basis of interaction with proteinases. It is fitting that this issue of Protein and Peptide Letters carries articles on different aspects of inhibitor structure and function by many experts in the field. This issue of Protein and Peptide Letters also represents a pivotal point in the history of standard mechanism serine proteinase inhibitors as it stands between the Michael- Laskowski-Jr. era and the post-Michael-Laskowski-Jr. era. Michael Laskowski Jr. died on August 2, 2004. He was a great pioneer in the field of serine proteinase inhibitors and made numerous remarkable contributions to the field during his fifty-plus years of research. This issue is also a tribute to this great scientist. Nine prominent research groups from all over the world have contributed articles in this issue. The international flavor is quite evident from the fact that there is one article each from Australia, France, Germany, Italy, New Zealand, and Poland, and three articles from different laboratories in the United States. The first article in this issue highlights some of the most important contributions which Michael Laskowski Jr. made to our understanding of serine proteinase inhibitors. The arrangement of other articles is arbitrary, although interested readers would be able to observe some logic in this arrangement. In the second article D. Krowarsch et al. have nicely summarized results obtained on the structure and inhibitory activity of a number of reactive site loop variants of BPTI. The next article by C. Kellenberger and A. Roussel deals with the structure and inhibitory activity of inhibitors belonging to the pacifastin family (the grasshopper family). The fourth article is by T. Komiyama; it describes mutational strategies for the design of inhibitors for furin, Kex2 and PC7 using eglin c as the wild type inhibitor. In the next article H. J. Schirra and D. J. Craik elegantly describe the structure, folding and intramolecular domain swapping in potato II proteinase inhibitors. The sixth article in this issue is by P. Ascenzi et al.; it reviews the mechanism and kinetics of inhibition by small synthetic suicide substrates for thrombin. The seventh article is a review on serine proteinase inhibitors of plant origin by J. Christeller and W. Laing. The next article is by W. Hohne and K. Hilpert and discusses the applications of substitutional analysis by cellulose-bound peptide spot synthesis for the characterization and optimization of several small polypeptide inhibitors. In this issue, which is heavily biased towards inhibitors of serine proteinases, the article by M. Sahin-Toth represents an interesting departure. Inhibitors are not always the winners, and occasionally enzymes use tricks to evade inhibition and sometimes even digest them. This scenario has been nicely reviewed by M. Sahin-Toth with special reference to mesotrypsin. The last article in this issue is by C. Kelly et al. and deals with the role of scaffolding in the structure, stability, and inhibitory activity of standard mechanism serine proteinase inhibitors. Although this issue is devoted to serine proteinase inhibitors and serine proteinases and would be of great interest to researchers in these areas, it should also be of interest to protein chemists involved in structure-function studies and to those who care about protein-protein interactions.

Michael Laskowski Jr. (1930-2004) was a pioneer in the field of Standard mechanism serine proteinase inhibitors. He made numerous important contributions in the field. This article highlights some of his most important contributions such as the discovery of the reactive site in serine proteinase inhibitors, the proposal of the Standard mechanism of inhibition, and the sequence to reactivity algorithm for the Kazal family of inhibitors.

We report our progress in understanding the structure-function relationships for the interaction between BPTI and serine proteases. We focused on extensive mutagenesis of four crucial positions from the protease binding loop of BPTI. Selected variants were characterized by determination of association constants, stability parameters and structures of protease-inhibitor complexes.

The members of the Pacifastin family are serine protease inhibitors found in insects and crustacean. They are either small inhibitors (made of one consensus cysteine-rich motif) or proteins (4-9 motifs). Some of these inhibitors are characterized by a species selectivity for the trypsin inhibition. Structural data discriminate the small inhibitors that apparently look very similar into two groups. Interestingly, the inhibitors that display species selectivity fall in the same structural group.

Potent inhibitors of the Kex2/furin family precursor processing proteases were developed by randomizing adventitious contact sites and screening for optimized affinity using inhibition assays in 96-well format [1]. In this review, the binding interactions of the developed inhibitors will be examined in light of the three dimensional structures of Kex2 and furin [2-4].

Potato type II serine proteinase inhibitors are proteins that consist of multiple sequence repeats, and exhibit a multidomain structure. The structural domains are circular permutations of the repeat sequence, as a result of intramolecular domain swapping. Structural studies give indications for the origins of this folding behaviour, and the evolution of the inhibitor family.

Thrombin is the last enzyme in the blood coagulation cascade. All pharmacological aspects support the use of thrombin inhibitors as antithrombotic agents. Here, we review the unusual inhibition behavior of the highly selective 'reversible suicide substrate' N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-α-azalysine p-nitrophenyl ester (Eoc-D-Phe-ProazaLys- ONp) targeted to the active center of human a-thrombin. Eoc-D-Phe-Pro-azaLys-ONp is an acylating agent, but its hydrolysis product 1(N-ethoxycarbonyl-D-phenylalanyl-L-prolyl)-2(4-aminobutyl) hydrazine behaves as a highly selective human α-thrombin competitive inhibitor.

Evidence that establishes the mechanism of the classes of plant proteinase inhibitors (PIs) is evaluated. Of the eight classes of PIs, six are unique to plants. Except for plant serpins, there is evidence that PIs from all other classes form tight binding complexes with their target proteinases, and that they follow the standard mechanism of inhibition.

The interaction of peptidic inhibitors with serine proteases is investigated using peptide spot synthesis on cellulose sheets. This method permits a highly parallel analysis of subsite specificities and an optimization of peptidic inhibitors towards higher affinity and specificity for a given target protease. Information from crystal structures of corresponding protease/inhibitor complexes may help to explain the observed binding pattern.

More than twenty years ago Rinderknecht et al. identified a minor trypsin isoform resistant to natural trypsin inhibitors in the human pancreatic juice. At the same time, Estell and Laskowski found that an inhibitor-resistant trypsin from the pyloric caeca of the starfish, Dermasterias imbricata rapidly hydrolyzed the reactive-site peptide bonds of trypsin inhibitors. A connection between these two seminal discoveries was made recently, when human mesotrypsin was shown to cleave the reactive-site peptide bond of the Kunitz-type soybean trypsin inhibitor, and degrade the Kazal-type pancreatic secretory trypsin inhibitor. These observations indicate that proteases specialized for the degradation of protease inhibitors are ubiquitous in metazoa, and prompt new investigations into their biological significance. Here we review the history and properties of human mesotrypsin, and discuss its function in the digestive degradation of dietary trypsin inhibitors and possible pathophysiological role in pancreatitis.

In single domain, “standard mechanism” protein inhibitors of serine proteinases, about a dozen residues make contact with the cognate enzyme. The remainder of the molecule, the scaffolding, holds the reactive site region of the inhibitor in a canonical conformation, improves the binding by about six orders of magnitude and protects it from proteolysis. However, the stability and global structure of the scaffolding is irrelevant to inhibition, provided that inhibition is measured much below the melting temperature,™.

In this paper, a 3D map of protein fold space was produced using Dali structure alignment and nonmetric multidimensional scaling. The fold space comprises four radial clusters, which correspond to the four classes of SCOP. The overall structure of the protein fold space is largely determined by three factors: secondary structure composition, topology of β sheet, and domain size.

The native metastability of serine protease inhibitors (serpins) is believed to facilitate the conformational change required for biological function. However, energetically unfavorable structural features that contribute to metastability of the native serpin conformation, such as buried polar groups, cavities, and over-packing of side-chains, also appear to hinder proper folding. Hence, folding of serpin polypeptides appears prone to error; in particular, the folding polypeptides are readily diverted toward a non-productive folding pathway culminating in a more stable but inactive conformation. In a survey of deficient serpin mutants, various folding defects, such as retarded protein folding, destabilized native conformation, and spontaneous conversion into more stable, inactive conformations such as the latent form and loop-sheet polymers, have been discovered.

The local fluorescence probes, 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) and NADPH were employed to detect urea-induced conformation changes at each active site of dihydrofolate reductase (DHFR), respectively. The results indicate that local conformation change at DHF/TNS could be superimposed by the conformation change calculated from the enzyme activity change with a three-state model; while at NADPH site it is lagged in the first transition. This difference is further supported by the different relative changes of Michaelis constants at 0, 1 and 1.8 M urea for each substrate. Our results suggest that local conformation at DHF site is more flexible than that at NADPH site, and the urea-induced unfolding could be ascribed to a four-state transition.

Human brain serine racemase (hSR) was expressed in large amounts in E. coli with N-terminal His-tag (HishSR). His-hSR expressed in inclusion body was solubilized and purified to homogeneity by Ni-NTA affinity column. Purified His-hSR was refolded in Tween 20/cycloamylose with ∼50% efficiency, and refolded His-hSR was isolated by Q Sepharose column chromatography. The refolding conditions are described in detail. His-hSR catalyzed the elimination of L-Ser as well as L-Ser-O-sulfate to form pyruvate.

In this study, we analyze the amino acid pairs in human protein C precursor to determine which amino acid pairs are more susceptible to 71 variants from missense mutant human protein C precursor. The results show 85.92% of 71 variants occur at randomly unpredictable amino acid pairs accounting for 61.96% of amino acid pairs in protein C.

Binding of reduced nicotinamide adenine dinucleotide to yeast alcohol dehydrogenase results in a hypsochromic shift of its absorbance maximum at 340 nm. Application of high hydrostatic pressure to the enzymenucleotide complex returns the absorbance maximum to longer wavelengths. This pressure-dependent bathochromic shift validates one of two assignments on the effects of pressure on the kinetics of the enzymatic oxidation of benzyl alcohol, namely the protein-ligand conformational change of the capturing form of enzyme.