Structural comparison of two serine proteinase-protein inhibitor complexes: Eglin-C-subtilisin Carlsberg and CI-2-subtilisin Novo

McPhalen, Catherine A.; James, Michael N.G.

doi:10.1021/bi00417a058

Cited by 333 publications

(247 citation statements)

References 61 publications

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“…The structure consists of a well defined rigid core, with an cl-helix on one side, a four stranded B-sheet and a binding loop with high mobility. Eglin c is a member of the potato inhibitor family [25] where the homologous structure of the proteinase inhibitor CI-2 from barley seeds has been determined in complex with proteinases by X-ray crystallography [26]. The structure of eglin in the crystal is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Structure of the proteinase inhibitor eglin c with hydrolysed reactive centre at 2.0 Å resolution

et al. 1993

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The inhibition of serine proteinases by both synthetic and natural inhibitors has been widely studied. Eglin c is a small thermostable protein isolated from the leech, Hirudo medicinalis. Eghn c is a potent serine proteinase inhibitor. The three-dimensional structure of native eghn and of its complexes with a number of proteinases are known. We here describe the crystal structure of hydrolysed eghn not bound to a proteinase. The body of the eghn has a conformation remarkably similar to that in the known complexes with proteinases. However, the peptide chain has been cut at the 'scissile' bond between residues 45 and 46, presumed to result from the presence of subtilisin DY in the crystallisation sample. The residues usually making up the inhibiting loop of eghn take up a quite different conformation in the nicked inhibitor leading to stabilising contacts between neighbouring molecules in the crystal. The structure was solved by molecular replacement techniques and refined to a final R-factor of 14.5%.

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Section: Resultsmentioning

confidence: 99%

Structure of the proteinase inhibitor eglin c with hydrolysed reactive centre at 2.0 Å resolution

et al. 1993

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“…CPhilipp and Bender (1983);Carter et al (1989). dInferred from inspection of X-ray structures (Robertus et al, 1972;McPhalen & James, 1988) and from Morihara et al (1970). CCarter and Wells (1987).…”

Section: N F I B Itormentioning

confidence: 99%

Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution

Abrahmsén¹,

Tom²,

Burnier³

et al. 1991

Biochemistry

242

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Protein engineering techniques were used to construct a derivative of the serine protease subtilisin that ligates peptides efficiently in water. The subtilisin double mutant in which the catalytic Ser221 was converted to Cys (S221C) and Pro225 converted to Ala (P225A) has 10-fold higher peptide ligase activity and at least 100-fold lower amidase activity than the singly mutated thiolsubtilisin (S221C) that was previously shown to have some peptide ligase activity [Nakatsuka, T., Sasaki, T., & Kaiser, E. T. (1987) J. Am. Chem. SOC. 109, 3808-38101. A 1.5-A X-ray crystal structure of an oxidized derivative of the double mutant (S22 1C/P225A) supports the protein design strategy in showing that the P225A mutation partly relieves the steric crowding expected from the S221 C substitution, thus accounting for its improved catalytic efficiency. Stable and synthetically reasonable alkyl ester peptide substrates were prepared that rapidly acylate the S22 1C/P225A enzyme, and aminolysis of the resulting thioacyl-enzyme intermediate by various peptides is strongly preferred over hydrolysis. The efficiency of aminolysis is relatively insensitive to the sequence of the first two residues in the acyl acceptor peptide whose a-amino group attacks the thioacyl-enzyme.To obtain greater flexibility in the choice of coupling sites, a set of three additional peptide ligases were engineered by introducing mutations into the parent ligase (S22 1 C/P225A) that were previously shown to change the specificity of subtilisin for the residue nearest the acyl bond (the P, residue). The specificity properties of the parent ligase and derivatives of it paralleled those of wild type and corresponding specificity variants. The set of specific peptide ligases should be useful for blockwise synthesis or semisynthesis of proteins in aqueous solution.C h e m i c a l approaches for synthesis and engineering of proteins offer many advantages to recombinant methods in that one can incorporate nonnatural or selectively labeled amino acids. However, peptide synthesis is practically limited to small proteins (typically <50 residues) due to the accumulation of side products and racemization that complicate product purification and decrease yields [for recent reviews see Kaiser (1989) and Offord (1987)l.Proteolytic enzymes, in particular serine proteases, have been used as alternatives to synthetic peptide chemistry because of their stereoselective properties and mild reaction conditions [for reviews see Kullmann (1987) and Chaiken (1981)l. Such enzymes have also been used to complement chemical coupling methods and allow larger proteins to be synthesized by blockwise enzymatic coupling of synthetic fragments [Tnouye et al., 1979; for review see Chaiken (1981)l. However, the narrow substrate specificities and hydrolytic activities of serine proteases have limited their use in peptide synthesis.A central problem in the use of serine proteases for peptide synthesis is that hydrolysis of the acyl-enzyme intermediate is strongly favored over aminoly...

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“…subtilisin BPN' (Hirono et al, 1984;McPhalen et al, 1985;McPhalen and James, 1988;Heinz et al, 1991;Takeuchi et al, 1991a,b), subtilisin Carlsberg (Bode et al, 1986;McPhalen and James, 1988), subtilisin BL (Godette et al, 1992), high-alkaline protease (Betzel et al, 1992;Sobek et al, 1992; Van der Laan et al, 1992;Lange et al, 1994), mesentericopeptidase (Dauter The pro-nisin part is fully modified, as indicated by the modified residues. Dha, dehydroalanine; Dhb, dehydrobutyrine; Ala-S-Ala, lanthionine; Abu-S-Ala, β-methyllanthionine.…”

Section: Introductionmentioning

confidence: 99%

“…Residues in subtilases that are in contact with substrate or inhibitor have been identified from crystal structures and modelling of enzyme-inhibitor complexes (Hirono et al, 1984;McPhalen et al, 1985;Bode et al, 1987;Betzel et al, 1988bBetzel et al, , 1993McPhalen and James, 1988;Gros et al, 1989;Dauter et al, 1991;Heinz et al, 1991;Takeuchi et al, 1991a,b) and enzyme-substrate complexes (Wells et al, 1987a), and from protein engineering studies of subtilisin BPN' (Estell et al, 1986;Wells et al, 1987a,b). In each case the mode of substrate-inhibitor binding appears to be essentially the same, and this provides a sound basis for predicting enzyme-substrate interactions in other members of this family.…”

Section: Introductionmentioning

confidence: 99%

Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

Siezen¹,

Rollema²,

Kuipers³

et al. 1995

Protein Eng Des Sel

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Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 12-05-2018Protein Engineering vol.8 no.2 pp. [117][118][119][120][121][122][123][124][125] 1995 Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin ITo whom correspondence should be addressed A model is presented for the 3-D structure of the catalytic domain of the putative leader peptidase NisP of Lactococcus lactis, and the interaction with its specific substrate, the precursor of the lantibiotic nisin. This homology model is based on the crystal structures of subtilisin BPN' and thermitase in complex with the inhibitor eglin. Predictions are made of the general protein fold, inserted loops, Ca2+ binding sites, aromatic interactions and electrostatic interactions of NisP. Cleavage of the leader peptide from precursor nisin by NisP is the last step in maturation of nisin. A detailed prediction of the substrate binding site attempts to explain the basis of specificity of NisP for precursor nisin. Specific acidic residues in the SI sub site of the substrate binding region of NisP appear to be of particular importance for electrostatic interaction with the PI Arg residue of precursor nisin after which cleavage occurs. The hydrophobic S4 subsite of NisP may also contribute to substrate binding as it does in subtilisins. Predictions of enzyme-substrate interaction were tested by protein engineering of precursor nisin and determining susceptibility of mutant precursors to cleavage by NisP. An unusual property of NisP predicted from this catalytic domain model is a surface patch near the substrate binding region which is extremely rich in aromatic residues. It may be involved in binding to the cell membrane or to hydrophobic membrane proteins, or it may serve as the recognition and binding region for the modified, hydrophobic C-terminal segment of precursor nisin. Similar predictions for the tertiary structure and substrate binding are made for the highly homologous protein EpiP, the putative leader peptidase for the lantibiotic epidermin from Staphylococcus epidermidis, but EpiP lacks the aromatic patch. Based on these models, protein engineering can be employed not only to test the predicted enzyme-substrate interactions, but also to design lantibiotic leader peptidases with a desired specificity.

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Structural comparison of two serine proteinase-protein inhibitor complexes: Eglin-C-subtilisin Carlsberg and CI-2-subtilisin Novo

Cited by 333 publications

References 61 publications

Structure of the proteinase inhibitor eglin c with hydrolysed reactive centre at 2.0 Å resolution

Structure of the proteinase inhibitor eglin c with hydrolysed reactive centre at 2.0 Å resolution

Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution

Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

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