Deciphering the Role of the Electrostatic Interactions Involving Gly70 in Eglin C by Total Chemical Protein Synthesis

Lü, Wei; Starovasnik, Melissa A.; Dwyer, John J.; Kossiakoff, Anthony A.; Kent, Stephen B. H.; Lu, Wuyuan

doi:10.1021/bi992292q

Cited by 36 publications

(44 citation statements)

References 32 publications

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“…Second, loops are in general flexible, assuming a low free energy barrier to the transition state of the productive enzyme-substrate complex. Thus, conformational rigidity in the loop region of a protein is critical for "slowing down" otherwise efficient proteolytic cleavage (45). Understandably, fur-…”

Section: Discussionmentioning

confidence: 99%

The Conserved Salt Bridge in Human α-Defensin 5 Is Required for Its Precursor Processing and Proteolytic Stability

Rajabi

Leeuw

Pazgier

et al. 2008

Journal of Biological Chemistry

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Mammalian ␣-defensins, expressed primarily in leukocytes and epithelia, play important roles in innate and adaptive immune responses to microbial infection. Six invariant cysteine residues forming three indispensable disulfide bonds and one Gly residue required structurally for an atypical ␤-bulge are totally conserved in the otherwise diverse sequences of all known mammalian ␣-defensins. In addition, a pair of oppositely charged residues (Arg/Glu), forming a salt bridge across a protruding loop in the molecule, is highly conserved. To investigate the structural and functional roles of the conserved Arg 6 -Glu 14 salt bridge in human ␣-defensin 5 (HD5), we chemically prepared HD5 and its precursor proHD5 as well as their corresponding salt bridge-destabilizing analogs E14Q-HD5 and E57Q-proHD5. The Glu-to-Gln mutation, whereas significantly reducing the oxidative folding efficiency of HD5, had no effect on the folding of proHD5. Bovine trypsin productively and correctly processed proHD5 in vitro but spontaneously degraded E57Q-proHD5. Significantly, HD5 was resistant to trypsin treatment, whereas E14Q-HD5 was highly susceptible. Further, degradation of E14Q-HD5 by trypsin was initiated by the cleavage of the Arg 13 -Gln 14 peptide bond in the loop region, a catastrophic proteolytic event resulting directly in quick digestion of the whole defensin molecule. The E14Q mutation did not alter the bactericidal activity of HD5 against Staphylococcus aureus but substantially enhanced the killing of Escherichia coli. By contrast, proHD5 and E57Q-proHD5 were largely inactive against both strains at the concentrations tested. Our results confirm that the primary function of the conserved salt bridge in HD5 is to ensure correct processing of proHD5 and subsequent stabilization of mature ␣-defensin in vivo.Defensins are expressed predominantly in leukocytes and epithelial cells as a family of cationic antimicrobial peptides that play important roles in innate and adaptive immune responses to microbial infection (1-5). In humans, defensins are classified into ␣-and ␤-families, differing by amino acid composition, cellular origin, and disulfide connectivity. To date, six human ␣-defensins have been identified, including four primarily from neutrophils, also known as human neutrophil peptides 1-4 or HNP1-4 2 (6 -10) and two mainly from intestinal Paneth cells, also known as human defensins 5-6 or 12). Although more than 30 putative ␤-defensin genes have been described using genomics tools (13-15), only a handful of ␤-defensins have been isolated and characterized at the protein level (16).Human ␣-defensins are synthesized in vivo as significantly larger precursor molecules consisting of an N-terminal pro domain of 43-49 amino acid residues and a C-terminal Cysrich defensin domain of 29 -33 amino acid residues. The pro domain in proHNPs, important for correct folding, sorting, and trafficking of defensins (17-20), is proteolytically excised by yet-to-be-identified enzyme(s) prior to the storing of mature HNPs in the azurophilic g...

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Section: Discussionmentioning

confidence: 99%

The Conserved Salt Bridge in Human α-Defensin 5 Is Required for Its Precursor Processing and Proteolytic Stability

Rajabi

Leeuw

Pazgier

et al. 2008

Journal of Biological Chemistry

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“…The experimental data were subjected to a six-parameter nonlinear regression analysis by using an equation published in ref. 34 that was derived from a two-state protein denaturation model, yielding the free energy change of unfolding, ⌬G°(kcal͞mol), at zero GuHCl concentration.…”

Section: Methodsmentioning

confidence: 99%

Total chemical synthesis of N-myristoylated HIV-1 matrix protein p17: Structural and mechanistic implications of p17 myristoylation

Alexandratos

Ericksen

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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The HIV-1 matrix protein p17, excised proteolytically from the N terminus of the Gag polyprotein, forms a protective shell attached to the inner surface of the plasma membrane of the virus. During the late stages of the HIV-1 replication cycle, the N-terminally myristoylated p17 domain targets the Gag polyprotein to the host-cell membrane for particle assembly. In the early stages of HIV-1 replication, however, some p17 molecules dissociate from the viral membrane to direct the preintegration complex to the host-cell nucleus. These two opposing targeting functions of p17 require that the protein be capable of reversible membrane interaction. It is postulated that a significant structural change in p17 triggered by proteolytic cleavage of the Gag polyprotein sequesters the N-terminal myristoyl group, resulting in a weaker membrane binding by the matrix protein than the Gag precursor. To test this ''myristoyl switch'' hypothesis, we obtained highly purified synthetic HIV-1 p17 of 131 amino acid residues and its N-myristoylated form in large quantity. Both forms of p17 were characterized by circular dichroism spectroscopy, protein chemical denaturation, and analytical centrifugal sedimentation. Our results indicate that although N-myristoylation causes no spectroscopically discernible conformational change in p17, it stabilizes the protein by 1 kcal͞ mol and promotes protein trimerization in solution. These findings support the premise that the myristoyl switch in p17 is triggered not by a structural change associated with proteolysis, but rather by the destabilization of oligomeric structures of membranebound p17 in the absence of downstream Gag subdomains.

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“…From the structures of intact inhibitors bound to proteases, it has widely been thought that the rigidity of the enzyme-inhibitor complexes effectively blocks the catalytic mechanism before the formation of the tetrahedral intermediate or acyl-enzyme. An alternative, or supplementary, model suggests that the acyl-enzyme intermediate forms readily, but that the peptide bond is more rapidly reformed, so that the intact form seen in crystal structures predominates (26)(27)(28)(29). This model is supported by experiments directly demonstrating formation of an acyl-enzyme intermediate by subtilisin and chymotrypsin inhibitor II (29) and analysis of the pH dependence of inhibitor hydrolysis rates (45).…”

Section: (Pdb Id Code 2age) (E) the Tetrahedral Intermediate For Hydmentioning

confidence: 99%

“…Because the products of hydrolysis remain physically associated, the resynthesis of these inhibitors is an intramolecular reaction and is, therefore, much more favorable than the equivalent intermolecular reaction at modest reactant concentrations. Despite the prevalence of Laskowski inhibitors (25), the mechanisms by which they resist proteolysis remain poorly understood (26)(27)(28)(29).…”

mentioning

confidence: 99%

Structure of a serine protease poised to resynthesize a peptide bond

Zakharova

Horvath

Goldenberg

2009

Proc. Natl. Acad. Sci. U.S.A.

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The serine proteases are among the most thoroughly studied enzymes, and numerous crystal structures representing the enzymesubstrate complex and intermediates in the hydrolysis reactions have been reported. Some aspects of the catalytic mechanism remain controversial, however, especially the role of conformational changes in the reaction. We describe here a high-resolution (1.46 Å) crystal structure of a complex formed between a cleaved form of bovine pancreatic trypsin inhibitor (BPTI) and a catalytically inactive trypsin variant with the BPTI cleavage site ideally positioned in the active site for resynthesis of the peptide bond. This structure defines the positions of the newly generated amino and carboxyl groups following the 2 steps in the hydrolytic reaction. Comparison of this structure with those representing other intermediates in the reaction demonstrates that the residues of the catalytic triad are positioned to promote each step of both the forward and reverse reaction with remarkably little motion and with conservation of hydrogen-bonding interactions. The results also provide insights into the mechanism by which inhibitors like BPTI normally resist hydrolysis when bound to their target proteases.trypsin ͉ bovine pancreatic trypsin inhibitor ͉ enzyme mechanisms S erine proteases are found throughout all 3 domains of cellular life and function in a wide range of physiological processes, including digestion, protein maturation and turnover, hemostasis, and immune responses (1). Approximately 0.6% of human protein-encoding genes are predicted to specify serine proteases, and this family is even more prevalent in other organisms, notably the arthropods (2, 3). A large body of biochemical and structural data have established a 2-step mechanism for hydrolysis of peptide bonds by this class of proteases (4), as shown in Scheme 1.The first step of the reaction is a nucleophilic attack by the catalytic serine residue (Ser-195 in trypsin and other members of the chymotrypsin, or S1, family) on the carbonyl carbon atom of the residue labeled P1, generating a covalent acyl-enzyme intermediate and a new peptide amino terminus, on the P1Ј residue. A second nucleophilic attack, by a water molecule, leads to hydrolysis of the acyl-enzyme, releasing the new carboxyl group and restoring the catalytic Ser residue to its initial state.

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Deciphering the Role of the Electrostatic Interactions Involving Gly70 in Eglin C by Total Chemical Protein Synthesis

Cited by 36 publications

References 32 publications

The Conserved Salt Bridge in Human α-Defensin 5 Is Required for Its Precursor Processing and Proteolytic Stability

The Conserved Salt Bridge in Human α-Defensin 5 Is Required for Its Precursor Processing and Proteolytic Stability

Total chemical synthesis of N-myristoylated HIV-1 matrix protein p17: Structural and mechanistic implications of p17 myristoylation

Structure of a serine protease poised to resynthesize a peptide bond

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