Binding of amino acid side chains to preformed cavities: Interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P<sub>1</sub> residues

Bigler, Theresa L.; Lu, Wuyuan; Park, Soon Jae; Tashiro, Misao; Wieczorek, Maciej; Wynn, Richard; Laskowski, M.

doi:10.1002/pro.5560020509

Cited by 60 publications

(60 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Several techniques for site-specific unnatural modifications of proteins are currently in practice, including total chemical synthesis (32,38,39), in vitro translation (40 -42), auxotrophic bacterial expression (43)(44)(45)(46), enzymatic semisynthesis (47)(48)(49), native chemical ligation (30,50), expressed protein ligation (51), and, more recently, expression in Escherichia coli by using novel orthogonal tRNA͞synthetase pairs (52,53). Among these methods, total chemical synthesis coupled with native chemical ligation has proven to be a robust and versatile technique for the production of small-to medium-sized proteins (54).…”

Section: Myristoylation Of P17 Results In Little Structural Change Anmentioning

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.

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Myristoylation Of P17 Results In Little Structural Change Anmentioning

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.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Inhibitor binding by subtilisins and chymotrypsins has been studied most extensively at the structural level by using the inhibitor eglin c (Read & James, 1986; Bode & Huber, 1992), and at the kinetic level by using avian ovomucoid third domains (Laskowski et al, 1987;Bigler et al, 1993). It is clear from both types of study that the binding is equivalent, but certain distinctions are evident.…”

Section: Discussionmentioning

confidence: 99%

“…It is supported by solution kinetic data showing that members of both families are inhibited, often with very similar binding constants, by the same protein inhibitors (Read & James, 1986;Bigler et al, 1993;Greagg et al, 1994). Therefore, the subtilisins and chymotrypsins not only have a similar mechanism of catalysis, they also have a similar mechanism of binding to inhibitors, such as eglin c, that obey the standard mechanism of inhibition (Laskowski & Kato, 1980).…”

mentioning

confidence: 90%

“…Therefore, the subtilisins and chymotrypsins not only have a similar mechanism of catalysis, they also have a similar mechanism of binding to inhibitors, such as eglin c, that obey the standard mechanism of inhibition (Laskowski & Kato, 1980). When comparing subtilisins with chymotrypsins, there has been a general focus on standard mechanism protein inhibitors that include members of the Kazal, eglin, and Streptomyces subtilisin inhibitor families (Read & James, 1986;Takeuchi et al, 1991;Bode& Huber, 1992;Bigler et al, 1993). There has been very little focus on the serpins, which may not operate by the standard mechanism and which have been studied almost exclusively with the chymotrypsin family.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Interaction of subtilisins with serpins

et al. 1996

View full text Add to dashboard Cite

Serpins are well-characterized inhibitors of the chymotrypsin family serine proteinases. We have investigated the interaction of two serpins with members of the subtilisin family, proteinases that possess a similar catalytic mechanism to the chymotrypsins, but a totally different scaffold. We demonstrate that a'proteinase inhibitor inhibits subtilisin Curlsberg and proteinase K, and alantichymotrypsin inhibits proteinase K, but not subtilisin Carlsberg. When inhibition occurs, the rate of formation and stability of the complexes are similar to those formed between serpins and chymotrypsin family members. However, inhibition of subtilisins is characterized by large partition ratios where more than four molecules of each serpin are required to inhibit one subtilisin molecule. The partition ratio is caused by the serpins acting as substrates or inhibitors. The ratio decreases as temperature is elevated in the range 0-45 "C, indicating that the serpins are more efficient inhibitors at high temperature. These aspects of the subtilisin interaction are all observed during inhibition of chymotrypsin family members by serpins, indicating that serpins accomplish inhibition of these two distinct proteinase families by the same mechanism.

show abstract

“…All peptides were purified to homogeneity by RP-HPLC and their molecular weights verified by electrospray ionization MS (ESI-MS). ␣-aminobutyric acid (Abu) is often considered to be isosteric to Cys (29,30) and therefore was our preferred amino acid used to remove disulfide bridges in hBD3. When assessed by circular dichroism spectroscopy, [Abu]-hBD3, i.e., disulfide-devoid hBD3 in which Abu replaces all six Cys residues, was found largely unstructured in aqueous solution.…”

Section: Methodsmentioning

confidence: 99%

Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human β-defensin 3

Hoover²,

Yang³

et al. 2003

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

Self Cite

411

431

View full text Add to dashboard Cite

Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into ␣ and ␤ families. Structures of several ␤-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., Cys 1 -Cys 5 , Cys 2 -Cys 4 , and Cys 3 -Cys 6 . We found that human ␤-defensin 3 (hBD3), a recently described member of the growing ␤ family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of Cys 1 -Cys 5 and of Cys 1 -Cys 6 , we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native ␤ pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coli, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by ␣-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human ␤-defensins and the design of novel peptide antibiotics.

show abstract

Binding of amino acid side chains to preformed cavities: Interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P₁ residues

Cited by 60 publications

References 53 publications

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

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

Interaction of subtilisins with serpins

Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human β-defensin 3

Contact Info

Product

Resources

About

Binding of amino acid side chains to preformed cavities: Interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P1 residues

Cited by 60 publications

References 53 publications

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

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

Interaction of subtilisins with serpins

Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human β-defensin 3

Contact Info

Product

Resources

About

Binding of amino acid side chains to preformed cavities: Interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P₁ residues