Disulfide bridges in interleukin-8 probed using non-natural disulfide analogues: Dissociation of roles in structure from function

Krishna Rajarathnam, Brian D. Sykes, Beatrice Dewald, Marco Baggiolini, Ian Clark-Lewis

Research output: Contribution to journalArticle

68 Scopus citations

Abstract

The structural and functional roles of the two disulfide bridges in interleukin-8 (IL-8) were addressed using IL-8 analogues with covalently modified disulfide bridges. The analogues Were prepared using chemical synthesis by replacement of a cysteine for either homocysteine, penicillamine, or selenocysteine and on folding resulted in a covalently modified disulfide. Deletion of either of the two disulfide bridges by replacement of either cysteine pair with alanine resulted in loss of both structure and function. In contrast, all of the analogues with modified disulfide bridges had native tertiary fold as determined by nuclear magnetic resonance spectroscopic methods. Their structural similarity provided a rational basis for assessing the functional effects of the changes to the disulfide. Modification to the disulfide bridge between cysteines 9 and 50 had only a modest effect on IL-8 function. In contrast, alterations to the 7- 34 disulfide bridge resulted in a dramatic reduction in biological potency. Thus, although both disulfide bridges are required for maintenance of the native tertiary fold, their role in determining IL-8 activity is distinct. We propose that 7-34 disulfide has a direct role in determining receptor binding and activation, whereas the 9-50 was not directly involved. The synthesis of nonnatural disulfide analogues is a novel general approach to structure- activity relationships of disulfide bridges. The demonstration that the participation of disulfide bridges in function can be dissociated from their effects on the stability of the tertiary structure suggests that this method Will lead to increased understanding of the roles of disulfide bridges in proteins.

Original languageEnglish (US)
Pages (from-to)7653-7658
Number of pages6
JournalBiochemistry
Volume38
Issue number24
DOIs
StatePublished - Jun 15 1999
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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