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

The structural and functional roles of the two disulfide bridges in interle ukin-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 seleno cysteine and on folding resulted in a covalently modified disulfide. Deleti on of either of the two disulfide bridges by replacement of either cysteine pair with alanine resulted in loss of both structure and function. In cont rast, all of the analogues with modified disulfide bridges had native terti ary fold as determined by nuclear magnetic resonance spectroscopic methods. Their structural similarity provided a rational basis for assessing the fu nctional effects of the changes to the disulfide. Modification to the disul fide bridge between cysteines 9 and 50 had only a modest effect on IL-8 fun ction. In contrast, alterations to the 7-34 disulfide bridge resulted in a dramatic reduction in biological potency. Thus, although both disulfide bri dges are required for maintenance of the native tertiary fold, their role i n determining n-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 analog ues is a novel general approach to structure-activity relationships of disu lfide bridges. The demonstration that the participation of disulfide bridge s in function can be dissociated from their effects on the stability of the tertiary structure suggests that this method will lead to increased unders tanding of the roles of disulfide bridges in proteins.