Abstract
The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (Le-Nguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway.
Highlights
The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif
Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (LeNguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165–174)
This knotted topology was originally determined for kalata B1 using NMR data [11] but the corresponding disulfide connectivity of other members of the cyclotide family has since been confirmed by synthetic methods involving directed disulfide bond formation [7, 15]
Summary
A third subfamily has been proposed based on a determination of the three-dimensional structure of MCoTI-II [13] This macrocyclic trypsin inhibitor was discovered in the seeds of a plant from the Cucurbitaceae, or squash, family [10], and its structure contains the CCK motif [13, 14] seen in the previously reported cyclotides despite having no sequence homology except for the six cysteine residues. In the current study we have examined the oxidative refolding and reductive unfolding of kalata B1 and characterized a major folding intermediate The properties of this intermediate, when compared with a topologically similar intermediate characterized for EETI-II [22], reveal fundamental differences in the folding pathways of cystine knot proteins with cyclic backbones relative to those with conventional, acyclic, backbones. Part of the study required the synthesis of a mutant in which a single disulfide bond is removed and we show that this adopts a native-like fold, consistent with expectations from the identified folding intermediate
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