Conformational transitions of the intrinsically disordered protein sic1 from the yeast saccharomyces cerevisiae. Towards structural and functional characterization of the whibitory complex with CDK1-CLB5

Many naturally occurring proteins have been shown to lack rigid three-dimensional structure, existing instead as dynamic ensembles of inter-converting conformations. Many of these proteins acquire an ordered structure only upon binding to specific intracellular partners. In isolation, these proteins exhibit a highly dynamic structure that is resembling more the denatured rather than native state of “normal” globular proteins. Intrinsically disordered proteins (IDPs) have attracted a great deal of interest since it became clear that their lack of structural specificity is of physiological importance. Indeed, structural disorder characterizes a broad class of regulatory proteins, which all share the feature to bind multiple interactors. Intrinsic disorder is a common property of many protein types especially among the proteins involved in signal transduction and transcription regulation. The dynamic properties of IDPs are considered to be instrumental to adaptation to different interaction surfaces and to favor rapid formation and dissociation of the complexes, as required for efficient intracellular regulatory networks. In the yeast Saccharomyces cerevisiae, Sic1 is a central regulatory protein of the cell cycle, acting as inhibitor of the cyclin-dependent protein kinases by forming ternary complexes with kinases and their cognate cyclins. A well established role of Sic1 is to control the timing of entrance in S phase during the yeast cell cycle by inhibiting the complex Cdk1-Clb5/6, whose activity is required for the G1-to-S transition. The inhibition is released upon ubiquitin-dependent Sic1 degradation, which is, in turn, triggered by Sic1 phosphorylation at multiple sites in its N-terminal region. Previous work has shown that Sic1 is disordered in its whole length, with some intrinsic propensity to form ordered helical structure. The Sic1 functional kinase-inhibitor domain (KID) corresponds to the C-terminal 70, out of 284, amino acids forming the native protein. This Sic1 C-terminal region is structurally and functionally related to the mammalian p21 and p27 KIDs that are located, instead, at the protein N-terminus. A detailed structural characterization of the mammalian proteins have been achieved, both on their unbound states and on the complexes with their partners. Structural characterization of proteins in disordered conformation is technically difficult, but it is important to better understand folding transitions to ordered states. In this work, an in-depth description of Sic1 in the absence of interactors, both in terms of secondary and tertiary structure, is presented. A multiparametric analysis, which employed a set of complementary methods sensitive to distinct structural features, has been used to guarantee the description of such a highly dynamic and heterogeneous molecular ensemble. First of all, a novel tool for extracting structural information from electrospray-ionization mass spectrometry (ESI-MS) data has been developed. It has been shown that the extent of protein ionization under nondenaturing conditions correlates with the solvent-accessible surface area (SASA), for either folded or unfolded proteins. Therefore, the technique has been employed to estimate the SASA of Sic1. Fragments corresponding to the N- and C-terminal moieties of Sic1 have been produced, and circular dichroism (CD) data showed that the little content in secondary structure is distributed quite uniformly throughout the chain length, although the C-terminus is slightly more ordered than the N-terminus. Consistent with such evidence, conformational analysis by ESI-MS suggested that the Sic1 C-terminal domain is more structured than its complementary N-terminal domain. Thus, altogether, functional and structural features pointed to a modular organization of this protein, despite its disordered nature. A more detailed description of the Sic1 KID has been achieved by integrating an array of biophysical data with all-atom molecular dynamics (MD) simulations. Highly dynamic helical elements are detected by Fourier-transformed infrared (FT-IR) spectroscopy, while protein tertiary structure has been probed by nondenaturing gel filtration, ESI-MS, and electrospray-ionization ion-mobility mass-spectrometry (ESI-IM-MS). The molecular ensemble of the isolated KID fragment has been found to interconvert between collapsed states of different compactness, with a small fraction of the population found in a highly compact state. MD simulations results has suggested a predominant role of electrostatic interactions in promoting the compaction in the Sic1 inhibitory domain. Moreover, comparison to the full-length protein hinted to a critical role of chain length in determining the overall compaction of Sic1. Since no structural data are available for the Sic1-Cdk1-Clb5/6 ternary complex, the molecular mechanism by which Sic1 inhibits S-Cdk1 activity remains unclear. In this work, the results about the heterologous expression and purification of Cdk1 and Clb5 are also presented. Cdk1 has been overexpressed as a histidine-tagged protein in Escherichia coli cells and CD analysis has revealed a similar fold to mammalian homologues. The full-length Clb5, on the contrary, underwent proteolytic degradation when expressed in E. coli cells, probably because of the high degree of disorder in the N-terminal region of the amino-acids sequence. The deletion of the first 156 residues has allowed the expression of the protein as inclusion bodies (IBs). A purification procedure has been then developed, so that IBs have been solubilized and refolded by on-column removal of denaturing conditions. The results concerning the expression and the characterization of Cdk1 and Clb5 represent a starting points for the production of a functional Cdk1-Clb5 complex and so, ultimately, for the complete description of Sic1 binding mechanism.