The heterogeneous and dynamic nature of IDPs makes structural characterization of their unbound state highly challenging. Although MD force fields have been developed to simulate protein folding, they have also proven useful to characterize the conformational ensembles of IDPs and unfolded proteins (Espinoza-Fonseca, 2009a
; Cino et al., 2011
; Arrigoni et al., 2012
; Ganguly et al., 2012
; Lindorff-Larsen et al., 2012
; Knott and Best, 2012
), especially when computational results are complemented by biophysical data. These studies contribute to enforce the applicability of classical MD to complex molecular ensembles. Nevertheless, analysis of dynamic and heterogeneous systems such as IDPs has to face limits in force field accuracy and sampling efficacy (Esteban-Martin et al., 2012
). Thus, while helping description of globular IDP states, classical MD simulations are not adequate to describe the actual equilibrium between extended and compact conformations. This complementary information can be provided by experimental assessment of species distributions, for instance by MS (Kaltashov and Abzalimov, 2008
) or NMR investigation (Esteban-Martin et al., 2012
; Schneider et al., 2012
We employ here atomistic, explicit-solvent MD simulations integrated by experimental data (Brocca et al., 2011b
) to provide a first atomic-level description of the conformational ensemble of compact states of the isolated Sic1 KID fragment. The results indicate that, in spite of its strong propensity for structural disorder, Sic1 KID can explore compact conformations, with considerable secondary and tertiary structure. The extents of secondary structure and solvent accessibility derived by the simulations are in good agreement with experimental results obtained by FT-IR spectroscopy and ESI-MS (Brocca et al., 2011b
). The conformational ensemble of Sic1 KID reveals a highly dynamic behavior, populating several different conformations. Also local conformations, such as helical IFSUs, are likely to be dynamic.
The present results could also be interpreted in the light of the structural and functional relation to the mammalian p21 and p27 KID domains (Barberis et al., 2005
). In fact, it has been shown that p27 can replace Sic1 in yeast cells (Barberis et al., 2005
) and that Sic1 KID can functionally interact with mammalian Cyclin A-Cdk2 inhibiting its kinase activity. The interaction between p27 and cyclin A/Cdk2 has been investigated suggesting a two-site, sequential binding process, in which p27 KID first interacts at one end with cyclin A (sub-domain D1) and then binds to Cdk2 by the other end (sub-domain D2), wrapping the central helical region (sub-domain LH) around the cyclin/kinase complex (Sivakolundu et al., 2005
; Galea et al., 2008
; Espinoza-Fonseca, 2009b
; Otieno et al., 2011
). The present results point to the stretch between E223 and L238 as the most persistent α-helix of Sic1 KID, while a shorter and transiently populated α-helix approximately maps between residues I244 and I248. Although it is difficult to identify the exact boundaries of the helical regions by MD simulations of such a highly heterogeneous system, these α-helical regions of Sic1 correspond to the p27 LH sub-domain (residues 38–60), according to the structural alignment of the two KID domains (Barberis et al., 2005
). This sub-domain has been identified as an IFSU also in p27 (Sivakolundu et al., 2005
) and it is thought to play a role tethering the D1 to the D2 sub-domain and enhancing the overall ΔG of binding (Otieno et al., 2011
). The corresponding and structurally similar region of Sic1 KID might, therefore, promote binding to the cyclin/kinase complex by a similar mechanism. More studies will be needed to test this hypothesis, and further biochemical investigation will be needed to characterize the physiological intermediates of Sic1 binding.
Moreover, according to the MD scenario reported here, electrostatic interactions seem to be the major determinant of structural compaction in the isolated Sic1 KID. This result of the MD simulations is supported by experimental evidence by ESI-MS. Furthermore, this conclusion is consistent with the low mean hydropathy and low mean net charge per residue of this protein (Brocca et al., 2009
) and is in agreement with the current view on the importance of charged residues defining IDP structural and functional properties (Uversky et al., 2000
Electrostatic interactions could also be relevant in vivo
, in relation to the multiple phosphorylation events that regulate Sic1 interactions (Nash et al., 2001
; Mittag et al., 2008
; Koivomagi et al., 2011
). By altering short- and long-range electrostatic interactions, phosphorylation could effectively modulate the conformational properties of this IDP, even far from the site of the modification (Johnson and Lewis, 2001
; Arrigoni et al., 2012
In particular, the present analysis points out that the globular states of Sic1 KID are stabilized by interconnected networks of electrostatic interactions with a few hub residues common to different conformations and involved in multiple paths. R270, K268, E267, E245, R261, and D265 emerge as the most relevant ones. These residues represent good targets for mutagenesis experiments to further explore the role of such networks in Sic1 KID structure. Although our results do not hint to a major role of hydrophobic residues in intramolecular networks, these could still contribute to global compaction of the domain. Further experiments will be necessary to investigate their structural role.
In conclusion, the here provided experimental and computational evidence indicates that Sic1 KID, though highly disordered, can acquire transient secondary and tertiary structure populating compact conformations.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.