doi: 10.1016/j.str.2010.01.020.
Structure/function implications in a dynamic complex of the intrinsically disordered Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase
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- PMID:20399186
- PMCID: PMC2924144
- DOI: 10.1016/j.str.2010.01.020
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Structure/function implications in a dynamic complex of the intrinsically disordered Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase
Tanja Mittag et al. Structure..
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Abstract
Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations using experimental nuclear magnetic resonance and small-angle X-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated.
Copyright 2010 Elsevier Ltd. All rights reserved.
Figures
Residual dipolar couplings (RDCs,1DHN) measured in PEG/hexanol alignment medium and paramagnetic relaxation enhancements (PREs) for Sic1 and pSic1. (A, B) Experimental1DHN couplings (black) for Sic1 and pSic1 (A and B, respectively) and1DHN couplings calculated from a TraDES ‘coil’ ensemble (blue) or an ensemble containing fractions of secondary structure (magenta) reflecting experimental chemical shifts. Correlation coefficients between experimental and TraDES ‘coil’ RDCs are 0.29 and 0.24 (for Sic1 and pSic1, respectively) and between experimental RDCs and RDCs from secondary structure containing ensembles are 0.65 and 0.61. (C, D) Comparison between experimental1DHN couplings (black) and1DHN couplings calculated from final Sic1 (C, red), pSic1 (D, red) and pSic1-Cdc4 ensembles (D, green). Experimental uncertainties are estimated from duplicate or triplicate experiments; errors on calculated couplings are standard errors from triplicate ensembles. (E) PRE effects for Sic1 (black) and pSic1 (red) for six different single-cysteine mutants nitroxide spin-labeled at positions -1, 21, 38, 64, 83 and 90 (green arrows). PRE effects are calculated as differences between HNR2 rates of paramagnetic and diamagnetic samples. Large PRE effects sequence distant from the spin-labeled site report on transient tertiary contacts. Phosphorylation sites are indicated by red circles. See also Figure S2.
Small angle x-ray scattering (SAXS) data for Sic1 and pSic1. (A) SAXS scattering curves, (B) Kratky plots and (C) Porod plots for Sic1 (black) and pSic1 (red) confirm that both states are intrinsically disordered. The slopes in the Porod plots are d=−1.26 and d=−1.50 for Sic1 and pSic1, respectively, while they are expected to be d=−2 and d=−1 for a Gaussian chain and a rigid rod, respectively. (A) Experimental scattering curves (black and red solid lines) and those calculated from final ensembles (black and red dotted lines) are compared to scattering curves calculated for random coil ensembles generated with TraDES (blue dashed-dotted and dashed lines). (D, E)Rg distributions (mean value, solid lines) of Sic1 (D, black), pSic1 (E, red), and pSic1-Cdc4 ensembles (E, green) or of random coil ensembles (blue dashed-dotted and dashed lines). Dotted lines represent meanRg distributions plus or minus one standard error for triplicate ensembles.
Secondary structural properties of the Sic1 and pSic1 ensembles. (A-C) Fraction of conformers in broad α- (red) or broad β-region (blue) of the Ramachandran plot or cooperatively formed α-helix (green) as a function of the residue number for Sic1 (A), pSic1 (B) and the pSic1-Cdc4 complex (C). Each of the three individually calculated ensembles is represented separately. (D and E) Comparison of experimental SSP scores (black) and excess of conformers in the broad α versus the broad β region (red; green for dynamic complex model) in Sic1 (D) and pSic1 (E) ensembles. SSP scores are calculated from a comparison of experimental and random coil chemical shifts and chemical shifts for fully formed secondary structural elements and are an estimate of the fraction of backbone torsion angles ‘excess’ population in the α- or β-region. Phosphorylation sites are marked with red circles. See also Figure S1.
Fractional contact plots and cluster analysis of final ensembles. (A) Fractional contact plots for Sic1 (top left) and pSic1 (bottom right). The plot color-codes the fraction of conformers with heavy atom distances shorter than 6 Å. Positions of phosphorylation sites (Thr-5, Thr-33, Thr-45, Ser-69, Ser-76, Ser-80) are indicated by solid black lines and contacts involving CPDs are marked by ovals. Positions of spin labels are indicated by red dashed lines. (B, C) The conformers from the three combined final ensembles for (B) Sic1 and (C) pSic1 are partitioned into 8 and 5 clusters, respectively, based on Cα-RMSDs. Fractional contact plots and one representative conformer from each cluster are depicted for ascendingRg (meanRg for the cluster presented). Conformers are shown as rainbow-colored cartoons from blue to red from N- to C-terminus. See also Table S1 and Figure S4.
Hydrophobic and electrostatic interactions in the Sic1 and pSic1 ensembles. Number of atoms within a 10 Å radius from each Cα atom for (A) all residue types, (B) hydrophobic and (C) hydrophilic and charged residues. Values for experimentally-restrained ensembles (solid lines) and random-coil ensembles (dashed lines) are compared for Sic1 (black) and pSic1 (red). (D) Average electrostatic field as a function of residue number for the Sic1 (black) and pSic1 (red) ensembles (solid lines) and respective random coil ensembles (dashed lines). The field is calculated as a sum of fields from charged atoms within a radius of 10 Å from each Cα atom as described in Methods. Phosphorylation sites are depicted by magenta circles, positively charged residues by blue pluses.
(A) Structural model of the dynamic pSic1-Cdc4 complex. Of the 14 or 15 conformers within each complex ensemble, 2 or 3 conformers bind to Cdc4 via phosphorylation site pThr-5, 1 through pThr-33, 2 though pThr-45 and 3 conformers each bind via pSer69, pSer76 and pSer-80. pSic1 conformers are depicted as cartoons, with phosphorylated residues represented as sticks. Cdc4 is depicted as a ribbon diagram. The complex ensembles utilized NMR line-broadening data to restrain the fraction of individual CPDs bound to Cdc4 and structural restraints of free pSic1. CPD conformations are based on the structure of Cdc4 bound to a pSer-76/pSer-80 Sic1 phosphopeptide (S.O., X.T., F.S. and M.T., unpublished data) and on models calculated from the PDB coordinates 1NEX (Orlicky et al., 2003) using Modeller (Martí-Renom et al., 2000). (B) Structural model of pSic1 bound to the SCFCdc4 dimer/Cdc34 complex. The pSic1-Cdc4 dynamic complex ensemble is superimposed on a structural model of the SCFCdc4 ubiquitin ligase dimer (the E3) bound to Cdc34 (the E2) (Tang et al., 2007). Cdc4 is depicted in red, Cdc34 in magenta, the other subunits Skp1, Cdc53/Cul1 and Rbx1 in gray. One pSic1 ensemble is in blue, with the pSic1 ensemble binding to the other Cdc4 subunit shown in green. Lysine residues 32, 36, 50, 53, 84 and 88 in pSic1, each a possible site of ubiquitin conjugation, are shown in a space-filling representation. The catalytic site cysteine residue in Cdc34 is shown with a space-filling representation (gold). The gap of 64 Å between the target binding site on Cdc4 and the Cdc34 catalytic cysteine is easily spanned by extended conformations of pSic1. Figures are made using the program PyMOL (DeLano, 2002). See also Figure S5.
Comment in
- The meandering of disordered proteins in conformational space.Konrat R.Konrat R.Structure. 2010 Mar 14;18(4):416-9. doi: 10.1016/j.str.2010.03.003.Structure. 2010.PMID:20399178
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