Dynamic equilibrium engagement of a polyvalent ligand with a single-site receptor

Intrinsically disordered proteins play critical but often poorly understood roles in mediating protein interactions. The interactions of disordered proteins studied to date typically entail structural stabilization, whether as a global disorder-to-order transition or minimal ordering of short linear motifs. The disordered cyclin-dependent kinase (CDK) inhibitor Sic1 interacts with a single site on its receptor Cdc4 only upon phosphorylation of its multiple dispersed CDK sites. The molecular basis for this multisite-dependent interaction with a single receptor site is not known. By NMR analysis, we show that multiple phosphorylated sites on Sic1 interact with Cdc4 in dynamic equilibrium with only local ordering around each site. Regardless of phosphorylation status, Sic1 exists in an intrinsically disordered state but is surprisingly compact with transient structure. The observation of this unusual binding mode between Sic1 and Cdc4 extends the understanding of protein interactions from predominantly static complexes to include dynamic ensembles of intrinsically disordered states.

[1]  Peter E Wright,et al.  Interaction of the TAZ1 Domain of the CREB-Binding Protein with the Activation Domain of CITED2 , 2004, Journal of Biological Chemistry.

[2]  T. Gibson,et al.  Systematic Discovery of New Recognition Peptides Mediating Protein Interaction Networks , 2005, PLoS biology.

[3]  István Simon,et al.  Preformed structural elements feature in partner recognition by intrinsically unstructured proteins. , 2004, Journal of molecular biology.

[4]  James E. Ferrell,et al.  Substrate Competition as a Source of Ultrasensitivity in the Inactivation of Wee1 , 2007, Cell.

[5]  T. Pawson,et al.  Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. , 1994, Biochemistry.

[6]  Mike Tyers,et al.  F-Box Proteins Are Receptors that Recruit Phosphorylated Substrates to the SCF Ubiquitin-Ligase Complex , 1997, Cell.

[7]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[8]  Robin S. Dothager,et al.  Random-coil behavior and the dimensions of chemically unfolded proteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[9]  L. Iakoucheva,et al.  The importance of intrinsic disorder for protein phosphorylation. , 2004, Nucleic acids research.

[10]  P. Tompa,et al.  Fuzzy complexes: polymorphism and structural disorder in protein-protein interactions. , 2008, Trends in biochemical sciences.

[11]  H. Dyson,et al.  Intrinsically unstructured proteins and their functions , 2005, Nature Reviews Molecular Cell Biology.

[12]  H. Dyson,et al.  Mechanism of coupled folding and binding of an intrinsically disordered protein , 2007, Nature.

[13]  J Wade Harper,et al.  Structure of a Fbw7-Skp1-cyclin E complex: multisite-phosphorylated substrate recognition by SCF ubiquitin ligases. , 2007, Molecular cell.

[14]  Mike Tyers,et al.  A Mechanism for Cell-Cycle Regulation of MAP Kinase Signaling in a Yeast Differentiation Pathway , 2007, Cell.

[15]  J. Gerhart,et al.  The theory of facilitated variation , 2007, Proceedings of the National Academy of Sciences.

[16]  C. Dobson,et al.  Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques. , 1999, Biochemistry.

[17]  Peter S. Swain,et al.  An Entropic Mechanism to Generate Highly Cooperative and Specific Binding from Protein Phosphorylations , 2006, Current Biology.

[18]  Haruki Nakamura,et al.  Structural basis of the KcsA K(+) channel and agitoxin2 pore-blocking toxin interaction by using the transferred cross-saturation method. , 2003, Structure.

[19]  Christian Griesinger,et al.  Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients , 1999 .

[20]  Tony Pawson,et al.  Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication , 2001, Nature.

[21]  S. Carr,et al.  Phosphorylation of Sic1p by G1 Cdk required for its degradation and entry into S phase. , 1997, Science.

[22]  Tony Pawson,et al.  Mathematical Modeling Suggests Cooperative Interactions between a Disordered Polyvalent Ligand and a Single Receptor Site , 2003, Current Biology.

[23]  P. Cohen,et al.  The regulation of protein function by multisite phosphorylation--a 25 year update. , 2000, Trends in biochemical sciences.

[24]  H. Chan,et al.  Polyelectrostatic interactions of disordered ligands suggest a physical basis for ultrasensitivity , 2007, Proceedings of the National Academy of Sciences.

[25]  I. Shimada NMR techniques for identifying the interface of a larger protein-protein complex: cross-saturation and transferred cross-saturation experiments. , 2005, Methods in enzymology.

[26]  Kim Nasmyth,et al.  The B-type cyclin kinase inhibitor p40 SIC1 controls the G1 to S transition in S. cerevisiae , 1994, Cell.

[27]  István Simon,et al.  BIOINFORMATICS ORIGINAL PAPER doi:10.1093/bioinformatics/btm035 Structural bioinformatics Local structural disorder imparts plasticity on linear motifs , 2022 .

[28]  R. Deshaies,et al.  SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities. , 1997, Molecular biology of the cell.

[29]  P. Tompa The interplay between structure and function in intrinsically unstructured proteins , 2005, FEBS letters.

[30]  A Keith Dunker,et al.  Characterization of molecular recognition features, MoRFs, and their binding partners. , 2007, Journal of proteome research.

[31]  Christopher J. Oldfield,et al.  Intrinsic disorder and functional proteomics. , 2007, Biophysical journal.

[32]  Marc S. Cortese,et al.  Coupled folding and binding with α-helix-forming molecular recognition elements , 2005 .

[33]  Mike Tyers,et al.  A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin. , 2004, Biochimica et biophysica acta.

[34]  R. Deshaies,et al.  A Complex of Cdc4p, Skp1p, and Cdc53p/Cullin Catalyzes Ubiquitination of the Phosphorylated CDK Inhibitor Sic1p , 1997, Cell.

[35]  P. Wright,et al.  Packing, specificity, and mutability at the binding interface between the p160 coactivator and CREB‐binding protein , 2004, Protein science : a publication of the Protein Society.

[36]  M. Tyers,et al.  Structural Basis for Phosphodependent Substrate Selection and Orientation by the SCFCdc4 Ubiquitin Ligase , 2003, Cell.

[37]  Lorna J. Smith,et al.  Long-Range Interactions Within a Nonnative Protein , 2002, Science.

[38]  A. Fink Natively unfolded proteins. , 2005, Current opinion in structural biology.

[39]  J. Forman-Kay,et al.  CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices , 2007, Nature Structural &Molecular Biology.

[40]  Donald Bashford,et al.  Disordered p27Kip1 exhibits intrinsic structure resembling the Cdk2/cyclin A-bound conformation. , 2005, Journal of molecular biology.

[41]  J. Marsh,et al.  Sensitivity of secondary structure propensities to sequence differences between α‐ and γ‐synuclein: Implications for fibrillation , 2006 .

[42]  L. Kay,et al.  Variable Control of Ets-1 DNA Binding by Multiple Phosphates in an Unstructured Region , 2005, Science.

[43]  James E. Ferrell,et al.  Tuning Bulk Electrostatics to Regulate Protein Function , 2007, Cell.

[44]  T. Pawson,et al.  Assembly of Cell Regulatory Systems Through Protein Interaction Domains , 2003, Science.

[45]  I. Shimada,et al.  Determination of the interface of a large protein complex by transferred cross-saturation measurements. , 2002, Journal of molecular biology.

[46]  L. Kay,et al.  Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. , 1989, Biochemistry.