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Dynamic protein complexes in the ubiquitination pathway

Figure 1: Sic1regulates the G1/S transition in the yeast cell cycle. Phosphorylation by a CDK activity leads to recognition by Cdc4, subsequent ubiquitination, degradation and cell cycle progression.

Figure 2: Dynamic complex of pSic1 with Cdc4. Each binding motif generated by a phosphorylation event interacts with the binding site in Cdc4, one at a time, in a dynamic equilibrium (3).

Figure 3: ‘Poly-electrostatic’ interaction model for intrinsically disordered proteins (4). Conformational averaging in the disordered state ensemble and resulting electrostatic averaging creates a mean electrostatic field that reflects the net charge of the disordered region, ql. The free binding energy is the sum of a local contact energy and a long-range electrostatic interaction between the charge of the binding site of the folded protein, qr, and the net charge of the disordered region, ql. The ‘poly-electrostatic’ model provides an explanation how Cdc4 can ‘count’ phosphorylation

Intrinsically disordered proteins are ubiquitous in all kingdoms of life with higher occurrences in multi-cellular eukaryotes. Indeed, 30% of human proteins contain intrinsically disordered stretches of at least 30 contiguous residues. This fraction rises to 61% and 79% for proteins associated with cardiovascular disease and cancer, respectively. Apparently, intrinsically disordered proteins are highly enriched in signaling processes and one of their major functions is the mediation of protein interactions, but our understanding of the underlying molecular processes is still very limited.

Intrinsically disordered proteins defy the common protein structure-function paradigm because they do not require a folded structure to carry out their biological function. They rather consist of an ensemble of diverse, rapidly interconverting, flexible and largely unfolded conformers. Until recently, these proteins were generally believed to require an interaction partner to adopt a uniquely folded structure. However, it has become increasingly clear that intrinsically disordered proteins do not necessarily undergo a global disorder-to-order transition but can remain partially or largely disordered even in complex (1). We are interested in the physiological functions of disorder and why dynamic and significantly disordered complexes are often beneficial over uniquely folded complexes in signal transduction.

An example of a highly dynamic interaction is binding of the intrinsically disordered cyclin-dependent kinase (CDK) inhibitor Sic1 with the folded protein Cdc4, the substrate recognition subunit of an E3 ubiquitin ligase. The interaction leads to ubiquitination and subsequent degradation of Sic1. Yeast cells can only commit to a new round of replication and enter into S-phase in the absence of Sic1. Thus, in late G1 phase, Sic1 is phosphorylated by a CDK leading to recognition by Cdc4, and to subsequent ubiquitination and degradation by the proteasome. Interestingly, the interaction of Sic1 with Cdc4 depends on multiple phosphorylation events, each of which creates an individual binding motif (2). Only if roughly six sites are phosphorylated the interaction is strong enough in order to lead to subsequent ubiquitination (2).

Each of the binding motifs of Sic1 interacts with a single binding site in Cdc4, one at a time, in a dynamic equilibrium (3). Each binding motif is only transiently ordered while the rest of Sic1 remains disordered. This dynamic complex obviously requires Sic1 to be very flexible and largely disordered (3). Sic1 and Cdc4 thus interact via a dynamic complex.

The benefits of this dynamic complex are in the ability to fine-tune the affinity based on the number of phosphorylated sites. The 'poly-electrostatic' interaction model suggests that even unbound phosphates in Sic1 can contribute to binding through favorable electrostatic interactions (4). These effects require the rapid interconversion of diverse conformers to create a mean electrostatic field rather than presenting fixed charges in 3D space as in a folded protein. In the Sic1-Cdc4 interaction, the 'poly-electrostatic' effects allow for 'counting' of phosphorylation sites and an ultrasensitive response to the levels of active kinase, an important mechanism to maintain genetic stability in yeast (4).

Figure 4: Structural model of the dynamic complex ensemble (5). The ensemble was generated with the ENSEMBLE algorithm. Cdc4 is depicted as space-filling model whereas pSic1 conformers are depicted as ribbons with color-coding from cyan (N-terminus) to magenta (C-terminus). Phosphorylation sites are depicted as sticks.

Although this is the first dynamic complex described with residue-level resolution, we expect that many intrinsically disordered proteins exploit their dynamic properties to create similar complexes with currently unexplored possibilities for exquisite control. The lab studies dynamic interactions related to ubiquitination since this pathway is critically important for regulating many processes e.g. progression of the cell cycle. We use NMR spectroscopy and other biophysical and biochemical techniques. Our understanding of the molecular basis of cell cycle control, especially of important checkpoints is crucial for novel insights into mechanisms of cancer development. In addition, general inhibitors of the ubiquitin-proteasome system appear to selectively kill transformed tumor cells. Understanding protein interactions associated with the ubiquitin-proteasome system on a molecular level may thus lead to the identification of new drug targets. Ultimately, our work will help shift the current paradigm of folded protein complexes to include more dynamic alternatives.

• 1. Mittag T, Kay LE, Forman-Kay JD. Protein dynamics and conformational disorder in molecular recognition. J Mol Recognit 23:105-116, 2010.

• 2. Nash P, Tang X, Willems A, Ters M, Sicheri F. Multiside phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. Nature 414: 514-521, 2001.

• 3. Mittag T, Orlicky S, Choy WY, Tang X, Lin H, Sicheri F, Kay LE, Tyers M, Forman-Kay JD. Dynamic equilibrium engagement of a polyvalent ligand with a single site receptor. Proc Natl Acad. Sci USA 105:17772-17777, 2008.

• 4. Borg M, Mittag T, Pawson T, Tyers M, Forman-Kay JD, Chan HS. Poly-electrostatic interactions of disordered ligands suggest a physical basis for ultrasensitivity. Proc Natl Acad. Sci USA 104:9650-9655, 2007.

• 5. Mittag T, Marsh J, Grishaev A, Orlicky S, Lin H, Sicheri F, Tyers M, Forman-Kay JD. Structure-function implications in a dynamic complex of the intrinsically disordered Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase. Structure 18:494-506, 2010. (Preview: R. Konrat (2010) The Meandering of Disordered Proteins in Conformational Space. Structure 18:416-419.)