Figure 1. Structured domains and intrinsically disordered regions (IDRs) are two fundamental classes of functional building blocks of proteins. The synergy between disordered regions and structured domains increases the functional versatility of proteins. Adapted with permission from ref 50.

The evolution of disordered proteins or regions of proteins differs from ordered proteins because of the differences in their sequence composition, intramolecular contacts and function. Recent assessments of disordered protein evolution at the sequence, structural and functional levels support this hypothesis. Disordered proteins have a different pattern of accepted point mutations, exhibit higher rates of insertions and deletions, and generally, but not always, evolve more rapidly than ordered proteins. Even with these high rates of sequence evolution, a few examples have shown that disordered proteins maintain their flexibility under physiological conditions, and it is hypothesized that they maintain specific structural ensembles.

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The interaction between the flagellum specific sigma factor, sigma 28, and its inhibitor, FlgM, was examined using multidimensional heteronuclear NMR. Here we observe that free FlgM is mostly unfolded, but about 50% of the residues become structured when bound to sigma 28. Our analysis suggests that the sigma 28 binding domain of FlgM is contained within the last 57 amino acids of the protein while the first 40 amino acids are unstructured in both the free and bound states. Genetic analysis of flgM mutants that fail to inhibit sigma 28 activity reveal amino acid changes that are also isolated to the C-terminal 57 residues of FlgM. We postulate that the lack of structure in free and bound FlgM is important to its role as an exported protein.

Levels of residual structure in disordered interaction domains determine in vitro bindingaffinities, but whether they exert similar roles in cells is not known. Here, we show that increasing residual p53 helicity results in stronger Mdm2 binding, altered p53 dynamics, impaired target gene expression and failure to induce cell cycle arrest upon DNA damage.These results establish that residual structure is an important determinant of signaling fidelity in cells.Intrinsically disordered protein domains often mediate protein-protein interactions that result in disorder-to-order transitions via coupled folding and binding reactions 1 . In addition, many disordered interaction domains exhibit defined levels of transient secondary structure resembling their complex-bound states when free in solution 2 . These levels of residual structure affect binding energies, also by reducing the loss of conformational entropy associated with disorderto-order transitions 3 . Accordingly, higher levels of residual structure in disordered interaction domains increase in vitro binding affinities 4,5 . Here we ask to what extent residual structure contributes to protein binding affinities in cells and whether engineered changes to residual structure affect protein function at the cellular level. To answer these questions, we designed p53 mutants with higher residual helicity within their disordered, N-terminal transactivation domains (TADs) and investigated their effects on cellular Mdm2 binding and p53's ability to induce 2 target gene expression and cell cycle arrest. p53 is activated by many forms of cellular stress, including DNA double-strand breaks (DSBs) and functions as a major tumor suppressor and cell cycle regulator 6 . In the absence of DNA damage, cellular p53 levels are kept low by targeted proteasomal degradation mediated by the E3 ubiquitin ligase Mdm2, which interacts with p53TAD and subsequently ubiquitinates p53's C-terminal regulatory domain 7 . Upon DNA damage, post-translational modifications of p53TAD and Mdm2, together with Mdm2 degradation, disrupt the p53-Mdm2 complex. This leads to p53 accumulation and the expression of p53 target genes that regulate DNA repair, cell cycle arrest, senescence or apoptosis 8,9 . One of these target genes is Mdm2, whose expression establishes a negative feedback loop that shapes cellular p53 dynamics and thereby controls cell fate decisions 10 .In its free form, p53TAD exists in equilibrium between disordered and partially helical conformations 11-13 , whereas residues 19-25 form a stable amphipathic α-helix in the Mdm2 complex 14 (Fig. 1a). To increase the binding affinity between p53 and Mdm2 without altering the binding interface, we designed p53TAD mutants with higher levels of residual helicity by mutating conserved proline residues flanking the Mdm2 binding site (i.e., Pro12, Pro13 or Pro27) to alanines (Supplementary Results, Supplementary Fig. 1a). Using NMR spectroscopy, we determined that wild-type (WT) p53TAD helicity (28%) increased to 64% when we replaced Pro27 with ...