No abstract

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 ...

Allosteric communication between two ligand-binding sites in a protein is a central aspect of biological regulation that remains mechanistically unclear. Here we show that perturbations in equilibrium picosecond-nanosecond motions impact zinc (Zn)-induced allosteric inhibition of DNA binding by the Zn efflux repressor CzrA (chromosomal zinc-regulated repressor). DNA binding leads to an unanticipated increase in methyl side-chain flexibility and thus stabilizes the complex entropically; Zn binding redistributes these motions, inhibiting formation of the DNA complex by restricting coupled fast motions and concerted slower motions. Allosterically impaired CzrA mutants are characterized by distinct nonnative fast internal dynamics "fingerprints" upon Zn binding, and DNA binding is weakly regulated. We demonstrate the predictive power of the wild-type dynamics fingerprint to identify key residues in dynamics-driven allostery. We propose that driving forces arising from dynamics can be harnessed by nature to evolve new allosteric ligand specificities in a compact molecular scaffold. Technological advances in structural biology have permitted insights (3-5) into how changes in protein structure and flexibility contribute to allostery (6-9). Allostery likely employs a continuum of mechanisms, from domain or subunit rearrangements to predominantly side-chain and backbone dynamics (6-8, 10, 11), to affect biological regulation (1). Although these motions clearly impact site-site communication via defined molecular pathways (9) or energy level perturbations at distant sites (12), an allosteric effect without conformational change remains largely a theoretical postulate (10,13,14). In this context, changes in dynamics upon ligand binding (8,(15)(16)(17)(18)(19)(20) have long been predicted to impact allostery (5, 14, 17, 21); however, obtaining a quantitative experimental demonstration of the role of conformational entropy in allosteric systems remains challenging. Here we test these ideas in the context of heterotropic linkage and pinpoint fast internal dynamics as a primary contributor to functional, structure-encoded dynamics. We report an example of allostery where side-chain rotamer degeneracy is largely responsible for coupling two ligand-binding events through perturbations in a dynamic network that is required for both entropic and enthalpic driving forces.Our model system for studying heterotropic allostery is the transcriptional regulator CzrA (chromosomal zinc-regulated repressor) from the bacterial pathogen Staphylococcus aureus (22-25) ( Fig. 1 and Fig. S1A). Zinc homeostasis is critical to the virulence of S. aureus (26) and of many other microbial pathogens, and allows the organism to adapt to host-imposed zinc toxicity or limitation (27, 28). CzrA is a member of the ubiquitous arsenic repressor (ArsR) family of metalloregulatory proteins (25, 29), individual members of which are capable of sensing a wide array of metal, metalloid, and nonmetal inducers on distinct sites on a relatively simple, homodimeric wi...

A group 14 atom bonded to three mesityl groups (2,4,6-trimethylphenyl) and to one allyl group serves as a novel precursor to tricoordinate group 14 cations, the analogues of the carbocation. The double bond of the allyl group provides an accessible reaction site that is located beyond the ortho methyl groups. Reaction of various electrophiles with the double bond releases the allyl group and leads to formation of the group 14 cations. The mesityl groups then are of sufficient steric bulk to protect the tricoordinate metal center from attack by nucleophiles. This approach is used herein with silicon, germanium, and tin as the central atom. The 29Si chemical shift (δ 225) indicates full cationic character for the silicon system. The 119Sn chemical shift (δ 806) indicates less than full cationic character for the tin system. The positive charge for the germanium system has been assessed by examination of the aromatic 13C chemical shifts. These results provide the highest current cationic character for silylium and stannylium ions.