NMR chemical shift and relaxation measurements provide evidence for the coupled folding and binding of the p53 transactivation domain

The interaction between the acidic transactivation domain of the human tumor suppressor protein p53 (p53TAD) and the 70 kDa subunit of human replication protein A (hRPA70) was investigated using heteronuclear magnetic resonance spectroscopy. A (1)H–(15)N heteronuclear single quantum coherence (HSQC) titration experiment was performed on a (15)N-labeled fragment of hRPA70, containing the N-terminal 168 residues (hRPA70(1–168)) and p53TAD. HRPA70(1–168) residues important for binding were identified and found to be localized to a prominent basic cleft. This binding site overlapped with a previously identified single-stranded DNA-binding site, suggesting that a competitive binding mechanism may regulate the formation of p53TAD–hRPA70 complex. The amide (1)H and (15)N chemical shifts of an uniformly (15)N-labeled sample of p53TAD were also monitored before and after the addition of unlabeled hRPA70(1–168). In the presence of unlabeled hRPA70(1–168), resonance lineshapes increased and corresponding intensity reductions were observed for specific p53TAD residues. The largest intensity reductions were observed for p53TAD residues 42–56. Minimal binding was observed between p53TAD and a mutant form of hRPA70(1–168), where the basic cleft residue R41 was changed to a glutamic acid (R41E), demonstrating that ionic interactions play an important role in specifying the binding interface. The region of p53TAD most affected by binding hRPA70(1–168) was found to have some residual alpha helical and beta strand structure; however, this structure was not stabilized by binding hRPA70(1–168). (15)N relaxation experiments were performed to monitor changes in backbone dynamics of p53TAD when bound to hRPA70(1–168). Large changes in both the transverse (R(2)) and rotating frame (R(1ρ)) relaxation rates were observed for a subset of the p53TAD residues that had (1)H–(15)N HSQC resonance intensity reductions during the complex formation. The folding of p53TAD upon complex formation is suggested by the pattern of changes observed for both R(2) and R(1ρ). A model that couples the formation of a weak encounter complex between p53TAD and hRPA70(1–168) to the folding of p53TAD is discussed in the context of a functional role for the p53–hRPA70 complex in DNA repair

Conserved Helix-Flanking Prolines Modulate Intrinsically Disordered Protein:Target Affinity by Altering the Lifetime of the Bound Complex.

Appropriate integration of cellular signals requires a delicate balance of ligand-target binding affinities. Increasing the level of residual structure in intrinsically disordered proteins (IDPs), which are overrepresented in these cellular processes, has been shown previously to enhance binding affinities and alter cellular function. Conserved proline residues are commonly found flanking regions of IDPs that become helical upon interacting with a partner protein. Here, we mutate these helix-flanking prolines in p53 and MLL and find opposite effects on binding affinity upon an increase in free IDP helicity. In both cases, changes in affinity were due to alterations in dissociation, not association, rate constants, which is inconsistent with conformational selection mechanisms. We conclude that, contrary to previous suggestions, helix-flanking prolines do not regulate affinity by modulating the rate of complex formation. Instead, they influence binding affinities by controlling the lifetime of the bound complex

Disorder Predictors Also Predict Backbone Dynamics for a Family of Disordered Proteins

Several algorithms have been developed that use amino acid sequences to predict whether or not a protein or a region of a protein is disordered. These algorithms make accurate predictions for disordered regions that are 30 amino acids or longer, but it is unclear whether the predictions can be directly related to the backbone dynamics of individual amino acid residues. The nuclear Overhauser effect between the amide nitrogen and hydrogen (NHNOE) provides an unambiguous measure of backbone dynamics at single residue resolution and is an excellent tool for characterizing the dynamic behavior of disordered proteins. In this report, we show that the NHNOE values for several members of a family of disordered proteins are highly correlated with the output from three popular algorithms used to predict disordered regions from amino acid sequence. This is the first test between an experimental measure of residue specific backbone dynamics and disorder predictions. The results suggest that some disorder predictors can accurately estimate the backbone dynamics of individual amino acids in a long disordered region

Contribution of proline to the pre-structuring tendency of transient helical secondary structure elements in intrinsically disordered proteins

Background: IDPs function without relying on three-dimensional structures. No clear rationale for such a behavior is available yet. PreSMos are transient secondary structures observed in the target-free IDPs and serve as the target-binding active motifs in IDPs. Prolines are frequently found in the flanking regions of PreSMos. Contribution of prolines to the conformational stability of the helical PreSMos in IDPs is investigated. Methods: MD simulations are performed for several IDP segments containing a helical PreSMo and the flanking prolines. To measure the influence of flanking-prolines on the structural content of a helical PreSMo calculations were done for wild type as well as for mutant segments with Pro→Asp, His, Lys, or Ala. The change in the helicity due to removal of a proline was measured both for the PreSMo region and for the flanking regions. Results: The α-helical content in ~70% of the helical PreSMos at the early stage of simulation decreases due to replacement of an N-terminal flanking proline by other residues whereas the helix content in nearly all PreSMos increases when the same replacements occur at the C-terminal flanking region. The helix destabilizing/terminating role of the C-terminal flanking prolines is more pronounced than the helix promoting effect of the N-terminal flanking prolines. General significance: This work represents a novel example demonstrating that a proline is encoded in an IDP with a defined purpose. The helical PreSMos presage their target-bound conformations. As they most likely mediate IDP-target binding via conformational selection their helical content can be an important feature for IDP function. Keywords: Flanking proline; Intrinsically disordered protein (IDP); Molecular dynamics simulation; PreSMo (Pre-Structured Motif). Copyright © 2013 Elsevier B.V. All rights reserved

Disorder for Dummies: Functional Mutagenesis of Transient Helical Segments in Disordered Proteins

Most cytosolic eukaryotic proteins contain a mixture of ordered and disordered regions. Disordered regions facilitate cell signaling by concentrating sites for posttranslational modifications and protein–protein interactions into arrays of short linear motifs that can be reorganized by RNA splicing. The evolution of disordered regions looks different from their ordered counterparts. In some cases, selection is focused on maintaining protein binding interfaces and PTM sites, but sequence heterogeneity is common. In other cases, simple properties like charge, length, or end-to-end distance are maintained. Many disordered protein binding sites contain some transient secondary structure that may resemble the structure of the bound state. α-Helical secondary structure is common and a wide range of fractional helicity is observed in different disordered regions. Here we provide a simple protocol to identify transient helical segments and design mutants that can change their structure and function

Unfolding and Disassociation of the Flgm/ σ28 Complex are Thermodynamically Coupled

FlgM and cr28 regulate the transcription of flagella and chemotaxis genes in E. coli and S. typhimurium. In an attempt to determine the strength of the interaction between FlgM and cr28 thermal unfolding studies were performed using circular dichroism spectroscopy (CD). A single, cooperative, melting transition was observed for the FlgM/cr28complex suggesting that unfolding and disassociation of the complex occur simultaneousl

Sequence Properties of an Intramolecular Interaction that Inhibits p53 DNA Binding

An intramolecular interaction between the p53 transactivation and DNA binding domains inhibits DNA binding. To study this autoinhibition, we used a fragment of p53, referred to as ND WT, containing the N-terminal transactivation domains (TAD1 and TAD2), a proline rich region (PRR), and the DNA binding domain (DBD). We mutated acidic, nonpolar, and aromatic amino acids in TAD2 to disrupt the interaction with DBD and measured the effects on DNA binding affinity at different ionic strengths using fluorescence anisotropy. We observed a large increase in DNA binding affinity for the mutants consistent with reduced autoinhibition. The ΔΔG between DBD and ND WT for binding a consensus DNA sequence is −3.0 kcal/mol at physiological ionic strength. ΔΔG increased to −1.03 kcal/mol when acidic residues in TAD2 were changed to alanine (ND DE) and to −1.13 kcal/mol when all the nonpolar residues, including W53/F54, were changed to alanine (ND NP). These results indicate there is some cooperation between acidic, nonpolar, and aromatic residues from TAD2 to inhibit DNA binding. The dependence of DNA binding affinity on ionic strength was used to predict excess counterion release for binding both consensus and scrambled DNA sequences, which was smaller for ND WT and ND NP with consensus DNA and smaller for scrambled DNA overall. Using size exclusion chromatography, we show that the ND mutants have similar Stokes radii to ND WT suggesting the mutants disrupt autoinhibition without changing the global structure