Anchor Residues Govern Binding and Folding of an Intrinsically Disordered Domain

Abstract

Minimal protein mimics have yielded novel classes of protein-protein interaction inhibitors; however, this success has not been extended to targeting intrinsically disordered proteins, which represent a significant proportion of important therapeutic targets. We sought to determine the requirements for binding an intrinsically disordered region (IDR) by its native binding partner as a prelude to developing minimal protein mimics that regulate IDR interactions. Our analysis reinforces the hypothesis that IDRs reside on a fulcrum between unfolded and folded states and that a handful of key binding residues on partner protein surfaces dictate their folding. Our studies also suggest that minimal mimics of protein surfaces may not offer specific ligands for IDRs and that it would be more judicious to target the globular protein partners of IDRs.

Figure 1.

(A) Minimal mimics of protein surfaces have yielded an attractive class of protein-protein interaction inhibitors, but synthetic protein mimics that target intrinsically disordered proteins have not been reported. (B) Intrinsically disordered regions can adopt different conformations in complex with different partners. The HIF-1α C-terminal activation domain (CTAD) illustrates this conformational promiscuity of IDRs in complex with Factor Inhibiting HIF (FIH) (PDB code: 1H2K) and the TAZ1 domain of p300/CBP (PDB code: 1L8C).

Figure 2.

(A) Cartoon representation of the complex between HIF-1α CTAD (red) and p300/CBP TAZ1 (blue). The CTAD consists of two short helical regions termed αA and αB. TAZ1 adopts a pyramidal geometry with three orthogonal helices (helices 1-3). The blue spheres on TAZ1 represent critical binding residues as determined by computational and experimental alanine scanning mutagenesis analysis. The critical TAZ1 binding residues are clustered in three different zones to engage CTAD helices. (B-E) Computational and experimental analysis of TAZ1 binding residues that engage HIF-1α CTAD. (B) Molecular dynamics simulation to probe impact of TAZ1 histidine-20 mutation to alanine. The results suggest that mutations in any hotspot regions reduces folding of HIF 1-α CTAD. Panel B depicts results with His-20, which is critical in engaging the CTAD αB domain. Mutation of this single residue leads to a significant loss of intrachain helical hydrogen bonding in αB (but not αA). (C-D) Binding analysis of TAZ1 mutants and fluorescein-labeled HIF1-α CTAD in a fluorescence polarization assay. (E) 2D NMR analysis of resonance shifts observed in ¹⁵N-labeled HIF-1α CTAD in complex with H20A TAZ1 versus WT TAZ1. Green bars indicate calculated peak shifts and red bars indicate free-protein state peaks that undergo major shifts or disappear upon complexation. Graphs for other mutants, as well as the raw ¹H/¹⁵N HSQC NMR spectra are included in the Supporting Information.

Figure 3.

(A) Summary of 2D NMR results for mutants in each of the three binding zones as defined in Figure 2. Mutation of residues Q69 and S72, which engage the HIF-1α CTAD αA Zones 1 and 2, completely abrogates folding of HIF 1α CTAD as judged by HSQC NMR (Supporting Information). Substitution of H20 with alanine leads to unfolding of αB while the αA domain retains its native configuration. (B) (left) TAZ1 H20 engages HIF-1α CTAD αB domain through hydrophobic and ionic interactions with L35, L38 and D39 (PDB code 1L8C). (right) Substitution of H20 to alanine is expected to abrogate the interactions with this anchor residue (molecular model derived from PDB code 1L8C).

Figure 4.

(A) The HIF-1α CTAD αA sequence adopts a helical or extended conformation depending on the partner protein. Three solvent exposed HIF-1α CTAD residues Y14, V18, and N19 in the TAZ1 complex become buried at the FIH surface and adopt a strand conformation. (B) Examples of complexes with intrinsically disordered proteins (blue ribbon) that feature binding grooves (left, PDB code: 1YCR) or extended interfaces (right, PDB codes: 2LWW and 1JSU)