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

• Published in: Biochemistry (Easton) 2017-05, Vol.56 (18), p.2379-2384
• Main Authors: Crabtree, Michael D, Borcherds, Wade, Poosapati, Anusha, Shammas, Sarah L, Daughdrill, Gary W, Clarke, Jane
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; binding capacity; Binding Sites; Cloning, Molecular; Conserved Sequence; dissociation; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Histone-Lysine N-Methyltransferase - chemistry; Histone-Lysine N-Methyltransferase - genetics; Histone-Lysine N-Methyltransferase - metabolism; Humans; Intrinsically Disordered Proteins - chemistry; Intrinsically Disordered Proteins - genetics; Intrinsically Disordered Proteins - metabolism; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mice; Models, Molecular; Mutation; Myeloid-Lymphoid Leukemia Protein - chemistry; Myeloid-Lymphoid Leukemia Protein - genetics; Myeloid-Lymphoid Leukemia Protein - metabolism; Phosphoproteins - chemistry; Phosphoproteins - genetics; Phosphoproteins - metabolism; proline; Proline - chemistry; Proline - metabolism; Protein Binding; Protein Conformation, alpha-Helical; Protein Folding; Protein Interaction Domains and Motifs; proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Tumor Suppressor Protein p53 - chemistry; Tumor Suppressor Protein p53 - genetics; Tumor Suppressor Protein p53 - metabolism
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/acs.biochem.7b00179

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.