Primary Sequence Controls the Specificity and Affinity of a Small Molecule Binding to the Intrinsically Disordered Protein c-Myc

Intrinsically disordered proteins (IDPs) are characterized by high flexibility and low hydrophobic to charged residue ratio. The transcription factor c-Myc is an IDP deregulated in many forms of cancer. The protein undergoes coupled folding and binding with its obligate dimerization partner Max, which is also a disordered monomer, to form a basic helix-loop-helix leucine zipper (bHLHZip). A small molecule, 10058-F4, binds specifically within an 11 amino acid region of the bHLHZip of c-Myc, stabilizing the disordered monomer. The affinity determining residues of the 10058-F4 binding site on c-Myc were distinguished by mutating individual residues to alanine and subsequently measuring binding of 10058-F4. Mutation of both hydrophobic and certain hydrophilic residues attenuated binding of the small molecule to c-Myc. The affinity determining residues may affect binding through direct, energetically favorable contact with the small molecule or via a conformational influence on the IDP which favors binding. Within the proteome (SLiMSearch3), only two proteins are identified with five affinity determining residues. Six affinity determining residues are enough to specify c-Myc. A minimal set of these key residues were introduced into Max, which does not normally interact with 10058-F4. The novel protein, ModMax, binds 10058-F4. Although the alanine scan distinguished the necessary amino acids for binding, conservative mutations of some affinity determining residues demonstrated that 10058-F4 affinity is tunable. Upon substitution of Y to W, the affinity was improved by an order of magnitude. On the other hand, the affinity decreased five-fold upon substitution of E to N. Primary sequence alone, without extended secondary and tertiary structure, is sufficient to confer both specificity and affinity of a small molecule-IDP interaction.