FC0074
c-Myc  -  Bin1 SH3 domain

Biological function
The c-Myc oncoprotein (Myc) is a transcription factor that regulates a wealth of genes involved in a wide-variety of biological activities including apoptosis, differentiation as well as proliferation. As a critical regulator of both normal and tumor cells, Myc is highly controlled at many levels and is at the center of an extensive interactome.

Domain organization/sequence features
The N-terminal transactivation domain of Myc (Myc TAD, residues 1–143;) is essential for Myc-mediated transformation, differentiation and apoptosis. This region serves as an interaction platform for proteins involved in chromatin and histone modification as well as ubiquitination and subsequent degradation. TAD also controls protein expression through mRNA translation as well as direct regulation of DNA replication. Interactions with Myc homology box I (MBI, residues 47–63) govern the cellular stability of the protein thereby setting a time window for its activity, whereas Myc homology box II (MBII, residues 128–143) coordinates interactions with the transcriptional regulatory machinery.

Structural evidence
Myc occupies a very heterogeneous conformational space, transiently structured regions can be found in residues 22–33 and in the Myc homology box I (MBI; residues 45–65); both these regions are conserved in other members of the Myc family. Binding of Bin1 to Myc-1–88 as assayed by NMR and surface plasmon resonance (SPR) revealed primary binding to the S62 region in a dynamically disordered and multivalent complex, accompanied by population shifts leading to altered intramolecular conformational dynamics. Dynamic properties of Myc-1–88 on a rapid time scale (ps–ns) were studied in the absence and presence of ligand binding. Binding maintains overall intrinsic disorder in Myc-1–88 with no large-scale evidence for stable structure in the complex. Residues in the more flexible middle and C-terminal region show very minor or insignificant differences between bound and free forms. For residues 1–30, 49–56 and 61–82 in the transiently ordered segments of the bound form compared to the free state suggest that fewer long-range interactions are sampled by these regions in the conformational ensemble of the disordered protein domain.

Biochemical evidence
Myc-1–88 may harbor two binding sites for Bin1–SH3 with different affinities. K_Ds of 33μM and 200μM, with k_on/k_off of 590 M^-1s^-1/0.019 s^-1 and 6.7 M^{- 1}s^-1/0.0013 s^-1 . In aggressive lymphomas, mutations at or near T58, which disturb its phosphorylation, lead to accumulation and retention of Myc in its activated, S62-phosphorylated state. the affinity of unphosphorylated Myc-55–68 for Bin1– SH3 was significant (K_D 4.2 μM), and unaltered by phosphorylation on T58, the same peptide phosphorylated at S62 was unable to bind Bin1–SH3 even at micromolar concentrations. Bin1 binding could retain Myc in its S62-unphosphorylated, inactive state, which is indirectly supported by liberated Myc cell proliferative activity when Bin1 expression is inhibited by the adenovirus E1A oncoprotein.

Structure/Mechanism
No folding-on-binding occurs in Myc-1–88 when it binds Bin1–SH3; instead disorder is maintained throughout Myc-1–88, also within transiently structured regions. In fact, dynamics on the millisecond level is even increased for Myc-1–88 as a whole in complex with Bin1– SH3. Binding to the previously identified Bin1–SH3 site, centered on P59–P60–L61–S62–P63, is indeed observed by small CSPs and localized small changes in dynamics, but these effects are not limited to the Bin1–SH3 binding site, and do not result in stabilized secondary structure or conformational restriction at the binding site or anywhere else in Myc-1–88. In addition, multivalent Bin1 binding appears to affect significantly larger regions of Myc than those directly targeted by Bin1– SH3 binding. Bin1–SH3 binding disrupts transient interactions involving these segments. The condensed ensemble is likely stabilized by electrostatic interactions the Myc-1–49 region is predominantly negatively charged with a pI of 3.5, while the pI of Myc-50–88 is 9.2. Multivalent Bin1–SH3 binding to Myc-1–88 hinders the formation of such intramolecular interactions resulting in a shift in the conformational ensemble toward less compact states, retaining local dynamics on the ps-ms time scale. In the free state, fluctuating secondary structure elements in Myc residues 22–33 and 48–68, as well as adjacent conserved hydrophobic clusters at residues 71–81 and 5– 15 loosely interact, thus shifting the population in the conformational ensemble toward more compactly disordered condensed states

Mechanism category
Tethering, Competitive binding

Posttranslational modification
Myc-1–88, where hierarchical phosphorylation of S62 and T58 regulates activation and destruction of the Myc protein. Myc has a short protein half-life of 20–30min, which is largely controlled through phosphorylation of T58 and S62. While phosphorylation at S62 by ERK or CDK kinases is critical for Myc transforming ability and transiently increases Myc cellular stability, subsequent phosphorylation of T58 by GSK3b triggers dephosphorylation of S62 by protein phosphatase 2 A (PP2A) and subsequent ubiquitin-mediated degradation through SCF-Fbwx7.

Significance
Fuzziness and complex dynamics in Myc together with multivalent interactions within Myc-1–88 and between Myc-1–88 and Bin1–SH3 may be critical for achieving rapid yet accurate response to cellular signals in gene regulation. The underlying role of the dynamics of Myc-1–88 both in isolation and in its multivalent complex with Bin1–SH3 could be to prevent stable binding to its critical regulatory interaction sites.

Medical relevance
Overexpression of c-Myc is associated with many human cancers. In aggressive lymphomas, c-Myc lifetime is prolonged owing to decreased/hampered T58 phosphorylation. Regulators of Myc homology box I were also shown to be oncogenes and tumor suppressors. Small molecules were demonstrated to efficiently inhibit the interaction of c-Myc with Max.

Further reading
15992821

Submitted by
Maria Sunnerhagen   marsu@ifm.liu.se