N and C-terminal Sub-regions in the c-Myc Transactivation Region and their Joint Role in Creating Versatility in Folding and Binding

The proto-oncogene c-myc governs the expression of a number of genes targeting cell growth and apoptosis, and its expression levels are distorted in many cancer forms. The current investigation presents an analysis by proteolysis, circular dichroism, fluorescence and Biacore of the folding and ligand-binding properties of the N-terminal transactivation domain (TAD) in the c-Myc protein. A c-Myc sub-region comprising residues 1–167 (Myc_1–167) has been investigated that includes the unstructured c-Myc transactivation domain (TAD, residues 1–143) together with a C-terminal segment, which appears to promote increased folding. Myc_1–167 is partly helical, binds both to the target proteins Myc modulator-1 (MM-1) and TATA box-binding protein (TBP), and displays the characteristics of a molten globule. Limited proteolysis divides Myc_1–167 in two halves, by cleaving in a predicted linker region between two hotspot mutation regions: Myc box I (MBI) and Myc box II (MBII). The N-terminal half (Myc_1–88) is unfolded and does not alone bind to target proteins, whereas the C-terminal half (Myc_92–167) has a partly helical fold and specifically binds both MM-1 and TBP. Although this might suggest a bipartite organization in the c-Myc TAD, none of the N and C-terminal fragments bind target protein with as high affinity as the entire Myc_1–167, or display molten globule properties. Furthermore, merely linking the MBI with the C-terminal region, in Myc_38–167, is not sufficient to achieve binding and folding properties as in Myc_1–167. Thus, the entire N and C-terminal regions of c-Myc TAD act in concert to achieve high specificity and affinity to two structurally and functionally orthogonal target proteins, TBP and MM-1, possibly through a mechanism involving molten globule formation. This hints towards understanding how binding of a range of targets can be accomplished to a single transactivation domain.

Introduction

The proto-oncogene c-myc is critical for growth and development by regulating the expression of a large number of genes involved in cell cycle progression, proliferation and apoptosis.1, 2, 3 This potency requires highly controlled expression of the c-myc gene in normal cells. Deregulation of c-myc expression affects the initiation and expansion of a number of human cancers, many of them aggressive.⁴ The underlying molecular mechanisms include amplification of c-myc by translocation to other promoters, often together with mutations or post-translational modifications in the N-terminal transactivation domain (TAD) of the expressed c-Myc protein. In particular, virtually all patients suffering from Burkitt's lymphoma have a translocation of c-myc causing deregulated expression and development of early pre-B cell lymphomas.⁵

Specific anchoring of c-Myc to DNA, and thereby to gene regulatory activity, is housed in its C-terminal bHLH-Zip domain (residues 354–434), which is linked to the N-terminal TAD domain by a suggested flexible linker region. The bHLH-Zip region has little structure by itself,⁶ but by heterodimerization with Max, another bHLH-Zip protein, high-affinity binding to E-box elements associated with c-Myc target genes is achieved.⁷ The crystal structure of c-Myc and Max bHLH-Zip regions with DNA shows a dimer of heterodimers, suggesting a DNA looping mechanism to allow for simultaneous binding to sequentially arranged E-boxes.⁸

The c-Myc transactivating activity, which is housed in its N-terminal TAD region, is regulated by binding to a range of proteins, including coactivators, cytoplasmic proteins, transcriptional regulators and tumor suppressors.3, 9, 10, 11 Two highly conserved regions in c-Myc TAD, Myc box I (MBI) and Myc box II (MBII), spanning residues 41–66 and 128–143, respectively (Figure 1), have been suggested as recognition sites for regulatory proteins interacting with c-Myc. Most of these appear to require the presence of both MBI and MBII (TRRAP, P-TEFb, NF-Y, TBP, Bin1, p107, Yaf2 and p21; reviewed by Oster et al.³) MBI and MBII contain the major sites of c-Myc phosphorylation, and their importance is further emphasised by mutations in Burkitt's and AIDS-related lymphomas, clustering around the “hotspot” residues Glu39, Thr58, Ser62 and Phe138.12, 13 Moreover, conserved hydrophobic c-Myc residues in Myc box II (W135, F138) have been shown to be required both for interaction with regulatory proteins¹⁴ and for transformation.15, 16 Indeed, close interplay between these two regions is suggested by an MBI mutant (P57S) which overrides decreased transformation and proliferation ability caused by mutations in MBII.¹⁶

Little is known about the structure of the N-terminal c-Myc TAD or about biophysical mechanisms related to its ability to transactivate. Residues 1–143 of c-Myc are sufficient to provide transactivation activity in neoplastic transformation,¹⁷ and are required for binding to the basal transcription factor TBP.18, 19 The secondary structure of c-Myc 1–143, as investigated by circular dichroism (CD), has little structural content in the domain itself,¹⁸ which agrees with several other studies of transactivation domains, showing little or no structure in the absence of target protein.20, 21, 22 However, the complex conserved sequence pattern in c-Myc TAD (Figure 1) suggests that the transactivation mechanism requires more than an abundance of acidic residues and/or glutamine residues within the TAD, and the charged patterns alone do not bind TBP.¹⁹

The c-Myc TAD region is of particular biophysical interest because of its capability of functional and specific binding to a wide range of target proteins. However, special care needs to be taken in defining the active region in partly folded proteins, and the notorious resistance of c-Myc TAD towards biochemical and structural analysis³ could be a result of poor domain boundary definitions for the c-Myc TAD. Indeed, the homology between c-Myc and the smaller single-domain protein b-Myc covers residues 1–165 of c-Myc,²³ suggesting a larger region of c-Myc might be required for functional and biophysical independence. To this end, it is notable that studies of c-Myc cellular degradation gave contradictory results if fusion proteins were attached to the N terminus¹³ or to the C terminus²⁴ of c-Myc TAD 1–147 and 1–149 constructs, respectively.

The current investigation explores the folding and binding properties of an extended c-Myc TAD region, comprising residues 1–167, and of its N and C-terminal sub-regions, using limited proteolysis, CD, fluorescence and Biacore. The biological functionality of the protein sub-regions were assayed by binding to two proteins with major structural and functional differences: the TATA box-binding protein (TBP), a major target in transactivation with a known β-fold,²⁵ and MM-1, a predictedly helical c-Myc binder with anti-tumor activity presumably arising from hindering transactivation.²⁶ The results have implications for further structural and functional studies of c-Myc, and add to our understanding of transactivation in general.