Limits...
Transient structure and dynamics in the disordered c-Myc transactivation domain affect Bin1 binding.

Andresen C, Helander S, Lemak A, Farès C, Csizmok V, Carlsson J, Penn LZ, Forman-Kay JD, Arrowsmith CH, Lundström P, Sunnerhagen M - Nucleic Acids Res. (2012)

Bottom Line: We have characterized in detail the intrinsically disordered properties of Myc-1-88, where hierarchical phosphorylation of S62 and T58 regulates activation and destruction of the Myc protein.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.These findings expand the increasingly recognized concept of intrinsically disordered regions mediating transient interactions to Myc, a key transcriptional regulator of major medical importance, and have important implications for further understanding its multifaceted role in gene regulation.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biotechnology, Department of Physics, Chemistry and Biology, Linköping University, SE-58183 Linköping, Sweden.

ABSTRACT
The crucial role of Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of Myc function. The N-terminal region of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of Myc-1-88, where hierarchical phosphorylation of S62 and T58 regulates activation and destruction of the Myc protein. By nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements and NOE analysis, we show that although Myc occupies a very heterogeneous conformational space, we find transiently structured regions 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. These findings expand the increasingly recognized concept of intrinsically disordered regions mediating transient interactions to Myc, a key transcriptional regulator of major medical importance, and have important implications for further understanding its multifaceted role in gene regulation.

Show MeSH

Related in: MedlinePlus

NMR analysis of Myc-1–88 in the absence and presence of Bin1–SH3. Omitted histogram bars correspond to missing or overlapped residues and prolines. (A) Myc-1–88 CSPs at a Myc-1–88:Bin1–SH3 ratio of 1:1.5. The cut-off value for significant CSPs is shown as a dashed line and calculated as described in ‘Materials and methods’ section. (B) Ratios of peak intensities with and without Bin1, derived from HNCO experiments at a Myc-1–88:Bin1–SH3 ratio of 1:1.5. In the absence of interactions, or if interactions are the same in free and bound forms, the intensity ratio would be 1 (gray line). Gain/loss of interactions in the bound state lead to decreased/increased peak intensity ratio, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3401448&req=5

gks263-F6: NMR analysis of Myc-1–88 in the absence and presence of Bin1–SH3. Omitted histogram bars correspond to missing or overlapped residues and prolines. (A) Myc-1–88 CSPs at a Myc-1–88:Bin1–SH3 ratio of 1:1.5. The cut-off value for significant CSPs is shown as a dashed line and calculated as described in ‘Materials and methods’ section. (B) Ratios of peak intensities with and without Bin1, derived from HNCO experiments at a Myc-1–88:Bin1–SH3 ratio of 1:1.5. In the absence of interactions, or if interactions are the same in free and bound forms, the intensity ratio would be 1 (gray line). Gain/loss of interactions in the bound state lead to decreased/increased peak intensity ratio, respectively.

Mentions: The complex was analyzed at Myc-1–88:Bin1–SH3 ratios of 1:1.5, where the high-affinity site should be up to ∼88% saturated (Figures 5 and 6). Titration of unlabeled Bin1–SH3 into labeled Myc-1–88 resulted in complete disappearance of residue S62 (Figure 5) already at 10% saturation of Bin1. This is consistent with chemical exchange on the millisecond time scale, in agreement with the kinetic parameters of association determined by SPR. The early disappearance of S62 from the spectra suggests that this residue experiences a large chemical shift change for 15N and/or 1HN upon binding of Bin1–SH3 giving rise to intermediate exchange broadening; this provides strong evidence that S62 is indeed involved in Bin1–Myc recognition. Smaller but significant CSPs are observed for residues 56, 58, 61, 64, 66, 68 and 69 in fast exchange on the NMR time scale (Figures 5 and 6A). These Myc-1–88 CSP values are maintained in a 1:10 excess of Bin1–SH3, suggesting high saturation of this site already in the 1:1.5 complex (Supplementary Figure S2). Concomitantly reduced peak ratios for residues 55, 58, 61, 64, 65 and 66 are observed on 1:1.5 complex formation (Figure 6B), but the reduction is smaller than would be expected for tight binding (60) to residues 55–66 as suggested previously (24). A tendency toward reduced intensity ratios without significant CSPs is also observed for residues in the region 37–48, although this part of the plot has more limited information due to overlap and missing assignments. This region contains a proline-rich segment (P42–P43–A44–P45) not previously identified as a Bin1-binding element but that shares sequence characteristics of SH3 domain targets. An additional region of broadening upon binding is observed at the extreme C-terminus; while no canonical SH3-binding sequence is apparent, binding may be to non-canonical sequences or the broadening may be due to transient contacts with other segments that do bind Bin1. Taken together, CSP and intensity ratios are consistent with Myc anchoring into the binding groove of Bin1–SH3 at S62–P63 (24), but in agreement with SPR measurements also suggest concomitant and multivalent binding both to the adjacent P59–P60 di-peptide recognition element and to the proline-rich P42–P43–A44–P45 segment. The presence of reduced intensity ratios already at a Myc-Bin ratio of 1:1.5 supports the relevance of multivalent Bin1 binding.


Transient structure and dynamics in the disordered c-Myc transactivation domain affect Bin1 binding.

Andresen C, Helander S, Lemak A, Farès C, Csizmok V, Carlsson J, Penn LZ, Forman-Kay JD, Arrowsmith CH, Lundström P, Sunnerhagen M - Nucleic Acids Res. (2012)

NMR analysis of Myc-1–88 in the absence and presence of Bin1–SH3. Omitted histogram bars correspond to missing or overlapped residues and prolines. (A) Myc-1–88 CSPs at a Myc-1–88:Bin1–SH3 ratio of 1:1.5. The cut-off value for significant CSPs is shown as a dashed line and calculated as described in ‘Materials and methods’ section. (B) Ratios of peak intensities with and without Bin1, derived from HNCO experiments at a Myc-1–88:Bin1–SH3 ratio of 1:1.5. In the absence of interactions, or if interactions are the same in free and bound forms, the intensity ratio would be 1 (gray line). Gain/loss of interactions in the bound state lead to decreased/increased peak intensity ratio, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401448&req=5

gks263-F6: NMR analysis of Myc-1–88 in the absence and presence of Bin1–SH3. Omitted histogram bars correspond to missing or overlapped residues and prolines. (A) Myc-1–88 CSPs at a Myc-1–88:Bin1–SH3 ratio of 1:1.5. The cut-off value for significant CSPs is shown as a dashed line and calculated as described in ‘Materials and methods’ section. (B) Ratios of peak intensities with and without Bin1, derived from HNCO experiments at a Myc-1–88:Bin1–SH3 ratio of 1:1.5. In the absence of interactions, or if interactions are the same in free and bound forms, the intensity ratio would be 1 (gray line). Gain/loss of interactions in the bound state lead to decreased/increased peak intensity ratio, respectively.
Mentions: The complex was analyzed at Myc-1–88:Bin1–SH3 ratios of 1:1.5, where the high-affinity site should be up to ∼88% saturated (Figures 5 and 6). Titration of unlabeled Bin1–SH3 into labeled Myc-1–88 resulted in complete disappearance of residue S62 (Figure 5) already at 10% saturation of Bin1. This is consistent with chemical exchange on the millisecond time scale, in agreement with the kinetic parameters of association determined by SPR. The early disappearance of S62 from the spectra suggests that this residue experiences a large chemical shift change for 15N and/or 1HN upon binding of Bin1–SH3 giving rise to intermediate exchange broadening; this provides strong evidence that S62 is indeed involved in Bin1–Myc recognition. Smaller but significant CSPs are observed for residues 56, 58, 61, 64, 66, 68 and 69 in fast exchange on the NMR time scale (Figures 5 and 6A). These Myc-1–88 CSP values are maintained in a 1:10 excess of Bin1–SH3, suggesting high saturation of this site already in the 1:1.5 complex (Supplementary Figure S2). Concomitantly reduced peak ratios for residues 55, 58, 61, 64, 65 and 66 are observed on 1:1.5 complex formation (Figure 6B), but the reduction is smaller than would be expected for tight binding (60) to residues 55–66 as suggested previously (24). A tendency toward reduced intensity ratios without significant CSPs is also observed for residues in the region 37–48, although this part of the plot has more limited information due to overlap and missing assignments. This region contains a proline-rich segment (P42–P43–A44–P45) not previously identified as a Bin1-binding element but that shares sequence characteristics of SH3 domain targets. An additional region of broadening upon binding is observed at the extreme C-terminus; while no canonical SH3-binding sequence is apparent, binding may be to non-canonical sequences or the broadening may be due to transient contacts with other segments that do bind Bin1. Taken together, CSP and intensity ratios are consistent with Myc anchoring into the binding groove of Bin1–SH3 at S62–P63 (24), but in agreement with SPR measurements also suggest concomitant and multivalent binding both to the adjacent P59–P60 di-peptide recognition element and to the proline-rich P42–P43–A44–P45 segment. The presence of reduced intensity ratios already at a Myc-Bin ratio of 1:1.5 supports the relevance of multivalent Bin1 binding.

Bottom Line: We have characterized in detail the intrinsically disordered properties of Myc-1-88, where hierarchical phosphorylation of S62 and T58 regulates activation and destruction of the Myc protein.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.These findings expand the increasingly recognized concept of intrinsically disordered regions mediating transient interactions to Myc, a key transcriptional regulator of major medical importance, and have important implications for further understanding its multifaceted role in gene regulation.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biotechnology, Department of Physics, Chemistry and Biology, Linköping University, SE-58183 Linköping, Sweden.

ABSTRACT
The crucial role of Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of Myc function. The N-terminal region of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of Myc-1-88, where hierarchical phosphorylation of S62 and T58 regulates activation and destruction of the Myc protein. By nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements and NOE analysis, we show that although Myc occupies a very heterogeneous conformational space, we find transiently structured regions 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. These findings expand the increasingly recognized concept of intrinsically disordered regions mediating transient interactions to Myc, a key transcriptional regulator of major medical importance, and have important implications for further understanding its multifaceted role in gene regulation.

Show MeSH
Related in: MedlinePlus