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Role of secondary structure in discrimination between constitutive and inducible activators
1Joslin Diabetes Center, Research Division, Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02138, USA.
Abstract:
We have examined structural differences between the proto-oncogene c-Myb and the cyclic AMP-responsive factor CREB that underlie their constitutive or signal-dependent activation properties. Both proteins stimulate gene expression via activating regions that articulate with a shallow hydrophobic groove in the KIX domain of the coactivator CREB-binding protein (CBP). Three hydrophobic residues in c-Myb that are conserved in CREB function importantly in cellular gene activation and in complex formation with KIX. These hydrophobic residues are assembled on one face of an amphipathic helix in both proteins, and mutations that disrupt c-Myb or CREB helicity in this region block interaction of either factor with KIX. Binding of the helical c-Myb domain to KIX is accompanied by a substantial increase in entropy that compensates for the comparatively low enthalpy of complex formation. By contrast, binding of CREB to KIX entails a large entropy cost due to a random coil-to-helix transition in CREB that accompanies complex formation. These results indicate that the constitutive and inducible activation properties of c-Myb and CREB reflect secondary structural characteristics of their corresponding activating regions that influence the thermodynamics of formation of a complex with CBP.
Insights
Structural differences in c-Myb and CREB proteins explain their distinct gene activation properties. Their interaction with CBP
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- Proto-oncogene c-Myb and cyclic AMP-responsive factor CREB regulate gene expression.
- Both proteins interact with the KIX domain of CREB-binding protein (CBP) via their activating regions.
- Understanding the structural basis for their distinct activation properties (constitutive vs. signal-dependent) is crucial.
Purpose of the Study:
- To investigate the structural differences between c-Myb and CREB that dictate their activation properties.
- To elucidate the role of specific residues and secondary structures in the interaction with CBP's KIX domain.
- To understand the thermodynamic basis for complex formation between these factors and CBP.
Main Methods:
- Comparative structural analysis of c-Myb and CREB activating regions.
- Site-directed mutagenesis to disrupt helicity and assess effects on KIX binding.
- Thermodynamic analysis (entropy and enthalpy measurements) of complex formation with KIX.
Main Results:
- Three conserved hydrophobic residues on an amphipathic helix are critical for c-Myb and CREB interaction with KIX.
- Disruption of helicity in this region abrogates binding to KIX.
- c-Myb binding to KIX is entropically driven, while CREB binding involves an entropic penalty due to a coil-to-helix transition.
Conclusions:
- Secondary structural features of the activating regions of c-Myb and CREB significantly influence their interaction thermodynamics with CBP.
- These structural and thermodynamic differences underlie the distinct constitutive and inducible gene activation properties of c-Myb and CREB.
- The findings provide insights into the regulation of gene expression by transcription factors.