The allosteric mechanism of the KIX domain

Allosteric regulation represents an effective mechanism of control in many key biological pro-cesses on a molecular level. Allostery requires that information about the presence of a binding partner is communicated between remote sites within a protein or protein complex, which is typically (but not always) accomplished through defined structural re-arrangements. While allosteric mechanisms of various dimeric or multi-domain protein systems have been characterized in detail, significantly less is known about the modes of communication between remote binding sites within single-domain proteins. The aim of this stand-alone project of the FWF is to provide a comprehensive description of the allosteric mechanism through which the KIX domain of the transcriptional co-activator CREB binding protein (CBP) communicates information internally between binding sites and regulates pairwise affinities for biological targets (transcription factors). Because allosteric communication is fundamentally dynamic in nature it can be characterized in a quantitative manner by nuclear magnetic resonance (NMR) spin relaxation techniques. Dynamic NMR spectroscopy provides various experimental tools to study conformational re-arrangement processes at atomic resolution, and can be employed to monitor directly the conformational transition through which KIX communicates information about the presence of biological targets internally. These experiments thus reveal information about the rate at which intra-moelcular communication between binding sites occurs, as well as the pathway through which information is transmitted. To provide further insights into mechanistic details of allosteric communication in KIX, dynamic NMR spectroscopy will be complemented by a variety of biophysical techniques, ranging from isothermal calorimetry and structure analysis to systematic mutational studies. The integration of diverse sets of experimental data will enable us to provide a quantitative mechanistic and structure-based description of how the KIX domain of CBP mediates cooperativity between transcription factors, and significatly contribute to a detailed understanding of the regulatory mechanisms of gene transcription.

In this project of the Austrian Science Fund FWF we characterized the molecular function of a protein at atomic resolution. In cells, proteins molecules perform most of the processes that form the basis of life. One specific function of proteins is, for example, the formation of larger groups of proteins to active "complexes", which then fulfill their biological function. Characterizing the mechanisms by which proteins form such complexes is of fundamental interest to biological sciences, in particular in light of the fact that the failure of proteins to do so has been linked to numerous prevalent diseases. An example for such protein complexes is RNA polymerase, which is responsible for the synthesis of RNA and thus for the transcription of genetic information.Using nuclear magnetic resonance (NMR) relaxation techniques in solution we were able to show that the protein KIX, which forms part of the multi-protein complex RNA polymerase, contributes significantly to the stabilitiy of this complex. Combination of complementary experimental approaches enabled us to quantitate the structural stability of various model complexes of KIX with interaction partners. We showed that KIX significantly contributes to the formation of these interactions through structural flexibility. Because our techniques enable the experimental characterization of protein flexibility at atomic resolution, a genuine description of the underlying mechanism could be provided. In addition, we characterized the three-dimensional structure of KIX in solution in complex with interaction partners. Our experiments showed that while the structural properties of this protein are in accordance with its flexibility, the flexibility itself appears to be time averaged in the three-dimensional structure. Our results thus point towards the importance of experimental studies of protein flexibility at atomic resolution for understanding interactions between protein molecules.