Conformational Control of the Binding of the Transactivation Domain of the MLL Protein and c-Myb to the KIX Domain of CREB

The KIX domain of CBP is a transcriptional coactivator. Concomitant binding to the activation domain of proto-oncogene protein c-Myb and the transactivation domain of the trithorax group protein mixed lineage leukemia (MLL) transcription factor lead to the biologically active ternary MLL∶KIX∶c-Myb complex which plays a role in Pol II-mediated transcription. The binding of the activation domain of MLL to KIX enhances c-Myb binding. Here we carried out molecular dynamics (MD) simulations for the MLL∶KIX∶c-Myb ternary complex, its binary components and KIX with the goal of providing a mechanistic explanation for the experimental observations. The dynamic behavior revealed that the MLL binding site is allosterically coupled to the c-Myb binding site. MLL binding redistributes the conformational ensemble of KIX, leading to higher populations of states which favor c-Myb binding. The key element in the allosteric communication pathways is the KIX loop, which acts as a control mechanism to enhance subsequent binding events. We tested this conclusion by in silico mutations of loop residues in the KIX∶MLL complex and by comparing wild type and mutant dynamics through MD simulations. The loop assumed MLL binding conformation similar to that observed in the KIX∶c-Myb state which disfavors the allosteric network. The coupling with c-Myb binding site faded, abolishing the positive cooperativity observed in the presence of MLL. Our major conclusion is that by eliciting a loop-mediated allosteric switch between the different states following the binding events, transcriptional activation can be regulated. The KIX system presents an example how nature makes use of conformational control in higher level regulation of transcriptional activity and thus cellular events. Author Summary: CBP (CREB-binding protein) is a transcriptional regulator of RNA polymerase II-mediated transcription. KIX is a domain of CBP. KIX binding to the activation domain of proto-oncogene protein c-Myb and the transactivation domain of the trithorax group protein mixed lineage leukemia (MLL) transcription factor forms the biologically active ternary MLL∶KIX∶c-Myb complex. This complex has been shown to play a key role in Pol II-mediated transcription. Experimental data show that the binding of the activation domain of MLL to KIX enhances c-Myb binding. Here, we studied the c-Myb∶KIX∶MLL ternary structure and models based on this structure, KIX-only, KIX-MLL, and c-Myb-KIX, by explicit solvent molecular dynamics (MD) simulations. MD simulations can help in figuring out allosteric events and thus functional mechanisms. Our in silico analysis of the dynamic behavior indicated that when MLL is bound, there is allosteric communication between the two KIX binding sites. We observed a shift in the conformational ensemble of KIX upon the binding of the MLL activation domain, leading to a pre-organization of the KIX∶c-Myb binding site and explaining the enhanced c-Myb binding observed experimentally. On the other hand, this is not the case if c-Myb binds to KIX, which suggests that KIX regulation is under conformational control.