Structure and dynamics of protein-protein and protein-ligand complexes studied by heteronuclear NMR spectroscopy in solution

Within the project it is planned to study the solution structures and dynamics of a group of proteins both in their apo-forms and when bound to their physiological targets. One of the proposed topics is concerned with the investigation of the solution structure of the EB1 protein. The EB1 family represents a highly conserved group of proteins, present in yeast through humans, that localize to spindle and cytoplasmic microtubules, and the human EB1 protein interacts physically with the adenomatous polyposis coli (APC) tumor suppressor protein. APC has been implicated in a number of cellular processes including the regulation of directed cell migration, and germline mutations in the APC gene lead to the inherited disease familial adenomatous polyposis (FAP) which results in the development of colorectal tumors in early adult life. The solution structures of both the apo-form of EB1 and in complex with CRP2, a protein whose level of expression is controlled by the oncogene v-myc, will thus provide crucial structural and functional information about EB1 and CRP2. A second topic is concerned with investigations of the binding determinants of the oncogenic transcription factor myc, which sequence-specifically binds double stranded DNA in homo-dimeric or in a a hetero-dimeric form with its authentic binding partner max (myc-associated x-factor). The monomeric form of myc exists as a partially folded protein comprising two alpha-helical domains, which become structured upon binding to max. The structural data of the complex will provide details of the structural reordering of monomeric myc upon formation of the transcriptionally active myc/max heterodimer. Finally, applications to dynamic studies of protein-ligand complexes will rely on a class of NMR experiments that exploit the measurement of cross-correlation between the fluctuations of two relaxation-active interactions. The focus of this part of the project will be the detailed characterization of intramolecular (micro-millisecond) protein fluctuation changes upon ligand binding. Specifically, we plan the structural and dynamical characterization of the lipocalin protein Q83 (a protein whose level of expression is strongly raised upon activation by the oncogene v- myc) ligated with a specific small molecular weigth binding partner. These studies will provide an important insight into the structural and dynamical changes occuring in secretory proteins upon binding small hydrophobic molecules.

Within the project the solution structures and dynamics of a group of proteins both in their apo-forms and when bound to their physiological targets were successfully determined. The EB1 family represents a highly conserved group of proteins, present in yeast through humans, that localize to spindle and cytoplasmic microtubules, and the human EB1 protein interacts physically with the adenomatous polyposis coli (APC) tumor suppressor protein. APC has been implicated in a number of cellular processes including the regulation of directed cell migration, and germline mutations in the APC gene lead to the inherited disease familial adenomatous polyposis (FAP) which results in the development of colorectal tumors in early adult life. The solution structures of both the apo-form of EB1 and in complex with CRP2 were determined, a protein whose level of expression is controlled by the oncogene v-myc. These data thus provides for the first time detailed structural and dynamical information about EB1 and CRP2. A second topic is concerned with investigations of the protein product (c-Myc) of the protooncogene c-myc which is a transcriptional regulator playing a crucial role in cellular growth, differentiation, and apoptosis. Myc-dependent interactions are highly regulated to sustain cellular homeostasis and deregulation of myc genes leads to cell transformation. Multi-dimensional NMR spectroscopy was used to provide for the first time structural and dynamical information about a heterodimeric helix-loop-helix oncogenic transcription factor complex before binding to its cognate DNA. NMR analysis revealed two stably formed -helical regions of v-Myc separated by a conformationally stable loop region, which is in stark contrast to monomeric apo v-Myc which exists as a partially folded state in solution and unambigiously demonstrated a heterodimeric v-Myc/Max protein complex. Additionally, unprecedented dielectric reflection spectroscopy data revealed significant macroscopic electric dipoles for the basic helix-loop-helix-leucine zipper (bHLHZip) protein family and provided evidence for an `anti- ferromagnetic` behaviour of dimeric transcription factors in solution. This finding is of great functional relevance as it provides a plausible (and novel) mechanism to upregulate gene expression involving widely separated E boxes, without Myc/Max heterotetramerization invoking direct and specific intermolecular interactions.