Structural basis for target search and recognition in protein complexes probed by NMR spectroscopy

Abstract

The protein-protein interaction is important in many biological processes, including enzyme catalysis, immune response, and cell signaling. It is important to understand the structural basis for the protein interactions, since there is a direct relationship between the structure and the biological function in cell. NMR spectroscopy has become a powerful analytical tool for the protein structure and dynamics at the molecular level and emphasizing the application in biomolecular systems. Many proteins have been studied on the determination of protein structure and the biological functions using NMR. In this thesis, I demonstrate that the target search process of a protein complex can be visualized by NMR paramagnetic relaxation enhancement (PRE), and that the specific target recognition can be achieved by unusual coupling of binding and unfolding. In Chapter I, the target search pathway via encounter complex ensemble is characterized between the N-terminal domain of enzyme I (EIN) and the heat-stable histidine phosphocarrier (HPr) of bacterial phosphotransferase system (PTS). The encounter complex ensemble represents short-lived and lowly-populated nonspecific complexes that quickly relax into the final specific complex. It has been difficult to experimentally detect the encounter complexes, but the recent application of PRE was enabled to prove the characterization of transient encounter complexes during protein-protein association. I employed rational mutations of EIN based on PRE data, which potentially perturbed the target search pathway, and examined how the mutations altered the encounter complex formation and also the equilibrium binding constant. If encounter complexes at specific region influences the protein association, they are classified as productive encounter, otherwise non-productive. Mapping the region of productive encounter complexes on the surface of EIN, I visually demonstrate the allowed and disallowed target search pathways between EIN and HPr. In Chapter II, the complex structure of fibronectin extradomain B (EDB) and a specific binding peptide (aptide, APT) was determined by NMR spectroscopy. EDB is a prominent marker of tumor angiogenesis, and APT is a small peptide (26 aa) with a beta-hairpin scaffold and two engineered target binding arms. APT recognizes EDB via an unusual beta strand replacement mechanism, in which APT permanently unfolds the entire C-terminal beta strand of EDB and forms a new intermolecular beta sheet within the complex. Thus the interaction interface of EDB is not located on the surface, but buried inside of the protein. Unfolding of EDB exposes the hydrophobic binding interface that is specifically recognized by APT. The unique binding mode of coupled binding and unfolding will broaden our understanding of the diverse protein interactions.