Structural biology studies of disease related protein-protein interactions by NMR spectroscopy

The receptor for advanced glycated end products (RAGE) is a multiligand receptor that is implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative disorders and inflammatory responses. There is very limited information available about molecular mechanisms underlying the physiological and pathological function of RAGE. The purpose of my work is to develop structural understanding of interactions between the RAGE receptor and some of its ligands, by using high resolution NMR spectroscopy.;As a pattern recognition receptor, RAGE can recognize advanced glycated end products (AGEs) formed by nonenzymatic glyoxidation. Structural mechanisms of AGE recognition was an enigma due to the diversity of chemical structures found in AGE modified proteins. By using NMR spectroscopy we identified three distinct surfaces of the immunoglobulin V-type domain of RAGE mediating AGE-RAGE domain interactions. The secondary structure elements of the interaction surfaces exhibit significant flexibility on the ms-μs time scale. Despite highly specific AGE-V domain interactions, the binding affinity of AGEs for an isolated V domain is low, ∼10 μM. We propose that constitutive oligomerization of RAGE is responsible for recognizing patterns of AGE modified proteins with affinities less than 100 nM.;With the ultimate goal of finding potential therapeutic agents for regulating RAGE activity, we developed a high-throughput in-cell NMR assay called SMILI- NMR (Screening of small Molecule I nteractor Library using In-cell NMR spectroscopy). The method relies on the formation of a well-defined biocomplex and utilizes in-cell NMR spectroscopy to identify the molecular surfaces involved in the interaction at atomic scale resolution. Utility of the method is demonstrated by screening a small dipeptide library against the FKBP-FRB protein complex involved in cell cycle arrest. The dipeptide identified by SMILI-NMR showed biological activity in the functional assay in yeast.;Calgranulin C (S100A12) is a member of the S100 family of proteins that undergoes a conformational change upon calcium binding allowing them to interact with target molecules and initiate biological responses; one such target is RAGE. The RAGE-calgranulin C interaction mediates a pro-inflammatory response to cellular stress and can contribute to the pathogenesis of inflammatory lesions. The soluble extracellular part of RAGE (sRAGE) was shown to decrease the inflammation response possibly by scavenging RAGE-activating ligands. By using high resolution NMR spectroscopy, we identified the sRAGE-calgranulin C interaction surface on both Ca2+ binding and Apo-form of calgranulin C and showed calgranulin C binds to the first immunoglobulin C-type domain of RAGE. By using native gel electrophoresis and chromatography, we established that sRAGE forms tetramers that bind to hexamers of Ca 2+-calgranulin C.