Impaired insulin signaling due to peripheral insulin resistance and failure of pancreatic insulin secretion is a key factor for the onset and manifestation of type 2 diabetes mellitus (T2D). Insulin receptor (IR) agonists are being developed to restore insulin signaling via direct activation of the cytoplasmic insulin receptor tyrosine kinase activity. Two classes of such compounds have been developed by the pharmaceutical companies Merck & Co., Inc., and Telik, Inc., but in patents and literature there is no data as to how these compounds activate the IR kinase activity. Attempts to solve the crystal structures of dimeric human IR kinase domains and complexes of these with IR agonists within this thesis failed. However, biophysical characterization of the full-length intracellular part of the IR containing the kinase domain demonstrated an intrinsic ability to dimerize independently of the enzyme-substrate type of interaction seen in IR autophosphorylation. This dimer formation was found to be enhanced in the presence of a distinct IR agonist. Crystals of various constructs and isoforms of the key antidiabetic target AMP-activated protein kinase (AMPK) were obtained. Despite substantial optimization trials these crystals were not sufficient for solving the structure of AMPK. However, the crystal structure of carnitine palmitoyltransferase 2 (CPT-2), a protein downstream of the metabolite signaling initiated by AMPK, was solved. CPT-1 and -2 facilitate the import of long-chain fatty acids into mitochondria. Modulation of the catalytic activity of the CPT system is currently under investigation for the development of novel drugs against diabetes mellitus. The crystal structure of the full-length mitochondrial membrane protein CPT-2 was solved at a resolution of 1.6 Å. The structure of CPT-2 in complex with the generic CPT-inhibitor ST1326 [(R)-N-tetradecylcarbamoyl-aminocarnitine], a substrate analog mimicking palmitoylcarnitine and currently in clinical trials for T2D treatment, was solved at 2.5 Å resolution. These structures of CPT-2 provide insight into the function of residues involved in substrate binding and determination of substrate specificity, thereby facilitating the rational design of novel antidiabetic drugs. A sequence insertion uniquely found in CPT-2 was identified that mediates membrane localization. Mapping of mutations described for CPT-2 deficiency, a hereditary disorder of lipid metabolism, implies effects on substrate recognition and structural integrity of CPT-2
