High resolution nuclear magnetic resonance studies of kringle containing proteins
The kringle is a name given to a sequence of seventy nine residues linked by three disulphide bridges, multiple copies of which have been found in a number of proteins involved in haemostasis. Kringles from plasminogen and prothrombin have been studied using high resolution nuclear magnetic resonance (NMR) spectroscopy. Preliminary experiments demonstrated the kringles to be globular proteins in aqueous solution having well defined tertiary folds. Resonances have been assigned in the NMR spectrum to all but two of the aromatic residues of kringle 4 of human plasminogen and in addition a number of aliphatic residues have also been assigned. The majority of these assignments have been made to specific protons in the protein with the aid of chemically modified kringle 4 derivatives and comparison with other kringle protein spectra. Nuclear Overhauser enhancement studies have demonstrated a number of structurally significant side chain interactions. The lysine binding site of kringle 4 has been explored using two ω-aminocarboxylic acids as ligands. Further structural probes have included copper and lanthanide ions as well as chemically modified kringle 4 species. Combining all the data has allowed a preliminary molecular model to be built of part of kringle 4 incorporating all the residues between the second and third disulphide bridges. Preliminary studies demonstrate the possibility of being able to refine parts of this structure to an accuracy approaching that of X-ray crystallographic studies. A similar number of assignments have been made in the spectrum of bovine prothrombin fragment 2. Comparison of the data on the kringles of both fragment 2 and kringle 4 shows the two proteins to have preserved structural elements, and in particular one of these involves the conserved residues Trp 25, Leu 45 and Trp 61. Furthermore, the two tryptophan residues have been shown to have quite unique spectral charateristics. Brief studies of human prothrombin fragment 2 reveals similar findings. The results discussed indicate that the kringle sequence gives rise to a well defined fold. In kringle 4 of human plasminogen this is seen to provide a highly specific binding site for certain ω-aminocarboxylic acids. The data is all in accord with kringles being autonomous units within the non protease portion of zymogen molecules and which are ideally suited to include the mediation of protein-protein interactions amongst their biological functions.