tailieunhanh - Báo cáo Y học: Molecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl-terminal domain

Lipoprotein lipase (LPL) plays a key role in lipid metabo-lism. Molecular modeling of dimeric LPL was carried out usingINSIGHT IIbased upon the crystal structures of human, porcine, and horse pancreatic lipase. The dimeric model reveals a saddle-shaped structure and the key heparin-binding residues in the amino-terminal domain located on the top of this saddle. | Eur. J. Biochem. 269 4701-4710 2002 FEBS 2002 doi Molecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl-terminal domain Yoko Kobayashi Toshiaki Nakajima and Ituro Inoue Division of Genetic Diagnosis Institute of Medical Science The University of Tokyo Tokyo Japan Lipoprotein lipase LPL plays a key role in lipid metabolism. Molecular modeling of dimeric LPL was carried out using INSIGHT II based upon the crystal structures of human porcine and horse pancreatic lipase. The dimeric model reveals a saddle-shaped structure and the key heparin-binding residues in the amino-terminal domain located on the top of this saddle. The models of two dimeric conformations - a closed inactive form and an open active form - differ with respect to how surface-loop positions affect substrate access to the catalytic site. In the closed form the surface loop covers the catalytic site which becomes inaccessible to solvent. Large conformational changes in the open form especially in the loop and carboxyl-terminal domain allow substrate access to the active site. To dissect the structure-function relationships of the LPL carboxyl-terminal domain several residues predicted by the model structure to be essential for the functions of heparin binding and substrate recognition were mutagenized. Arg405 plays an important role in heparin binding in the active dimer. Lys413 Lys414 or Lys414 regulates heparin affinity in both monomeric and dimeric forms. To evaluate the prediction that LPL forms a homodimer in a head-to-tail orientation two inactive LPL mutants - a catalytic site mutant S132T and a substrate-recognition mutant W390A W393A W394A - were cotransfected into COS7 cells. Lipase activity could be recovered only when heterodimerization occurred in a head-to-tail orientation. After cotransfection 50 of the wild-type lipase activity was recovered indicating that lipase activity is determined by the .