NEUTRON CRYSTALLOGRAPHIC EVIDENCE OF LIPASE/COLIPASE COMPLEX ACTIVATION BY A MICELLE.
Juan Hermoso1, David Pignol2, Simon Penel3, Michel Roth2, Catherine Chapus4 & Juan C. Fontecilla-Camps2
1Grupo de
Cristalografía Macromolecular y Biología Estructural, Intituto
"Rocasolano"; CSIC, Serrano 119, 28006-Madrid, Spain.
2 Laboratoire de Cristallographie et de Cristallogenese des
Protéines, Institut de Biologie Structurale Jean-Pierre Ebel,
CEA-CNRS, 41 Avenue des Martyrs, 38027 Grenoble Cedex 1,France.
3Large Scale Structures Group, Institut Laue-Langevin, BP 156,
38042 Grenoble, France.
4Laboratoire de Bioénergétique et d'Ingénierie des Protéines,
UPR 9036 CNRS, BP 71, 13402 Marseille Cedex 9, France.
e-mail: xjuan@iqfr.csic.es
Keywords: hydrolase,
neutron difraction, lipid, interfacial activation, ternary
complex.
The concept of lipase interfacial activation stems from the finding that the catalytic activity of most lipases depends on the aggregation state of their substrates. It is thought that activation involves the unmasking and structuring of the enzyme's active site through conformational changes requiring the presence of oil-in-water droplets. We have recently reported the X-ray structure of the porcine pancreatic lipase (PL)-colipase (CL) complex obtained in the presence of the C8E4 non-ionic detergent1. In that structure pancreatic lipase was found to be in the open conformation, result we attributed to the presence of detergent micelles in the crystallization medium. Here, we present the neutron structure of the activated lipase/colipase/micelle complex as determined using the D2O/H2O contrast variation low resolution diffraction method. In the ternary complex (Fig.1), the disk-shaped micelle interacts extensively with the concave face of colipase and the distal tip of the C-terminal domain of lipase2. This is , to our knowledge, the first time a micelle is visualized as a part of an enzymatic complex without displaying extensive hydrophobic interaction with the protein moieties, as in membrane proteines.
Since the micelle and substrate binding
sites concern different regions of the protein complex, we
conclude that lipase activation is not interfacial but occurs in
the aqueous phase and it is mediated by colipase and a micelle.
We believe that, in vivo, the formation of a complex between
inactive PL, CL and a mixed micelle (Fig. 2) activates the enzyme by stabilizing the open
conformation and exposing a large hydrophobic surface. This
surface should facilitate complex binding to the underlying
triglycerides of the emulsified duodenal oil particle.
Fig. 2. Schematic
depiction of PL activation in solution by CL and a duodenal mixed
micelle. The particle polar layer and underlaying substrates (not
drawn to scale) are depicted in gray and yellow respectively.
1. Hermoso, J., Pignol, D.,
Kerfelec, B., Crenon, I., Chapus, C. & Fontecilla-Camps, J.C.
J. Biol. Chem. 271 (1996) 18007-18016.
2. Hermoso, J., Pignol, D., Penel,
S., Roth, M., Chapus, C. & Fontecilla-Camps, J.C. EMBO
Journal 16 (1997), No.18, 5531-5536.