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.