Probing Conformational Changes and Interfacial Recognition Site of Lipases With Surfactants and Inhibitors

Abstract

Structural studies on lipases by X-ray crystallography have revealed conformational changes occurring in the presence of surfactants/inhibitors and the pivotal role played by a molecular “lid” of variable size and structure depending on the enzyme. Besides controlling the access to the enzyme active site, the lid is involved in lipase activation, formation of the interfacial recognition site (IRS), and substrate docking within the active site. The combined use of surfactants and inhibitors has been critical for a better understanding of lipase structure–function relationships. An overview of crystal structures of lipases in complex with surfactants and inhibitors reveals common structural features and shows how surfactants monomers interact with the lid in its open conformation. The location of surfactants, inhibitors, and hydrophobic residues exposed upon lid opening provides insights into the IRS of lipases. The mechanism by which surfactants promote the lid opening can be further investigated in solution by site-directed spin labeling of lipase coupled to electron paramagnetic resonance spectroscopy. These experimental approaches are illustrated here by results obtained with mammalian digestive lipases, fungal lipases, and cutinases.

Introduction

Lipases (EC 3.1.1.3; triacylglycerol hydrolases) play an important role in the uptake of fatty acids from fats and mobilization of triglycerides stored in various lipid bodies or transported by lipoproteins. They are found to occur in most organisms in the animal, plant, and microbial kingdoms (Aloulou et al., 2006). In order to catalyze the hydrolysis of ester bonds in triglycerides, they have to bind at the oil–water interface of lipid droplets, and their activity depends on the available surface (Benzonana & Desnuelle, 1965) and various interfacial parameters like surface tension (Verger, 1980). Some lipases also interact with smaller substrate aggregates like micelles and present a broader substrate specificity (monoglyceride lipase, phospholipase A1, galactolipase) (Bakala N'Goma et al., 2012, Eydoux et al., 2008). The biochemical characterization of lipases has always been associated with the use of surfactants, like gum Arabic to form and stabilize fine substrate emulsions (Tiss, Carriere, & Verger, 2001) or bile salts to solubilize lipolysis products (Hofmann, 1963). Surfactants also have a significant impact on the partitioning of lipase between the water phase and oil–water interface (Delorme et al., 2011). They can compete with lipases for adsorption (Bezzine et al., 1999) or promote lipase exchange between lipid droplets via lipase interaction with micelles in solution (Haiker, Lengsfeld, Hadvary, & Carrière, 2004). Last but not least, they are required for efficient lipase inhibition in solution, and these findings have led to various speculations on their interactions with lipase and/or inhibitors.

In early studies on pancreatic lipase inhibition by serine hydrolase inhibitors, it was shown that diethyl p-nitrophenyl phosphate (E600; Fig. 1) could be included into bile salt micelles and that pancreatic lipase inhibition rate was strongly dependent on the bile salt concentration and micellar concentration of the organophosphate, as well as on the presence of colipase (Rouard, Sari, Nurit, Entressangles, & Desnuelle, 1978). It was proposed that the “solubilization” of the poorly water-soluble organophosphate in micelles was a prerequisite for lipase inhibition after enzyme interaction with mixed micelles. 3D structures of lipases were unknown at that time, and there was no indication that bile salts could promote lipase inhibition by other mechanisms. The use of bile salts like sodium taurodeoxycholate (NaTDC; Fig. 1) and other surfactants like β-octylglucoside (BOG; Fig. 1) and tetraethylene glycol monooctyl ether (TGME; Fig. 1) has become a classical approach to promote lipase inhibition by poorly water-soluble inhibitors (Delorme et al., 2011, Hermoso et al., 1996, Moreau et al., 1991) and investigate structural changes by various means, including tryptophan fluorescence spectroscopy (Bourbon-Freie et al., 2009, Lüthi-Peng and Winkler, 1992, Yapoudjian, Ivanova, et al., 2002a, Yapoudjian, Ivanova, et al., 2002b).

When the first lipase 3D structures revealed the presence of a molecular lid blocking the access to the active site (Brady et al., 1990, Winkler et al., 1990), it was rapidly envisioned to cocrystallized lipases with surfactants and covalent inhibitors to unravel conformational changes necessary for lipase activation and inhibition (Brzozowski et al., 1991, van Tilbeurgh et al., 1993). The combined use of surfactants and inhibitors has thus been critical for elucidating the mechanism of lipase activation. As illustrated in this chapter, crystal structures of several lipases in complex with surfactants and inhibitors are today available and have revealed common structural features and shown how surfactants monomers can interact with the lid and stabilize its open conformation. The location of surfactants, inhibitors, and hydrophobic residues exposed upon lid opening provides insights into the interfacial recognition site (IRS) of lipases.

X-ray crystallography has been the method of choice for studying lipase conformational changes because most lipases are too large to be studied by nuclear magnetic resonance. Data on the mechanism of lid opening are however limited and mostly deduced from molecular dynamics simulations based on known X-ray structures of lipases (Barbe et al., 2009, Bordes et al., 2010, Jensen et al., 2002, Rehm et al., 2010, Santini et al., 2009, Selvan et al., 2010). Another approach for investigating the conformational changes in large proteins like lipases consists in the association of site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. EPR spectroscopy allows to document protein conformational changes at the residue level and provides dynamic information on structural transitions occurring within well-characterized structured proteins for which X-ray crystallography can only provide snapshots of the initial and final conformations as illustrated with lipases. Moreover, SDSL–EPR spectroscopy allows working in solution, at room/physiological temperature and with limited amounts of enzyme/protein (20 μL samples at 40–80 μM with pancreatic lipase). This approach is illustrated here by the study of pancreatic lipase lid opening in the presence of various bile salt concentrations (Belle et al., 2007).