Lipases hydrolyse long-chain fatty acid esters at water-oil interfaces through the mechanism of interfacial activation mediated by the movement of a lid subdomain that covers the active site. Studying lid movement is an area of active research in the field of protein dynamics. The lipase from Pseudomonas aeruginosa is a 29-kDa protein that was previously crystallized in the open conformation, and as expected, an approximately 20-residue lid subdomain was identified. In the present study, the authors report extensive molecular dynamics simulations of the P. aeruginosa lipase. They show that this protein has two lids covering the substrate-binding pocket. The first lid is the one proposed from the known crystal structure. The second lid, a much shorter one, lies over the binding pocket facing the first lid. Furthermore, using position-restrained simulations, these authors show that movement of the second lid may actually be a trigger for the movement of the first, and that this triggering action is driven by hydrophobic contacts between the two lids. This computational study paves a way for experimentalists to study the structure and dynamics of this protein in greater detail in order to understand coupled subdomain movements in a comprehensive fashion.

Lipases are enzymes that catalyze both the formation and cleavage of long-chain acylglycerols [1]. In recent years, crystal structures of several lipases—bacterial, fungal and mammalian—have been determined [2–8]. Industrially, lipases—especially those of bacterial and fungal origins—are seemingly attractive as biocatalysts due to their cofactor independence, broad substrate specificity, enantioselectivity, and stability in organic solvents [9]. In addition, there have been a number of reports implicating lipases in pathogenicity and biofilm formation [10,11].

All lipases, despite extensive sequence divergence, have a common fold known as the alpha/beta hydrolase fold [12]. The canonical alpha/beta hydrolase fold has eight parallel beta strands, with the second one anti-parallel. Strands 3–8 are connected by alpha helices, which pack on either side of the beta sheet. The active site of lipases is the classical catalytic triad composed of a serine, an aspartate/glutamate, and a histidine [13]. An interesting characteristic of lipases is their interfacial activation. One of the common modes by which this activation occurs is through a lid, also called flap: a stretch of amino acids that moves towards closing the hydrophobic binding pocket in an aqueous environment and opens in an oil-water interface [14]. For further details, see Protocol S1.

The lipase in this study—that from Pseudomonas aeruginosa—is a 29-kDa protein. This lipase differs from the canonical alpha/beta hydrolase fold in that the first two beta strands and one alpha helix are missing. The active site residues in this lipase are Ser82, Asp229, and His251. The loop containing the active-site histidine is stabilized by an octahedrally coordinated calcium ion. On top of the active site, a lid subdomain, comprising the alpha helix residues 125–148 and its surrounding loops, is present in an open conformation in the crystal structure [15].

Molecular dynamics (MD) studies have been used extensively in the study of protein folding [16] and enzyme catalysis [17]. MD simulations have been used to show that there are three mobile regions in the lipase from Rhizomucor miehei, one of which corresponds to the active-site lid [18]. The role of lids and protein orientation with respect to the lipid-water interface in lipase catalysis has been investigated using MD simulations [19,20]. Dependence of enantioselectivity of Candida antarctica lipase on temperature has also been studied using MD [21]. Our group has recently used MD simulations to explain pH-dependent enantioselectivity of C. rugosa lipase and has elucidated the role of the lid in the same [22]. A short introduction to MD simulations is provided in Protocols S2, S3, and S4.

Lipases were traditionally believed to have a single lid. By carrying out extensive molecular dynamics simulations, we infer the presence of a second lid in P. aeruginosa lipase. (The lid proposed from the crystal structure is henceforth called lid1, and the second lid as seen from our results, lid2.) We show that the movement of lid2 acts as a trigger for the movement of lid1. Using similar MD simulations of rational in-silico mutants of this lipase, we elucidate the importance of hydrophobic interactions in movement of the lids. Thus we provide novel insights into interfacial activation in P. aeruginosa lipase.

Comparisons of the crystal structures of the open and the closed forms of lipases have been used to conclusively identify mobile loops. However, the crystal structure of only the open form of the lipase from P. aeruginosa is available. Under these circumstances, it is imperative to use other techniques, such as MD simulations, to identify such mobile loops that are essential for the enzyme's activity. Simulations of the open and the closed form of Humicola lanuginose lipase, a fungal lipase, in water for 12 ns using a standard procedure has shown that the open form tends to move towards the closed conformation, while not closing entirely, whereas the closed form remains as such [19]. Ours is, to our knowledge, the first comprehensive study on the dynamics of P. aeruginosa lipase.

The open structure of P. aeruginosa lipase was obtained from the Protein Data Bank (PDB). This was solvated in explicit water and energy minimized. The energy-minimized structure was subjected to MD simulation for 20 ns. The backbone root mean square deviation (RMSD) as calculated after least square fit increased steadily up to about 2.5 ns and reached a plateau. The RMSD value started increasing again after about 4.5 ns and stabilized at around 7 ns. General simulation parameters are shown in Protocol S5.

Figure 1 Lid Movement in P. aeruginosa Lipase: Quantitative Measures

Figure 2 Lid Movement in P. aeruginosa Lipase: Qualitative Representation

The 10 ns structure does not differ much from the final 20-ns structure (lid1 and lid2 RMSDs are less than 0.1 nm) and hence was taken as the representative structure on the basis of which simulations involving the closed form were performed. Also, the third highly mobile region, residues 20–30, does not cover the binding pocket and hence is not included in further discussion. This movement is touched upon in Protocol S6. Moreover, restraining the movement of this third loop does not seem to affect lid movement in any manner.

A “back” simulation was performed using the closed structure in a water-octane interface. We observed that both the lids actually opened out, and the structure thus formed was nearly the same as the initial crystal structure. The RMSD variation of the lids during the course of the simulation is shown in Figure 3A. The open form obtained after 20 ns and the crystal structure are superimposed to a C-alpha RMSD of 0.08 nm and is shown in Figure 3B.

Figure 3 Simulation Results of Closed Form in Octane-Water Interface

Figure 4 Lid1 Movement is Triggered by Lid2, but Lid2 Movement Is Independent of Lid1

Figure 5 Helical Wheel Diagrams for Lid1 and Lid2

At the first level, we carried out MD studies on single mutants: the Phe214 and Ala126 were independently mutated to Ser in two different simulations. This was done so as to investigate the role of individual residues in domain movement. These mutations do not seem to affect the lid movement, which is almost the same as in the wild type. The RMSDs of the lids are similar in both the cases (Figure 6; for qualitative information, see Protocol S7).

Figure 6 Simulations with Lipase Single Mutants

Subsequently we performed a double mutation, Phe214Ser/Ala217Ser. After 20 ns of simulation, the lids did not close. The RMSD plot of the simulation is highly fluctuating and there is nearly no change in the distance between the lids (Figure 7). Thus the double mutation affected the lid movement to a great extent, establishing the robustness that multiple hydrophobic contacts confer to the function of a protein.

Figure 7 Simulation with Lipase Double Mutants

We have also looked at the importance of hydrophobicity in maintaining the closed structure. The closed form of the lipase was mutated at both residues Phe214 and Ala217 to Ser. This structure was simulated in both water and an octane-water interface. In both cases, the lids failed to open. This behaviour in water, where the wild-type lipase is expected to be in a closed form, shows that these hydrophobic contacts are essential only to drive the lid-closure process, but they do not affect the stability of the closed conformation. That the mutant is unable to open up in a water-octane interface, contrary to the wild type (Figure 8), shows that hydrophobicity appears to be essential to lid opening and hence interfacial activation as well. Possibly there is a greater hydrophobic affinity between octane and lid2 than between the lids; thus there appears to be an intricate role for hydrophobicity in the domain movement of lipase.

Figure 8 Simulation of Closed-Structure Double Mutant in Octane-Water Interface

The above standard procedure has been used by Jensen et al. [19] to simulate the pair of open and closed forms of H. lanuginose lipase in aqueous conditions. They show that, in 12 ns, the open form tends to move towards a closed conformation, while the closed conformation remains as such.

In order to generate the water-octane interface, a water box (~3,000 water molecules), similar to the one used for the aqueous simulations, was built around the protein. A second box containing ~200 molecules of octane was laid over the water box. Following this, standard simulation protocols were used. The protein molecule, with its lid facing the octane box, moved to the interface within the first nanosecond. All box dimensions are given in Protocol S8.

In appreciation of Prof. Varadachari Krishnan, JNCASR, Bangalore, India. We thank the Department of Biotechnology Government of India through the BioTechnology Information Services programme and the Department of Science and Technology (SP/SO/D-18/1999) for providing funds. One of us, SLC, thanks the Council of Scientific and Industrial Research for a studentship. We also thank the developers of GROMACS for making the software available under the GNU license. We thank the anonymous reviewers for their valuable comments and suggestions.