Structure determination

A particular focus of this research group is on membrane proteins in P. aeruginosa. Accordingly, a structure of Lnt from this opportunistic human pathogen (LntPae) was selected for investigation. Because so much was known about Lnt from E. coli (LntEco), it too was included in the study. The two proteins have 39% sequence identity. The pure proteins were functionally active as demonstrated in assays where product formation was quantified with the synthetic biotinylated lipopeptide, fibroblast stimulating ligand-1 (FSL-1-biotin), as the second substrate (Fig. 2). Crystallization trials were undertaken using the lipid cubic phase method25. Crystals that diffracted to ∼3 Å were obtained for both Lnt constructs. Seleno-methionine (Se-Met) labelling of LntEco for Se-single-wavelength anomalous diffraction (Se-SAD) phasing was used to solve the native LntEco structure to a resolution of 2.9 Å. The LntPae structure and that of an inactive Cys387Ala mutant of LntEco were solved by molecular replacement using the native LntEco structure as the search model. Data collection and refinement statistics are presented in Table 1. Representative electron density maps are shown in Supplementary Figs 2 and 3. The overall structure of the three constructs is similar. Co-evolutionary covariance analysis is consistent with the crystal structures26 (Supplementary Fig. 4). For purposes of the discussion that follows the focus will be on the wild type LntEco construct.

Figure 2: Lnt activity assay. (a–c) Lnt activity monitored as a shift in the SDS-PAGE mobility of FSL-1-biotin on N-acylation with quantitation by western blotting. Uncropped images of the western blots including marker lanes are shown in Supplementary Fig. 1. (d,e) Lnt activity monitored by the conversion of NBD-PE to NBD-lyso-PE (NBD-LPE) by thin layer chromatography with quantitation by fluorescence. (a) N-acyl transferase activity of LntPae and LntEco is evident as a band shift towards higher molecular weight values resulting from a conversion of FSL-1-biotin (open arrow head) to N-acylated FSL-1-biotin (full arrow head) in the presence of a lipid donor. (b) Time course experiment and lipid head group specificity of LntPae. After a 1 h incubation at 37 °C in the presence of both substrates and enzyme (20 nM), the FSL-1-biotin band appeared ∼3 mm higher in the gel than was observed for both negative control reactions without either DOPE or LntPae (lanes 1 and 2). The time dependence of the reaction is shown in lanes 3–5. When DOPE was replaced with DOPG or DOPC, the product was formed much less efficiently (lanes 6 and 7). (c) Densitometric analysis of the time-dependent data in b. (d) LntEco activity measurements for wild type (WT) and mutants E267Q, K335A and C387S. The reaction was stopped after 60 min. Data are shown for duplicate reaction measurements. (e) Time course of lyso-PE production catalysed by LntEco. (f) Densitometric analysis of the time-dependent data in e. Full size image

Table 1 Data collection and refinement statistics for Lnt. Full size table

Overall architecture

Lnt has two domains, a MD and a periplasmic ND with 230 and 278 residues, respectively (Fig. 3). The interfacial area between domains is 440 Å2. The active site, defined by the catalytic triad, resides in the ND slightly above the membrane surface with an opening for substrates and products that leads into the bulk membrane. The MD consists of eight transmembrane helices (H1–H8) with both N and C termini in the cytoplasm (Fig. 4). The first six helices are arranged cylindrically with helices forming the wall of the cylinder and coiled counter-clockwise. At its periplasmic end, opposing helices across the cylinder’s diameter are approximately equidistant giving the MD the appearance of an open ended cylinder (Fig. 4b). This periplasmic opening is covered by a highly conserved 27-residue long linker (L1) connecting H5 and H6 that includes two short helices, h1 and h2. The positioning of L1 across the top of the MD is due, in part, to Pro129 which introduces a kink in H5 causing it to bend away from H6 and to align with H4 (Fig. 4a). This enables conserved Arg139 on the periplasmic end of H5 to hydrogen bond with backbone carbonyls of highly conserved residues Phe146 and Trp148 in L1 thereby securing the loop as a periplasmic lid on the MD. The essential nature of Arg139 is borne out by mutational studies. A strong interaction between highly conserved Gly145 in L1 of the MD and Tyr388 (Fig. 5d) in the ND contributes to holding the two domains together where they meet at the periplasmic surface of the membrane. This, in turn, helps position the catalytic Cys387 for reaction (Fig. 5). Cys387 in the ND sits above the center of the MD about 13 Å from where L1 crosses its cylindrical opening (Fig. 3a). Mapping conserved residues onto the structure identifies this region, between the two domains, as the putative active site pocket (Supplementary Figs 5a,6).

Figure 3: Overall architecture of Lnt from E. coli. (a) View from the membrane plane. The protein has two domains, a membrane domain and a periplasmic nitrilase-like domain. The structure is shown in cartoon representation and rainbow colour coded from N (blue) to C terminus (red). The catalytic cysteine Cys387 side chain is shown in sphere representation (carbon, magenta; sulfur, yellow). The magenta arrow indicates the proposed substrate entry portal and identifies what is referred to as the front of the enzyme. Approximate location of the membrane boundaries are shown as horizontal lines. Cys387 sits ∼13 Å above the bulk membrane surface. (b) Schematic representation of the secondary structure elements in the LntEco structure. Colour coding follows that used in a. Full size image

Figure 4: Membrane domain of LntEco. (a) View from the membrane plane. (b) View from the periplasm. (c) View from the cytoplasm. Colour coding as in Fig. 3. Gly74 and Pro129 side chains shown as spheres. Full size image

Figure 5: Nucleophilic elbow in LntEco. (a) The nucleophilic elbow (orange) consisting of a β-strand-turn-helix (dashed box) shown in context of the overall Lnt structure (grey). (b) Expanded view of the boxed region in a showing the catalytic Cys387 in the turn. The oxyanion hole created by backbone amides in α3′ is indicated. (c) Coordination between residues in the turn and the α3′ helix. Dashed lines correspond to distances of ≤3.5 Å. (d) Residues in L1 are coordinated to the core of the MD via Arg139 in H5 and to the nucleophilic elbow via Tyr388. Full size image

On the opposite, cytoplasmic end of the MD, the same cylindrical arrangement of helices H1–H6 holds with one exception. H2 is tilted into the core of the cylinder effectively closing this end of the protein (Fig. 4c). Arg123 on the cytoplasmic end of H5 interacts with Thr48 on H2 and with other local residues creating a water-tight seal between the two sides of the membrane. H7 and H8 in the MD are bridged by the ND in the periplasm. The connection between the two domains is by way of two long linkers (L2, L3) that look and possibly act like braces crossing over one another at the back of the ND (Fig. 3a, Supplementary Fig. 7a). The H7/H8 helix pair sits to one end of the MD (hereafter referred to as the back end), run approximately parallel to one another, are oriented almost normal to the membrane plane and are separated from each other by the width of the MD. Arg438 on the ND has extensive interactions with Thr478 and Gly479 in the L3 brace to H8. Furthermore, highly conserved Thr481 on the L3 brace interacts with highly conserved Glu435 on the ND (Supplementary Fig. 7b). Collectively, these interactions presumably help poise the catalytic triad in the ND above the MD for reaction and contribute to Lnt’s overall structural integrity and function. Interestingly, a Thr481Arg mutation inhibited the S-acylation step in the transferase reaction consistent with an uncoupling of the two domains21.

Despite having a very high content of hydrophobic residues, H3 and H4 appear to extend above the membrane interface into the periplasm (Figs 3a and 4a, Supplementary Fig. 5b). H3 is bent at conserved Gly71 and coils over H4 in an anticlockwise direction. We speculate that the periplasmic surface of H3 and H4 forms one side of the portal for amphiphilic substrates to enter the active site. In addition to Gly71, H3 has three conserved glycines (Gly60, Gly64 and Gly66) in close proximity to one another. This suggests that H3 has considerable flexibility, possibly to accommodate and to orient into the active site the N-terminal diacylglyceryl–cysteine in differently sized and shaped lipoproteins. Crystallography and molecular dynamics simulations (MDS) data support this proposal (Supplementary Fig. 5c and d).

The ND is globular in shape and rests on the periplasmic surface of the MD (Figs 3a and 6). It emerges from the MD as a 277-long polypeptide connecting H7 and H8 (Supplementary Fig. 7). As noted, the two domains are linked by way of two brace-like linkers (L2, L3) at the back of the ND (Supplementary Fig. 7a). L2 and L3 connect H7 and H8 in the MD with the first (β1) and last β-strands (β12) in the ND, respectively. L3 includes a short helix (h4) towards the junction with H8. The ND is of the four-layer αββα sandwich fold type. Helices α1 and α2 form the upper layer that resides atop the protein in the periplasm with helices α3 and α4 forming the lower layer that sits on the periplasmic surface of the MD. α3 is really a pair of helices. The first is a single-turn helix (α3′) and is part of a β-strand-turn-helix motif termed a nucleophilic elbow in which resides the catalytic Cys387 (Figs 5 and 6). Helix α3′ includes highly conserved residues Tyr388 and Glu389 both of which were shown in mutational work to be essential24. The second helix in the pair (α3″) is two turns long. It is separated from the lower β-sheet of the ND and tilts into the core of the MD providing shape complementarity between the two domains. The two sheets of the nitrilase sandwich have different alternation of parallel and anti-parallel strands and, as usual, are twisted. The upper and lower αβ halves are connected on the front end of the ND by a 40 residue long loop with a short helix (α**) between β5 and β6 and on the back end by the MD tethering linkages L2 and L3 between β11 and β12. Layers within the sandwich are held together primarily by hydrophobic interactions. The back end of the domain consists of short loops and, as a result, it is relatively flat and featureless. By contrast, the front end includes several loops of varying length that extend away from the ND approximately parallel to the membrane plane arranged roughly in a ring around the catalytic triad. Together with the periplasmic H3 and H4 helical extensions, these loops create an opening to the membrane and a funnel-shaped pocket at the base of which sits the catalytic triad (Fig. 7a). They resemble arms that reach out from the active site creating a pocket for substrates to enter and for products to leave. For convenience of description these active site cavity surrounding loops are hereafter referred to as arms and are identified numerically clockwise around the catalytic Cys387 (Fig. 7a). Residues Pro353, Phe358 and Met362 on Arm3 and Trp237 on Arm7 have been identified in mutational studies as important or essential21. Highly conserved Gly447 resides at the end of Arm6. Given the location of these key residues and the fact that they do not interact notably with other parts of the protein we speculate that their essentiality arises from the critical roles the respective residues and the loops in which they reside play in guiding substrates and products into and out of the active site.

Figure 6: Nitrilase-like domain of LntEco. (a) View from the membrane plane as in Fig. 3a. Colour coded by secondary structure to highlight the αββα sandwich feature of the domain. Catalytic triad residues are shown in stick representation. The asterisks in α** and β5** indicate that the α-helix and β-strand secondary structure elements form in some structures (α-helix found in: LntEco C387A, chain A; LntPae WT. β-strand found in: LntPae WT) but not in others (α-helix absent in: LntEco WT, chains A and B; LntEco C387A, chain B). (b) Schematic representation of the secondary structure elements in the nitrilase-like domain. Colour coding follows that used in a. Helix α3 consists of two small helices, α3′ and α3″. The dashed lines around α** and β5 indicate that these elements are formed in some structures but not in others as in a. Full size image

Figure 7: Architecture of LntEco active site pocket. (a) View into the active site pocket from the membrane surface. Helices and loops, referred to as arms, extending from the MD and the ND radiate out creating a funnel-shaped entrance to the active site. Spheres are used to mark the reach of each arm. Reaches are connected to the catalytic Cys387 by dashed lines to communicate a sense of the funnelled nature of the entrance. A description of what constitutes the different arms follows. Arm 1. Periplasmic extensions of H3 and H4 and connecting loop, originating in the MD. Arm 2. L1 between H5 and H6 in the MD. Arm 3. The 40-residue long loop between β5 and β6 linking the two halves of the αββα sandwich in the ND. Arm 4. Long β-strands β3 and β4 and connecting loop in the upper half of the ND sandwich. Arm 5. Loop connecting β1 and α2 in upper half of ND sandwich. Arm 6. Loop between β9 and β10 in bottom half of ND sandwich. Arm 7. Loop between β1 and α1 in upper half of ND sandwich. Arm 8. Loop between β11 and β12, connecting the bottom and top halves of the sandwich. Arm 9. Loop between β8 and α4 in bottom half of the ND sandwich. (b) Expanded view into the active site showing the catalytic triad residues, E267, K335 and C387 along with other proximal conserved residues. Side chains are shown in stick representation. The orientation is similar to that in a. Dashed lines correspond to distances of ⩽3.2 Å. Full size image

A portal into and out of the active site pocket

A cleft exists between H4 and H5 in the periplasmic leaflet of the MD that extends out of the bulk membrane into the space between Arm 1 and Arm 3 (Fig. 7a, Supplementary Fig. 5b). This long hydrophobic opening leads into the active site pocket. We propose that it serves as the portal through which lipid and lipoprotein substrates enter the active site. In support of this, the structures of Lnt solved in the course of this study contain a varying number of structured monoolein lipid molecules from the mesophase in which crystallization occurs. Most lipids decorate the MD of Lnt roughly in a bilayer arrangement reminiscent of lipids in a native membrane (Fig. 8). Some however reside in the H4–H5 cleft and occupy positions between Arm 1 and Arm 3 and above Arm 2 that feed into the active site pocket. In the LntEco-Cys387Ala mutant structure, where lipid density is well defined and plentiful in this region of the protein, the lipids arrange themselves in single file along the hydrophobic pocket entrance into the active site (Fig. 8b,c). The apolar nature of many of the residues in the periplasmic extensions of H3 and H4 and the three arms is consistent with this finding (Supplementary Fig. 5b, Fig. 8c). These observations suggest that the positions occupied by the structured lipids define the route to the active site taken by the phospholipid and lipoprotein substrates of Lnt. MDS performed with Lnt in a model hydrated membrane, reveal that lipids may enter this region from the bulk membrane (Supplementary Fig. 8). This indicates that there is sufficient space for both substrates to enter the proposed catalytic site one at a time via this conduit. On the basis of the structured lipids in the crystal structures and simulations it was plausible to dock substrates and products individually into this binding pocket in a physico-chemically reasonable manner and that is consistent with the proposed ping-pong reaction mechanism. Collectively, these observations help explain how the lipid and lipoprotein substrates can effectively migrate out of the bulk membrane along the apolar conduit created by the cleft between H4 and H5 and the space between Arm1 and Arm3 to the active site Cys387 situated some 13 Å above the periplasmic surface of the membrane (Supplementary Fig. 8).

Figure 8: Bound lipids define substrate portal in and membrane around Lnt. (a) Structured monoolein lipids in LntEco-C387A decorate the surface of the protein in a manner reminiscent of the membrane bilayer and the portal between the bulk membrane and the enzyme active site. Lipid molecules in the portal line up in single file and are individually numbered from 1 to 4. Lipids are shown in stick representation. Cys387Ala is coloured magenta. (b) Expanded view of lipids arranged in single file in the portal facing into the active site and of lipids (shown in stick representation) at the surface of the enzyme. (c) Lipid binding to the surface and in the portal of the enzyme. Enzyme shown in surface representation with hydrophobic residues in light blue and polar residues in grey. Orthogonal views presented in left, middle and right panels. An expanded view of lipids (shown as spheres) in the portal is shown in the right panel. Full size image

N-acyltransferase mechanism

The N-acyltransferase reaction catalysed by Lnt is proposed to take place in two steps (Fig. 9, Supplementary Fig. 9). The first is an acyl transfer reaction where the acyl chain at the sn-1 position of the substrate, preferentially PE in the case of Lnt from E. coli, is transferred to form a thioester linkage with the γS of the catalytic Cys387 (Fig. 9a–c, Supplementary Fig. 9a–c). In the second step, the acyl chain is transferred from Cys387 to the α-amino group of the dagylated N-terminal cysteine of the lipoprotein (Fig. 9d–f, Supplementary Fig. 9d–f). The reaction follows a ping-pong mechanism where the first product departs the active site before the second substrate enters. The first step is proposed to involve proton abstraction from the γS of Cys387 by catalytic Glu267. This generates a thiolate which, in turn, attacks the ester linkage between the acyl chain and the glycerol backbone of the phospholipid substrate to form a tetrahedral intermediate (Fig. 9b, Supplementary Fig. 9b). A net negative charge on the oxygen attached to the tetrahedral carbon, the oxyanion, is stabilized by nearby Lys335, and by the proximal oxyanion hole created by the backbone amides of I390, I391 and L392 in α3′ (Fig. 5b). Lys335 has a predicted pK a some 2.25 units above that for a lysine containing model peptide (Supplementary Table 1). It should be charged under physiological conditions for effective oxyanion stabilization. Mutating Lys335 to Ala inactivated the enzyme consistent with the essential role played by this catalytic triad member (Fig. 2d). Glu343 is an invariant residue in the active site pocket. Its side chain carboxyl oxygens are proximal (2.8 Å) to the ɛ-amino group of Lys335 enhancing its cationic character for oxyanion stabilization. Collapse of the tetrahedral intermediate accompanied by proton abstraction from Glu267 releases the lipid product, lyso-PE. Lyso-PE exits the binding pocket at which point the protein is primed for step 2 which happens as soon as apo-lipoprotein substrate enters and forms the Michaelis complex. The reaction passes through a second proposed tetrahedral intermediate (Fig. 9d, Supplementary Fig. 9e) which forms when the α-amino group at the N terminus of the lipoprotein attacks at and bonds to the carbonyl carbon in the thioester linkage between Cys387 and the acyl chain that is about to undergo transfer. As with the tetrahedral intermediate in the first step of the reaction, the oxyanion is stabilized by Lys335 and the oxyanion hole in the nucleophilic elbow. Collapse of the tetrahedral intermediate gives rise to the mature triacylated lipoprotein product and a reformed enzyme. As soon as the lipoprotein departs and a PE molecule enters the binding pocket, the enzyme is reset for another round of catalysis. Several of the reaction states have been simulated by molecular dynamics and shown to be stable in silico (Supplementary Fig. 9, Supplementary Movies 1 and 2).

Figure 9: Proposed N-acyltransferase reaction mechanism in Lnt. Electron lone pairs shown as double dots. (a) First Michaelis complex with 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) substrate. (b) First tetrahedral intermediate. Tetrahedral carbon shown in stereochemical representation. The oxyanion bears a negative charge. (c) Second Michaelis complex with apo-lipoprotein substrate. (d) Second tetrahedral intermediate. (e) Product complex. (f) Empty active site ready to undergo another reaction cycle. Red curved arrows indicate electron flow. Dashed blue lines denote oxyanion stabilization. GPE, glyceryl-phosphoethanolamine; LP, apo-lipoprotein; DAG, diacylglyceryl; LPE, lyso-PE. Full size image

The structure of Lnt is entirely consistent with the proposed mechanism. The predicted pK a of the γS in the side chain of Cys387 in the Lnt model is 12.7, almost four pH units higher than that of a cysteine containing model peptide (Supplementary Table 1). Therefore, at pH 7.0, Cys387 is fully protonated. However, nearby Glu267 has a side chain pK a of 7.0 some two and a half pH units above that of Glu in a model peptide. This residue, which is 50% deprotonated at pH 7.0, is in a position to extract a proton from Cys387 thereby generating the highly nucleophilic thiolate. The closest distance between a side chain carboxyl oxygen on Glu267 and the γS of Cys387 is 4.1 Å (3.5 Å in LntPae; Fig. 7b). This is too far for direct interaction and suggests that proton abstraction may involve the mediation of a water molecule to relay transfer from Cys387 to Glu267. Such a water molecule was not seen in the structure perhaps due to resolution limits or disorder. It may also be that flexibility in local backbone and side chain conformation bring the Cys387 and Glu267 into proximity for direct proton exchange. The latter conjecture is supported by MDS, which suggests a spontaneous and consistent hydrogen bond arrangement between the γS proton of Cys387 and the side chain of Glu267 (Supplementary Movie 1). Furthermore, a Glu267Gln mutation inactivated the enzyme consistent with its proposed role in proton abstraction (Fig. 2d). Interestingly, His425 which is highly conserved and located in the active site pocket next to Cys387 (Fig. 7b) has a predicted pK a of 3.5, some 3 pH units lower than expected (Supplementary Table 1). At pH 7, it is fully deprotonated and should provide additional proton extracting power to increase the nucleophilic character of Cys387. In its protonated state, His425 may also provide stabilization for the oxyanion of the proximal tetrahedral intermediate that forms in each step. In addition to His425, catalytic Cys387 is in hydrogen bonding distance to conserved Ser411 (Fig. 7b). Ser411 is suitably positioned to stabilize the γS of Cys387 as it alternates between thiol and thiolate forms during the two-step transferase reaction.

In several of the crystal structures solved in the course of this work host lipid molecules are visible extending deep into the binding pocket and next to Cys387. In one of these (LntEco-C387A), the γS of Cys387 is 4.5 Å from the hydroxyl on the glycerol backbone of a structured monoolein (Fig. 8). This is compatible with a mechanism where a fatty acyl donor substrate, such as PE, accesses the site with its ester linked acyl chain at the sn-1 position of the glycerol backbone proximal to the nucleophilic thiolate for reaction. The first step in the reaction happens ‘spontaneously’ yielding an acylated-enzyme intermediate. Indeed, the Lnt enzyme in whole cells has been shown to exist in its acylated form27. However, the thioester linkage is labile and can deacylate by hydrolytic cleavage in the absence of second substrate. By contrast, the Cys387Ser mutant has been shown to acylate spontaneously but because the oxygen ester is more stable than the thioester, the subsequent N-acylation step is blocked even in the presence of lipoprotein substrate. We confirmed this in functional assays where the Cys387Ser construct proved to be inactive (Fig. 2d). Efforts aimed at capturing a structure of this intermediate were unsuccessful. In the absence of an actual structure, a model of the palmitoyl-enzyme intermediate has been created and shown to be stable in silico (Supplementary Fig. 10b).

A structure comparison of LntEco and LntPae

As noted, LntEco and LntPae have 39% sequence identity. The two structures are remarkably similar with RMSD values over 501 residues of 1.2 Å (Supplementary Fig. 11a). Despite having different residues in the active site pocket, the catalytic triad residues superpose almost exactly on one another (Supplementary Fig. 11c) consistent with the two enzymes catalysing the same N-acyltransferase reaction with similar substrates. Indeed, the phospholipid profile of E. coli and P. aeruginosa membranes are similar with PE and PG together making up 95% of membrane lipids. The rest is mainly cardiolipin. The fatty acids of the phospholipids are predominantly 16 and 18 carbons long. These similarities in membrane lipid composition help rationalize the near structural identity of the two enzymes. Relatedly, LntPae can complement an lnt depletion strain of E. coli and is dominant negative over LntEco at 37 °C (ref. 24). Thus, LntPae is functional in E. coli.

Sequence alignment shows LntEco to have an heptapeptide motif, YSYESAD (Tyr325-Asp331), that is absent in LntPae28. The heptapeptide insert forms a loop that clamps into the cleft between α2, β3 and β4 at the top of the ND (Supplementary Fig. 11a,b). Given its location at considerable remove from the active site, it is not obvious that this difference impacts in any way on catalytic activity or selectivity of the enzyme.

N-acyltransferase activity

Detailed characterization of the lipid head group specificity and kinetics of LntEco have identified PE as the preferred substrate ahead of PG. PC was the least favoured acyl donor of the lipids studied20. To likewise investigate the head group specificity of LntPae, a lipopeptide band shift assay was performed with LntPae, and with DOPE, DOPG or DOPC as acyl donors. FSL-1-biotin was used as the lipoprotein substrate (Fig. 2). Under these conditions and with PE as the lipid substrate, the reaction ran at the rate of one FSL-1-biotin molecule N-acylated every second. However, transfer rate dropped significantly with PG and PC. Qualitatively therefore, LntPae and LntEco share a similar head group specificity. The structural similarity of the two enzymes is consistent with this finding.