Structural basis for lipid binding and mechanism of the Mycobacterium tuberculosis Rv3802 phospholipase

Abstract

The Mycobacterium tuberculosis rv3802c gene encodes an essential enzyme with thioesterase and phospholipase A activity. Overexpression of Rv3802 orthologs in Mycobacterium smegmatis and Corynebacterium glutamicum increases mycolate content and decreases glycerophospholipids. Although a role in modulating the lipid composition of the unique mycomembrane has been proposed, the true biological function of Rv3802 remains uncertain. In this study, we present the first M. tuberculosis Rv3802 X-ray crystal structure, solved to 1.7 Å resolution. On the basis of the binding of PEG molecules to Rv3802, we identified its lipid-binding site and the structural basis for phosphatidyl-based substrate binding and phospholipase A activity. We found that movement of the α8-helix affords lipid binding and is required for catalytic turnover through covalent tethering. We gained insights into the mechanism of acyl hydrolysis by observing differing arrangements of PEG and water molecules within the active site. This study provides structural insights into biological function and facilitates future structure-based drug design toward Rv3802.

Introduction

In 2015, 10.4 million incident cases of tuberculosis were reported with 1.4 million people succumbing to the disease (1). Lack of antibiotic adherence has led to an increase in multiple and totally drug-resistant strains of Mycobacterium tuberculosis, the causative agent of tuberculosis (2). As such, improved antibiotics and novel targets for antibiotic development are of high importance. Isoniazid and ethambutol are two first-line antibiotics used to treat tuberculosis; both drugs inhibit cell wall biosynthesis (3). The M. tuberculosis cell wall remains an appealing target for antibiotic development (3, 4).

The M. tuberculosis cell envelope consists of four major components: an inner cytoplasmic membrane, a periplasmic region composed of the peptidoglycan and arabinogalactan, the outer membrane (mycomembrane), and the extracellular capsule (5). The hallmark fatty acids of mycobacteria are the mycolic acids (MA),³ which account for the majority of the mycomembrane (5). Secondary to MA, but of equal importance, are the essential phosphatidylinositol (PI)-based glycerophospholipids (6, 7). The major derivatives of PI are phosphatidylinositol mannoside (PIM) and the further glycosylated forms of PIM, lipoarabinomannan (LM) and mannose lipoarabinomannan (ManLM) (5). All three can be found within both the cytoplasmic membrane and mycomembrane, with the glycans of LM and ManLM extending into the extracellular capsule (5). Mycobacterial lipids and the subsequent modulation of the lipid composition have been associated with cell division, virulence, pathogenesis, and macrophage survival (5).

Residing within the gene cluster that encodes proteins responsible for MA and arabinogalactan biosynthesis is the rv3802c gene (8, 9). The biological function of rv3802c remains in question; however, the gene has been determined to be essential for M. tuberculosis viability (10–12). In addition to being required for general viability, the Mycobacterium avium ortholog of M. tuberculosis rv3802c has been shown to be associated with intestinal epithelium invasion (13). The rv3802c gene encodes an N-terminal translocation signal sequence that includes a predicted transmembrane region (14). Rv3802 was found only in cell wall extracts upon cellular lysis and maintained a molecular mass indicative of an uncleaved protein, suggesting that the enzyme remains anchored in the cytoplasmic membrane (14). Due to sequence similarities with cutinase-like enzymes, the rv3802c gene product was named CULP6 (cutinase-like protein 6), despite lacking cutinase activity (14). However, Rv3802 has been shown to possess both thioesterase and phospholipase A (PLA) activity utilizing residues Ser¹⁷⁵-His²⁹⁹-Asp²⁶⁸ as the catalytic triad (8, 14). The structure of the Mycobacterium smegmatis ortholog, MSMEG_6394, has been solved, revealing that the enzyme possesses an α/β-hydrolase fold (12). The apo structure presented two intact disulfides and a small cavity leading to the identified active-site residues (12). M. tuberculosis Rv3802 shares a high level of sequence identity with a variety of Mycobacterium homologs (68.8% sequence identity between encoded M. tuberculosis Rv3802 and MSMEG_6394) in addition to the Corynebacterium glutamicum ortholog NCgl2775 (11). Upon heat-induced cell stress, NCgl2775 was shown to play an essential role in the modulation of the mycomembrane composition (11). As a result, the total mycolate content increased, whereas glycerophospholipid content decreased (11). This elevated mycolate/glycerophospholipid ratio was also observed when both MSMEG_6394 and NCgl2775 were overexpressed under physiological conditions (11). Meniche et al. (11) therefore proposed naming this class of enzymes the “envelope lipids regulation factor,” or the ElrF family.

Possessing lipase activity, Rv3802 was shown to be susceptible to in vitro inhibition by tetrahydrolipstatin (THL), a Food and Drug Administration-approved covalent lipase inhibitor (8). THL is reported to have a K_i^app of high nanomolar affinity for Rv3802 (12). As such, a series of THL derivatives have been synthesized and tested, displaying improved in vitro and correlated in vivo activity against M. tuberculosis (15). It has been shown that up to 14 lipases undergo covalent modification by THL in Mycobacterium bovis (16). Although the M. bovis ortholog of Rv3802 was identified as being modified by THL, it was not indicated as one of the 14 statistically validated targets of the study (16).

Due to the essentiality and potential role of Rv3802 in cell wall modulation, we sought to further characterize the function and mechanism. We present the X-ray crystal structure of M. tuberculosis Rv3802 to 1.7 Å resolution. Due to binding of PEG molecules to both protein molecules within the asymmetric unit, we were able to characterize the lipid-binding site. Based on the location of the identified lipid-binding site and previous studies, further insights into a biological function for Rv3802 are gained as well as a basis for future structure-based drug design efforts.

Results

M. tuberculosis Rv3802-PEG structure

The M. tuberculosis Rv3802 crystal diffracted to 1.7 Å, having a space group of P2₁2₁2₁ with two molecules present in the asymmetric unit having a Cα root mean square deviation of 0.14 Å (X-ray diffraction and model refinement statistics are given in Table 1). Molecule A was used for structural analyses if not otherwise noted. As predicted by homology to the M. smegmatis ortholog, M. tuberculosis Rv3802 was found to have an α/β hydrolase fold (Fig. 1) (12). Rv3802 has 10 helices (nine α-helices and one 3₁₀-helix) and a single, central β-sheet composed of five parallel β-strands (Fig. 1). The nucleophilic Ser¹⁷⁵ is located on the aptly named nucleophilic elbow connecting the β3-strand to the α3-helix. The remaining catalytic triad residues, His²⁹⁹ and Asp²⁶⁸, are located on the α9-helix and a connecting loop between the β5-strand and α8-helix, respectively (Fig. 1). Two intact disulfides are present in the Rv3802 structure: Cys⁷²–Cys¹⁶⁴ and Cys²⁶⁴–Cys²⁷¹ (Fig. 1). These conserved disulfides are also present in the MSMEG_6394 structure (12).

Table 1.

X-ray diffraction data and structure refinement statistics. Values in parentheses are for the outer shell.

	Rv3802-PEG (PDB code 5W95)
Data collection
Space group	P 21 21 21
Cell dimensions
a, b, c (Å)	57.19, 91.30, 101.39
α, β, γ (degrees)	90, 90, 90
Resolution (Å)	44.32–1.72 (1.78–1.72)
Rmerge	0.09 (0.51)
I/σI	23.23 (6.08)
Completeness (%)	99.63 (96.24)
Redundancy	14.8 (14.8)
Refinement
Resolution (Å)	44.32–1.72
No. of reflections	56,631
Rwork/Rfree	0.1582/0.1830
No. of atoms
Protein	3921
Ligand/ion	64
Water	648
B-factors (Å2)
Protein	17.7
Ligand/ion	33.1
Water	28.0
Root mean square deviations
Bond lengths (Å)	0.007
Bond angles (degrees)	1.06
Ramachandran (%)
Favored	98.0
Outliers	0.4

Figure 1.

The X-ray crystal structure of M. tuberculosis Rv3802 with PEG bound. The enzyme has an α/β-hydrolase fold with the catalytic triad residues of Ser¹⁷⁶, His²⁹⁹, and Asp²⁶⁸ shown in addition to the two conserved disulfides, Cys⁷²–Cys¹⁶⁴ and Cys²⁶⁴–Cys²⁷¹. The two molecules of PEG found bound to Rv3802 are shown in spheres. Atom colors are CPK without carbon, which is white for protein and wheat for the two PEG molecules. Helices and β-strands are numbered and colored in increasing respective color intensity from N to C terminus.

During the early stages of model refinement, two regions of linear difference density converging at the active site were observed. This non-protein electron density is interpreted as two molecules of PEG found bound to each of the two protein molecules in the asymmetric unit. Difference density maps for PEG molecules in protein molecule A can be found in Fig. 2a, and the PEG molecules in protein molecule B are shown in Fig. S1. PEG 3350 (average molecular mass is 3350 kDa or ∼75 repeating monomer units) was present in the crystallization solution; however, the bound PEG polymers consist of only 5 ethylene glycol monomer units.

Figure 2.

The identified lipid tail (fatty acid)-binding site is highly conserved and hydrophobic. A, F_o − F_c likelihood weighted omit map contoured to 3σ for salmon-colored PEG molecules. Amino acid side chains within 4 Å of the PEG molecules are shown in cyan, and active-site residues are orange, with other non-carbon atoms in CPK color. These residues comprise the lipid tail-binding site. B, without ligand present, the amino acid side chains of the lipid tail-binding site form a wall of hydrophobic interactions, as seen in the apo MSMEG_6394 structure (PDB code 3AJA) (12). C, sequence alignment of the fatty acid-binding region from Mycobacteria. Residues of differing sequence are shaded, and starred residues are shown in A and B, whereas a red underline corresponds to secondary structure location.

Identification of lipid tail-binding site

Due to the presence of PEG molecules within Rv3802 and PEG resembling the fatty acid chains of lipids, the lipid-binding site of Rv3802 and presumptive orthologs have been structurally identified (Fig. 2). The fatty acid portion of the lipid-binding site begins at the active site, with the terminal oxygen of PEG1 positioned within hydrogen bonding distance to both Ser¹⁷⁵ and His²⁹⁹, 3.2 and 3.8 Å, respectively. The terminal oxygen of the PEG2 resides 6.1 Å away from the terminal oxygen of PEG1 and is positioned between the side chains of Tyr¹⁴² and Ile²⁷⁰. Both PEG molecules extend away from the active site in a linear fashion, flanked on both sides by mostly aliphatic amino acid side chains that are positioned on helices α6 and α8 as well as various loop regions (Fig. 2A). Without ligand present, the aliphatic side chains involved in PEG binding form a hydrophobic core of van der Waals interactions, as seen in the previously solved apo MSMEG_6394 structure (Fig. 2B) (12). Whereas Rv3802 and MSMEG_6394 have a relatively high level of sequence identity for all encoded residues, 68.8%, the residues that make up the identified fatty acid-binding region possess a higher level of conservation, 76.2% between M. tuberculosis and M. smegmatis specifically, as well as a higher level of conservation with other mycobacterial orthologs (Fig. 2C).

Identification of dynamic protein regions

As stated above, Rv3802 and the previously solved apo MSMEG_6394 ortholog share a common core scaffold with two disulfides present. However, a noticeable difference between the M. tuberculosis and M. smegmatis structures exists between residues 279 and 298 (Fig. 3A). To accommodate the two bound PEG molecules, α8-helix and flanking residues are shifted away from the adjacent loop in the Rv3802 structure.

Figure 3.

Superposition of M. tuberculosis Rv3802 (gray) and ortholog MSMEG_6394 (purple; PDB code 3AJA); residue numbering is given as Rv3802/MSMEG_6394 (12). A, the α8-helix and surrounding residues are found to be structurally dynamic upon substrate binding. B, the N-terminal of α8-helix moves ∼9 Å between the open and closed states; the region shown spans from residues 265/266 to 302/303. C, despite dynamic movement near the active site, catalytic and surrounding residues are positioned similarly between the open and closed states. The side chain of Asn¹⁷⁶ in Rv3802 was modeled in the most likely conformation as assessed by inspection of nearby hydrogen bond donors and acceptors, resulting in the discrepancy with the previously solved MSMEG_6394 Asn¹⁷⁷.

The N-terminal region of α8-helix is extended outward 8.8 Å upon PEG binding, when compared with the apo M. smegmatis ortholog (Fig. 3B). The opening of α8-helix alters a connecting loop to the neighboring 3₁₀-7-helix; however, this does not affect 7-helix positioning. The loop connecting α8 to the upstream catalytic His²⁹⁹ is unstructured. Due to poor difference density, this initially modeled region was ultimately removed in the deposited structure. Moreover, it is also unresolved in the M. smegmatis ortholog structure. This structurally dynamic region is the hinge point for α8-helix movement, being composed of mostly alanine and glycine residues in both enzymes.

Due to a high sequence similarity in the active site between Rv3802 and its ortholog, a direct active-site comparison between the two conformations is feasible. Throughout the remainder of this work, analogous residue numbers will be given with the Rv3802 number first and the MSMEG_6394 number second (e.g. Thr^83/84). The top of the α8-helix abuts the active site; despite significant movement between the open and closed states of α8-helix upon PEG binding, little movement is observed in the residues of the active site (Fig. 3C). However, the nucleophilic Ser¹⁷⁶ of MSMEG_6394 is positioned toward the presumed oxyanion hole consisting of the backbone amides of Thr^83/84 and Asn^176/177, whereas the nucleophilic Ser¹⁷⁵ of Rv3802 is hydrogen-bonded to the catalytically relevant His²⁹⁹. Uncertainty remains as to whether this difference is a function of the modest 2.9 Å resolution of the MSMEG_6394 structure or represents a true structural difference between the open, substrate-bound form and the closed, substrate-free form.

Structural insights into substrate binding and catalysis

When α8-helix is in the open conformation, a sizable substrate-binding site is apparent (Fig. 4A). However, in the closed state visualized in the M. smegmatis ortholog, only a small cavity leading to the nucleophilic serine is observed (12). Therefore, for a large substrate possessing hydrophobic, aliphatic character to bind, α8-helix must move to expose the substrate-binding site. It is expected that initial substrate binding positions the scissile ester/thioester bond in proximity to Ser¹⁷⁵, affording bifurcation of the active site into two distinct areas that accommodate either the hydrophobic tail or the hydrophilic headgroup. With the reasonable expectation that the PEG molecules bind within the lipid tail-binding site, the lipid head-binding site becomes apparent (Fig. 4B). The headgroup-binding site is unchanged from that observed in the closed state of M. smegmatis ortholog, being the observed small cavity described previously (12). This site is composed of the hydrophobic side chains of Trp⁸⁴, Leu¹⁰², Phe¹⁷⁴, Ala²⁹², and Ala³⁰⁰; the negatively charged residues or carbonyl backbones of Thr⁸³, Glu⁸⁵, and Ala³⁰⁰; and the positively charged side chain of Lys¹⁰⁰. These residues are fully conserved between Rv3802 and the M. smegmatis ortholog aside from Lys¹⁰⁰ and Ala³⁰⁰; these two residues are Leu and Asn in M. smegmatis, respectively.

Figure 4.

Structure-based insights on substrate-binding sites and arrangement for catalysis. A, identification of the Rv3802 lipid tail (fatty acid)-binding site results in the identification of the lipid head-binding site that is linear to that of the lipid tail. Additionally, a noticeable solvent channel leads to the active site directly intersecting the lipid tail and lipid head sites in a perpendicular manner. PEG molecules are wheat, and water molecules are red spheres, and cavity surfaces are shown in light blue. B, residues comprising the proposed lipid head-binding site adjacent to the catalytic triad are depicted; catalytic residues are in orange.

Perpendicular to and intersecting the substrate-binding site is a solvent channel leading to the active site (Fig. 4A). In both protein molecules within the asymmetric unit, ordered water molecules are found alongside His²⁹⁹, opposite of the lipid headgroup-binding site. In molecule A, the terminal PEG1 oxygen is 3.2 Å from the Ser¹⁷⁵ hydroxyl, whereas the Ser¹⁷⁵ hydroxyl is 3.0 Å from the ϵ nitrogen of His²⁹⁹ (Fig. 5A). Additionally, a water molecule is found 4.2 Å away from His²⁹⁹. In molecule B, the terminal PEG1 oxygen is pointing toward the plausible oxyanion hole, 3.4 Å equidistant to both backbone amides of Thr⁸³ and Asn¹⁷⁶. A water molecule is now found within hydrogen-bonding distance, 2.9 Å, to the ϵ nitrogen of His²⁹⁹ (Fig. 5B). It should be noted that, although not depicted in the figures, solvent molecules are positioned in a similar fashion in the lipid head-binding sites of both protein molecules A and B.

Figure 5.

Positioning of PEG and water in the active sites of both protein molecules in the asymmetric unit. The F_o − F_c likelihood weighted omit maps are contoured at 4σ. A, the terminal oxygen of PEG1, denoted by an asterisk, is within hydrogen-bonding distance of the catalytic Ser¹⁷⁵ in protein molecule A. His²⁹⁹ is also hydrogen-bonded with Ser¹⁷⁵. A nearby water molecule is positioned within the water channel. This active-site configuration resembles initial substrate binding and the activation of Ser¹⁷⁵ for nucleophilic attack on substrate. B, in protein molecule B, the terminal oxygen of PEG1 is pointed toward the presumptive oxyanion hole, whereas a water molecule is within hydrogen-bonding distance of His²⁹⁹. This configuration resembles water activation by the enzyme to hydrolyze the acyl-enzyme intermediate.

Influence of α8 movement on hydrolase activity

To investigate the effects that α8 movement has on catalysis and to validate the structural observations, M. tuberculosis Rv3802 was mutated to limit α8 movement. In the closed state, Asn¹³³ and Glu²⁸⁹ of the M. smegmatis ortholog are ∼5 Å apart; therefore, we mutated the corresponding residues in M. tuberculosis Rv3802, Asn¹³² and Asn²⁸⁸, to cysteine residues to form an engineered disulfide. The resulting N132C/N288C mutant would therefore have limited α8 helical movement due to covalent tethering. The Rv3802 N132C/N288C variant exhibited lower expression levels but was purified in an identical fashion to WT Rv3802. Both WT and N132C/N288C Rv3802 run as single monomeric bands on a non-reducing SDS-polyacrylamide gel, indicating the lack of intermolecular disulfide bonds (Fig. S2A). Additionally, the amount of free thiols present in both WT and N132C/N288C Rv3802 was quantified under non-reducing and reducing conditions using monochlorobimane. Non-reduced samples had levels of fluorescence equal to background, indicating that no free thiols are present in both WT and N132C/N288C mutant (Fig. S2B). The resulting ratio of quantified cysteines in reduced WT to N132C/N288C Rv3802 was 0.68 ± 0.04, consistent with the 4:6 ratio of cysteines present in WT and N132C/N288C mutant, suggesting that all cysteine residues were forming disulfides and were indeed intramolecular (Fig. S2C).

Initial enzymatic activity was assessed using a fluorescence-based assay that monitored the hydrolysis of 4-methylumbelliferyl heptanoate (4MH). Subsequent hydrolysis of 4MH produces the fluorescent molecule 4-methylumbelliferone and heptanoic acid. Michaelis–Menten kinetics were determined for both WT and N132C/N288C mutant using 4MH (Fig. 6B) (WT: K_m = 19.88 ± 1.18 μm, V_max = 0.51 ± 0.01 μm·min⁻¹, k_cat = 10.05 ± 0.17 min⁻¹; N132C/N288C: K_m = 37.21 ± 3.11 μm, V_max = 0.38 ± 0.01 μm·min⁻¹, k_cat = 5.04 ± 0.15 min⁻¹). The affinity of the N132C/N288C mutant for 4MH decreases by 53% and results in a 2-fold decrease in the k_cat when compared with WT enzyme.

Figure 6.

Influence of α8-helix movement on enzymatic hydrolysis. A, in the closed state, α8-helix blocks the lipid tail (fatty acid) portion of the lipid-binding site. Based on the proximity of Asn¹³³ and Glu²⁸⁹ in the MSMEG_6394 closed state, the corresponding amino acids in Rv3802, Asn¹³² and Asn²⁸⁸, were both mutated to cysteine to create an engineered disulfide and limit α8-helix movement. A surface rendering and gray schematic show Rv3802 with MSMEG_6394 depicted in purple (MSMEG_6394 PDB code 3AJA) (12). B, Michaelis–Menten kinetics of WT compared with N132C/N288C mutant using 4MH as the substrate. Both the affinity for 4MH and the k_cat decrease by half as a result of hindered helical movement. C, TLC plate of reaction products when the much larger PI is used as the substrate. A shift indicative of PLA1 or PLA2 activity is observed with WT Rv3802, reaction 1 (Rf value of 0.19). However, no acyl hydrolysis is observed with the disulfide mutant (reaction 2), or when enzyme is lacking (reaction 3).

The residual hydrolase activity of the N132C/N288C mutant toward 4MH indicated that N132C/N288C mutant retained sufficient dynamics or space near the active site for 4MH to bind, despite the hindered helical movement. Whereas 4MH has a 7-carbon alkyl chain, the substrate is significantly shorter than phosphatidyl-based substrates that could occupy the entire lipid tail-binding site. Therefore, PLA activity of WT and N132C/N288C mutant was tested using PI as a substrate. Following 1 h of incubation with PI, hydrolyzed product indicative of PLA activity was observed for WT (Fig. 6C, lane 1). However, no PLA activity was observed for the N132C/N288C mutant (Fig. 6C, lane 2).

Discussion

M. tuberculosis Rv3802 structure and PLA classification

Given the structural and enzymatic attributes of Rv3802, the Rv3802 family of enzymes most likely falls within the PLA₂ enzyme class (18). Rv3802 was previously determined to have PLA activity toward phosphatidyl species (phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine) (8). In contrast to thioesters, the ester moieties of phospholipids are generally stable in aqueous solutions, so the enzymatic activity observed in vitro is strongly suggestive of biological activity. The two general classes of PLAs differ with regard to specificity for either the sn-1 acyl chain (PLA₁) or sn-2 acyl chain (PLA₂) of the glycerol phosphate moiety of phosphatidyl species (17). For unknown reasons, PLA₂ enzymes possess numerous disulfides, whereas the PLA₁ enzymes typically lack disulfides (18). M. tuberculosis Rv3802 was found to possess two disulfides (Fig. 1) (8). These two disulfides are present in the M. smegmatis ortholog structure and were found to be conserved across all Rv3802 orthologs (12).

The first conserved disulfide, Cys⁷²-Cys¹⁶⁴, covalently links the N-terminal peptides to the base of α2-helix (Fig. 1). This is of potential structural importance, as the 41 residues preceding Val⁷⁵, Cys⁷² included, are predicted to be unstructured until the predicted N-terminal transmembrane region (Ile¹²–Ile³⁴) (secondary structure and transmembrane prediction in Fig. S4). Therefore, this disulfide may help retain proper protein fold of the structured catalytic hydrolase domain while being immediately preceded by a large, unstructured domain. The second conserved disulfide, Cys²⁶⁴–Cys²⁷¹, induces a kink in a loop to appropriately position the catalytically important Asp²⁶⁸ residue. This second disulfide therefore ensures the proper positioning of Asp²⁶⁸ relative to His²⁹⁹, maintaining the catalytic triad despite movement of the nearby α7-helix upon lipid binding.

Mechanism of catalysis

PLA₂ enzymes catalyze fatty acid hydrolysis through two general mechanisms: a calcium-dependent His/Asp mechanism or a calcium-independent mechanism (18). Given the identified catalytic triad and the lack of calcium-dependent enzyme activity, Rv3802 is a calcium-independent PLA₂ enzyme. Flanked by aromatic side chains (Trp⁸⁴, Phe¹⁷⁴) and displaying little movement of catalytic residues between open and closed enzymatic states, the active site of Rv3802 resembles that of a typical hydrolase enzyme (Fig. 3C) (19). These features observed in the Rv3802 active site are known to help promote substrate hydrolysis over transfer (19). These structural attributes help explain why Rv3802 has no reported transferase activity in the presence of acyl acceptors (8).

Based on the enzymatic activity, structure, and catalytic triad of Rv3802, catalysis most likely proceeds through a Ping-Pong reaction mechanism. The reaction can be broken down into two half-reactions. The first half-reaction proceeds through nucleophilic attack on the carbon of the ester/thioester carbonyl by Ser¹⁷⁵, which is deprotonated by His²⁹⁹. The terminal oxygen of PEG1 would therefore mimic the carbon of the carbonyl moiety of substrate before nucleophilic attack, depicted in Fig. 5A. Following attack, the plausible oxyanion hole consisting of backbone amide nitrogen atoms of Thr⁸³ and Asn¹⁷⁶ stabilizes the tetrahedral intermediate. Tetrahedral intermediate collapses yield the acyl-enzyme intermediate and the release of the now deacylated lipid at the end of the first half-reaction. The acyl-enzyme intermediate form of the enzyme is approximated by the terminal oxygen of PEG1 from protein molecule B (Fig. 5B). The second half-reaction would therefore proceed through the activation of a water molecule by His²⁹⁹ and subsequent nucleophilic attack on the acyl-enzyme intermediate. The nucleophilic water molecule is observed within hydrogen bonding distance to His²⁹⁹ in protein molecule B and is depicted in Fig. 5B. Tetrahedral collapse following this second nucleophilic attack results in the release of a free fatty acid and a regenerated enzyme.

Lipid binding

Based on the structural similarity of PEG to the short-chain fatty acids of various lipids, it is therefore logical that a similar repositioning of the α8-helix must occur for binding of natural substrate and subsequent catalysis to occur. The Rv3802 N132C/N288C mutant, with limited α8-helix movement, displayed a lower ability to bind 4MH, resulting in a significant loss in activity compared with WT enzyme (Fig. 6B). However, when the larger PI substrate was used, no PLA activity was observed for the N132C/N288C mutant (Fig. 6C). This loss in activity as a result of hindered helical movement highlights the importance of protein dynamics with respect to substrate binding. Given the large variety of possible structural forms of α8-helix and adjacent loops, it is difficult to fully assess how this dynamic interplay affects substrate recognition.

Initial substrate recognition may proceed through binding of the lipid head, as this site does not change between open and closed states of the enzyme. The MD simulation of apo Rv3802 suggests that the α8-helix quickly adapts the closed conformation and remains in that state without substrate present when in the aqueous phase (Fig. S3). Therefore, substrate binding may drive helical opening in a solvated environment. However, given the uncertainty of the true biological function of Rv3802, it is difficult to fully assess the mechanism of lipid binding based on the Rv3802-PEG structural observations and mutant kinetics. PLA₂ enzymes have been found to bind only the surface-exposed lipid headgroup, to reside partially within the membrane itself, and to bind the entire lipid or extract the lipid from the membrane for hydrolysis (18, 20).

When α8-helix is in the open state, a vast hydrophobic lipid-binding site is apparent (Fig. 2). In agreement with typical lipid-binding sites, the identified Rv3802 lipid-binding site is lined with aliphatic amino acid side chains (Fig. 2) (8, 21). The Rv3802 hydrophobic binding channel is relatively linear, extending away from the active site, therefore resulting in a site capable of binding two alkyl chains in a parallel linear arrangement similar to the arrangement of the two observed PEG molecules. Therefore, the binding of PEG molecules observed within Rv3802 provides a structural basis for the binding of the two acyl chains of the phosphatidyl substrate.

Structure-based insights into biological function

Meniche et al. (11) determined that Rv3802 orthologs NCgl2775 and MSMEG_6394 play an essential role in modulating lipid composition. Specifically, they noted the increase of the mycolic acid/glycerophospholipid ratio, suggesting that this family of enzymes plays a direct role in decreasing glycerophospholipid content within the mycomembrane (11). It should be noted that NCgl2775 is not essential under physiological growth, whereas the M. tuberculosis orthologs are essential (11, 12). This differing essentiality of Rv3802 orthologs may be attributed to the differing compositions of the outer membranes between mycobacteria and corynebacteria (22). For example, the inner layer of the mycomembrane of corynebacteria has a much higher composition of cardiolipin relative to other glycerophospholipids, when compared with mycobacteria (22).

PI species represent a major portion of the glycerophospholipids found within the plasma membrane and mycomembrane layers in both M. tuberculosis and M. smegmatis (23). Previously, Rv3802 was shown in vitro to hydrolyze a fatty acid from PIM2 upon treatment of M. smegmatis cell wall extracts with the enzyme (8). PIMs play an important role in the permeability of the M. tuberculosis cell wall, affect proper cell division, and influence pathogenesis (24–27). Interestingly, when a conditional knockout of MSMEG_6394 was cultured, cell morphology was jagged and elongated compared with WT (12). PIM2 has four acyl substituents, two on the phosphoglycerol, one on the inositol, and the fourth on the mannose moiety. The observed lipid-binding site of Rv3802 is significantly larger than the palmitoyl-binding pocket of other hydrolases known to hydrolyze esters of PIM (28). Specifically, the enzyme PatA resides within the PIM biosynthetic pathway and is responsible for the transfer of a palmitoyl group to the 6-position on the mannose sugar (28). Based on the larger Rv3802 lipid-binding site compared with PatA, a sensible biological function for Rv3802 is the hydrolysis of one or both of the fatty acids from the phosphoglycerol component of PIM or other PI species. PIM has been shown to be an antigen of CD1d-restricted T cells; however, PIM binding to CD1d is abolished upon the hydrolysis of the diacyl phosphoglycerol moiety following treatment with a PLA₂ enzyme (27). Whereas Rv3802 has the enzymatic ability to facilitate this chemistry, further in-depth studies are required to truly validate the biological context of such action.

Using the Rv3802-PEG structure as a basis, PI was modeled into the open form of the enzyme (Fig. 7). This model provides a reasonable structural basis for binding of phosphatidyl-based substrates to Rv3802 (8). Structurally, PI can be easily accommodated within the lipid-binding site of Rv3802 in a manner that promotes catalysis. As modeled, the sn-2 acyl chain would be subject to hydrolysis. Although little positive charge is present at this site to counter the negatively charged phosphodiester moiety, stabilization of interactions with the phosphate moiety may be afforded through solvent or by backbone amide nitrogen atoms of the highly dynamic loop upon restructuring as a function of substrate binding (Ala²⁹²–Glu²⁹⁶). Due to the proximity of this loop to the lipid head-binding site, this region may also be relevant to sugar binding of larger PI species, such as PIM.

Figure 7.

PI modeled into the lipid-binding site of Rv3802. The fatty acid chains are easily accommodated within the lipid-binding site, allowing the sn-2 ester to be positioned by the nucleophilic Ser¹⁷⁵, placing the inositol moiety within the proposed sugar-binding site. FA1, tuberculostearic acid, sn-1 position; FA2, palmitic acid, sn-2 position of glycerol.

The essential nature of Rv3802 to M. tuberculosis viability makes it an alluring new target for future drug development. Derivatives of THL exhibit enhanced in vitro inhibition against Rv3802 and related in vivo bioactivity (15). However, THL has been shown to target numerous M. tuberculosis lipases and directly affect MA content (16). Therefore, inhibition of M. tuberculosis by THL proceeds through a shotgun, polypharmacology strategy that has little specificity toward Rv3802. The identification of the lipid-binding site of Rv3802 highlights an extremely intriguing and potentially promising site for drug development. The presented structure and subsequent analysis can be utilized for designing inhibitors specific to Rv3802.

Experimental procedures

Molecular cloning

An Escherichia coli codon-optimized, synthetic gene encoding the periplasmic domain of M. tuberculosis Rv3802c was purchased from Integrated DNA Technologies. The bases of 5′-CGCTGTTCCAGGGACCT-3′ and 5′-GCGTCCGGATCCGAA-3′ were added to the 5′- and 3′-ends of the gene, respectively. The resulting gene was PCR-amplified using primers with sequences identical to those added to the gene and inserted into a pET32-derived plasmid linearized with PshAI using a Gibson Assembly^TM Master Mix. The protein expression construct produces protein with the following sequence: Met-His₆-Ser₂-Gly-rhinovirus 3C protease cut site-Rv3802c. The N132C/N288C mutant was cloned in pieces overlapping the site of mutation using the WT construct as a template. Using the 5′ WT amplify primer and 5′-TCAGAGGGCAGTGGAACTG-3′ generated the first fragment. 5′-CAGTTCCACTGCCCTCTGA-3′ and 5′-GCAAGGGTGCAAAGAGTCGTA-3′ were used to generate the middle fragment. 5′-TACGACTCTTTGCACCCTTGC-3′ and the 3′ WT amplify primer were used to generate the end fragment. The full gene was constructed by amplifying the front and middle fragments together, followed by the middle and end fragments. The two resulting fragments were then amplified together to produce a full-length mutant gene and inserted into an expression plasmid in an identical fashion to WT. Both WT and mutant plasmids were sequenced to confirm the desired DNA sequence.

Protein expression and purification

Protein expression and purification were adapted and modified from a methodology published previously (14). Both WT and N132C/N288C mutant protein were expressed and purified in an identical fashion. Chemically competent T7 Express E. coli cells (New England Biolabs) were transformed with plasmid encoding WT or mutant protein. Subsequent cultures were grown at 37 °C in lysogeny broth medium containing carbenicillin. Protein expression was induced for 3 h at 37 °C, following the addition of 1 mm isopropyl 1-thio-β-d-galactopyranoside once culture density reached an A_{600 nm} of 0.6. Induced cells were pelleted and resuspended in a 50 mm Tris, pH 7.5, 300 mm NaCl buffer and placed at −80 °C for storage.

Induced cells were thawed in a warm bath, followed by the addition of DNase I and lysozyme, and allowed to incubate on ice for half an hour. Resuspended cells were then sonicated, and the crude lysate was pelleted at 18,515 × g for 40 min at 4 °C. Supernatant was discarded, and the pellet was resuspended in 50 mm Tris, pH 7.5, 3 m NaCl buffer. Resuspended cells were again pelleted at 18,515 × g for 40 min at 4 °C, with the supernatant again discarded. The resulting pelleted inclusion bodies containing Rv3802 were solubilized in 50 mm Tris, pH 7.5, and 4 m guanidine HCl (GdnHCl) and pelleted at 18,515 × g for 40 min at 4 °C. The supernatant containing soluble Rv3802c was loaded onto an equilibrated 5-ml HiTrap Talon crude cobalt column (GE Healthcare) and was washed for 15 column volumes with 50 mm Tris, pH 7.5, 4 m GdnHCl. Protein was eluted over a 20-column volume gradient with increasing imidazole concentration. Rv3802 was refolded through the removal of GdnHCl as a result of extensive dialysis using a 50 mm Tris, pH 7.5, 300 mm NaCl buffer. Protein must be at a low concentration (∼0.25 mg/ml) within the dialysis bag for refolding, or significant precipitation is observed. Refolded protein was centrifuged at 18,515 × g for 1 h at 4 °C to remove any residual insoluble protein. Soluble, pure protein was dialyzed into either 100 mm HEPES, pH 7.5, for crystallization or 50 mm NaPO₄, pH 7.5, for cysteine quantification, 4MH, and PLA assays.

Crystallization and data collection

Purified Rv3802 was concentrated to 4.5 mg/ml for crystallization. Screening of Rv3802 against the index screen (Hampton Research) using the hanging drop method resulted in the formation of a single orthorhombic crystal. The crystal formed in a 1:1 ratio of well solution (0.1 m BisTris, pH 6.5, 25% (w/v) PEG 3350) to protein, requiring 1 month to grow with incubation at 16 °C. The resulting crystal was looped and flash-cooled in liquid nitrogen. Extensive efforts to crystallize the N132C/N288C mutant have been exerted; to date, diffraction quality crystals have not been obtained. X-ray diffraction data were collected using synchrotron radiation at the Advanced Photon Source, LS-CAT beamline F, at Argonne National Laboratory.

Structure determination and refinement

Diffraction data were indexed and scaled in a P2₁2₁2₁ space group using HKL2000 (29). The phase solution with two molecules in the asymmetric unit came from molecular replacement (PHASR-MR) using a model generated with SWISS-MODEL that was based on the previously solved MSMEG_6394 structure (PDB entry 3AJA) (30, 31). Atoms lacking 2F_o − F_c density were deleted, and the resulting model was subjected to rigid body and simulated annealing refinements (PHENIX Refine) (30). Deleted atoms were built back into corresponding F_o − F_c and 2F_o − F_c difference density maps using COOT (32). The progressing model was subjected to rounds of xyz coordinate, real-space, occupancy, and individual B-factor refinements (PHENIX-Refine) with manual modeling in COOT (30, 32). The four molecules of PEG were added using LigandFit and subjected to model refinement (33). Model refinement was complete once a final R_work/R_free of 0.1582/0.1830 was achieved. The 0.4% Ramachandran outlier is due to Val²³⁷ and is warranted, given the strong electron density for all atoms of the residue in both molecules within the asymmetric unit. MolProbity was utilized to validate the structure (34). Rv3802 model coordinates and structure factors were deposited in the PDB with accession code 5W95.

Cysteine quantification

To ensure proper disulfide formation of the engineered N132C/N288C mutant, thiols present in WT and mutant were quantified and compared using fluorescent labeling with a non-reversible thiol-reactive probe. Respective enzymes were compared in non-reduced and reduced states. A 5 μm concentration of respective enzyme in 50 mm sodium phosphate buffer, pH 7.5, was incubated with 0.3 mm tris(2-carboxyethyl)phosphine (reduced) or equivalent volume buffer (non-reduced) for 30 min at 37 °C (200-μl reaction volumes). Samples were titrated with 240 μm monochlorobimane (100 mm stock, DMSO), mixed, and incubated at 37 °C for 30 min. A standard curve to quantify free thiols was generated by serially dilution of l-cysteine in the presence of 0.3 mm tris(2-carboxyethyl)phosphine, reduction for a half hour at 37 °C, and modification with 240 μm monochlorobimane for 30 min at 37 °C (Fig. S2D). Fluorescent reads were conducted in triplicates using λ_ex = 390 nm and λ_em = 490 nm on a Synergy H4 plate reader (Biotek).

In vitro hydrolase assays

A fluorescence-based assay using 4-methylumbelliferyl heptanoate (4MH) (Sigma-Aldrich) was developed for Rv3802, which affords a sensitive method for activity determination and is amenable to drug screening, with a Z′ score of 0.84 (Z′ determination and methods shown in Fig. S5). In the presence of enzyme, the heptyl chain is hydrolyzed from 4MH, producing the now fluorescent 4-methylumbelliferone molecule and heptanoic acid. All assays were performed in triplicate in a 50 mm sodium phosphate, pH 7.5, buffer. For Michaelis–Menten kinetics, 50 nm WT Rv3802c and 75 nm N132C/N288C mutant were used with varying concentrations of serial diluted 4MH (20 mm DMSO stock). All fluorescent reads were conducted at 37 °C using λ_ex = 360 nm and λ_em = 450 nm on a Synergy H4 plate reader (Biotek). Background fluorescence as a consequence of 4MH hydrolysis was subtracted, and relative fluorescence units were converted to concentration of standard curve (Fig. S6). Reaction rates and Michaelis–Menten kinetic parameters were determined using PRISM version 7.

PLA activity

PLA activity was assessed using a modified version of a previously established Rv3802-PIM hydrolysis experiment (8). Briefly, soy PI (Avanti® Polar Lipids Inc.) was resuspended in 50 mm sodium phosphate, pH 7.5, forming a 2 mg/ml suspension of PI. Reactions consisted of 300 μg of PI suspension, 50 μg of respective enzyme or an equivalent volume of buffer and were brought to a final volume of 400 μl with 50 mm sodium phosphate, pH 7.5, buffer. Reactions were incubated for 1 h at 37 °C, being mixed every 10 min, and quenched with 200 μl of 50:50:0.3 CHCl₃/CH₃OH/HCl. The lower organic phase was extracted, spotted onto a silica TLC plate, and resolved using a mobile phase of 80:20:2 CHCl₃/CH₃OH/NH₄OH. Inositol-containing species were visualized by treating the TLC plate with 5% H₂SO₄ in methanol and charring.

Structural and bioinformatic analysis

X-ray crystal structure alignments, measurements, and subsequent analysis were performed using PyMOL (35). Sequence alignments were performed with ClustalW, and figures were generated through the use of T-COFFEE and BOXSHADE (36, 37). Jpred4 was used for secondary structure prediction (38). Transmembrane regions were predicted using the TMHMM server (39).

Author contributions

C.M.G., C.M.S., and S.D. formal analysis; C.M.G., C.M.S., and S.D. visualization; C.M.G. and S.D. writing-original draft; C.M.G., C.M.S., S.D., and D.R.R. writing-review and editing; D.R.R. conceptualization; D.R.R. supervision; D.R.R. funding acquisition; D.R.R. validation; D.R.R. project administration.