If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
The covalent attachment of a 14-carbon aliphatic tail on a glycine residue of nascent translated peptide chains is catalyzed in human cells by two N-myristoyltransferase (NMT) enzymes using the rare myristoyl-CoA (C14-CoA) molecule as fatty acid donor. Although, NMT enzymes can only transfer a myristate group, they lack specificity for C14-CoA and can also bind the far more abundant palmitoyl-CoA (C16-CoA) molecule. We determined that the acyl-CoA binding protein, acyl-CoA binding domain (ACBD)6, stimulated the NMT reaction of NMT2. This stimulatory effect required interaction between ACBD6 and NMT2, and was enhanced by binding of ACBD6 to its ligand, C18:2-CoA. ACBD6 also interacted with the second human NMT enzyme, NMT1. The presence of ACBD6 prevented competition of the NMT reaction by C16-CoA. Mutants of ACBD6 that were either deficient in ligand binding to the N-terminal ACBD or unable to interact with NMT2 did not stimulate activity of NMT2, nor could they protect the enzyme from utilizing the competitor C16-CoA. These results indicate that ACBD6 can locally sequester C16-CoA and prevent its access to the enzyme binding site via interaction with NMT2. Thus, the ligand binding properties of the NMT/ACBD6 complex can explain how the NMT reaction can proceed in the presence of the very abundant competitive substrate, C16-CoA.
Members of the acyl-CoA binding domain (ACBD)-containing protein family are involved in the maintenance of diverse cellular functions and can interact with a multitude of proteins implicated in a variety of cellular functions such as neural stem cell self-renewal, neurodegeneration, stress resistance, lipid homeostasis, intracellular vesicle trafficking, organelle formation, viral replication, and apoptotic response. Several ACBD members have been reported to associate with proteins such as GABAA (
A complex comprising phosphatidylinositol 4-kinase IIIbeta, ACBD3, and Aichi virus proteins enhances phosphatidylinositol 4-phosphate synthesis and is critical for formation of the viral replication complex.
). However, the requirement and role of the acyl-CoA ligand bound to the conserved N-terminal ACBD for the interaction with other proteins is poorly understood. Human ACBD6 is a modular protein with the N-terminal ACBD linked to a nonconserved and nonessential carboxy-terminal domain carrying two ankyrin-repeat motifs (
The role of ACBD6 in lipid metabolism of mammalian cells has not been established. Previous studies have shown that expression of the human ACBD6 protein was elevated in hematopoietic progenitor cells and erythrocyte precursors (
). In human cells infected with the bacterial pathogen Chlamydia trachomatis, ACBD6 protein was associated with cytosolic lipid droplets and was transferred to the parasitophorous vacuole resulting in its removal from the cytosol (
). Inside the vacuole, ACBD6 regulates the activity of a bacterial acyltransferase essential for lipid metabolism of the pathogen. A related fungal fatty acyl-CoA binding protein (ACBP) from the human pathogen Cryptosporidium parvum is required by the fungus for growth in human cells, as confirmed using chemical inhibition of acyl-CoA binding to this ACBD protein (
Co-translational N-myristoylation of the glycine +2 residue of nascent peptides, following removal of the initiator methionine by the human N-myristoyltransferase (NMT) enzymes (NMT1 and NMT2), can affect the association of the acyl-proteins with membranes (
). The addition of a 14-carbon long aliphatic tail can act as a lipid anchor and mediates oligomeric assembly and interaction with other proteins. Although amide linkage of the myristate to the glycine residue appears irreversible, a structural conformational property of some acyl-proteins, called the myristoyl-switch, can remove the aliphatic tail from the lipid bilayer and dissociate the proteins from their membrane-bound location (
). Chemicals inhibiting myristoylation of proteins are potent drugs against parasitic protozoa and fungi. The catalytic reaction is a multistep process initiated by binding of C14-CoA to apo-NMT, which triggers a conformational change exposing the peptide binding site (
). C14-CoA is then hydrolyzed and CoA is released. The aliphatic tail is transferred to the glycine +2 residue of the peptide, which is then released. NMT proteins can bind palmitoyl-CoA (C16-CoA), but a 16-carbon chain cannot be transferred to the glycine +2 residue by the myristoyl-transferase (
). It is unclear how NMT enzymes are able to perform myristoylation in vivo using a very rare acyl-donor (C14-CoA) in the presence of a very abundant acyl-CoA competitor (C16-CoA). Thus, a process to prevent binding or trigger release of this competitor from the C14-CoA binding site of NMT seems essential to warrant its activity in vivo.
We determined that ACBD6 was associated with the two human NMT enzymes in human cells, with a stronger interaction with NMT2 than NMT1. The C-terminal domain of ACBD6 interacted with a C-terminal region of NMT2. Our results further show that ACBD6 protected the enzyme from competitive binding by C16-CoA and that lipid-bound ACBD6 stimulated NMT2 activity toward its substrate, C14-CoA. Together, these results establish a physiological role for the binding property of a member of the human family of acyl-CoA carriers, and determine how NMT enzymes can perform their function in vivo.
MATERIALS AND METHODS
The No-Weigh format of disuccinimidyl suberate (DSS) was purchased from Thermo Fisher Scientific. Ellman's reagent [5,5′-dithio-bis-(2-nitrobenzoic acid)] (DTNB), acyl-CoAs, and fatty acids were from Sigma-Aldrich. All other compounds used were reagent grade. The peptide Hs pp60src#2-9 (GSNKSKPK, C-term amide; MW of 843.99) (
). Human NMT2 isoform 1 (NP_004799) was cloned by PCR from NM_004808.2 into pBIND vector and human ACBD6 was cloned into the pACT vector (Check-Mate Mammalian two-hybrid system; Promega). As previously described (
), the membrane targeting signal present in the first 97 residues of NMT2 was removed to produce an active cytosolic form of the protein in Escherichia coli (also known as ecNMT2, see below). Site-directed mutagenesis experiments were performed with the QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies) according to the manufacturer's instructions. Primers were designed with the QuikChange® primer design program. The presence of the intended nucleotide change(s) and the absence of unwarranted mutations were verified by full-length sequencing of the constructs. N-terminally tagged green fluorescent protein (GFP) and hemagglutinin (HA) fusion of full-length ACBD6 and full-length NMT2 were obtained in the pAcGFP1-C1 and pIRESneo vector (Clontech Laboratories, Inc.), respectively.
Protein expression and purification
All proteins and mutant forms were produced as hexahistidine fusion forms. ACBD1 and ACBD6 were expressed in the E. coli host BL21(DE3) cells (Novagen) and ecNMT2 was produced in RosettaDE3 cells (Novagen). Proteins were purified by affinity metal chromatography with the addition of 0.2% Triton X-100 in all the buffers for the ecNMT2 protein. The purified proteins were stored at −80°C in Tris-HCl [50 mM (pH 8.0)], NaCl (0.1 M), EDTA (5 mM), and glycerol [10% (v/v), with 0.2% Triton X-100 for ecNMT2]. Prior to isothermal titration calorimetry (ITC) measurements, proteins were dialyzed in the ITC buffer (see below).
Affinity purification MS
Affinity purification followed by LC-MS/MS was used to identify candidate protein interactions of ACBD6 following identical methods to those previously published (
). The cells were harvested and lysed, and the Strep-tagged protein was captured on StrepTactin Sepharose under native conditions, and then eluted with 1× desthiobiotin (IBA Technology, Gottingen Germany) as reported (
) against the Refseq NCBInr human database (downloaded July 29, 2015 from ftp://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/mRNA_Prot/) containing 99,857 sequences, concatenated with 99,857 additional randomized decoy sequences for calculation of the false discovery rate (FDR) (
). The FDR was <1%, using a protein score of 22, a peptide score of 15, a protein expectation value of 0.01, and a peptide expectation value of 0.001. Modifications allowed were: fixed carbamidomethylation of Cys and the following variable modifications: oxidation of Met, start-Met cleavage, oxidation of the N terminus, acetylation of the N terminus, and pyroglutamate formation from Gln.
Protein identifications from ACBD6 affinity-purification experiments were performed over four biological replicate experiments. Proteins were reported as specific interacting proteins if observed in at least three of four replicate experiments with more than one unique peptide in at least one experiment. These proteins were also compared with a list of high-frequency background proteins from >2,000 similar affinity purification MS experiments with Strep-tagged proteins (
), and excluded if the background frequency was >0.10. MS data for proteins meeting these criteria are reported in supplementary Table 1.
Protein interaction assays
Mammalian two-hybrid interaction assays were performed in HeLa and HEK293 cells grown in 96-well plates with the CheckMate system (Promega). Hela cells were transfected with TurboFect reagent (Thermo Scientific) and HEK293 cells with TransIT-LT1 transfection reagent (Mirus Bio). Luminescence was quantified with the Dual-Luciferase reporter assay of Promega, according to the manufacturer's instructions. The homobifunctional N-hydroxysuccinimide ester reagent, DSS, reacts with primary amine groups and was used to detect interaction of purified NMT2 and ACBD6 proteins. DSS was bought in individual sealed microtubes (Thermo Fisher Scientific) and stock solutions of 100 mM in DMSO (further diluted in DMSO as needed) were made fresh prior to each experiment. DSS was added in a 30-fold excess of the protein (mole/mole). Proteins were dialyzed in 10 mM potassium phosphate (pH 7.4) at 4°C. ACBD6 and NMT2 proteins were mixed at a 1:1 molar ratio (20 ¼M each) in 29 ¼l of 50 mM potassium phosphate (pH 7.4) at 37°C for 20 min. When acyl-CoAs were used, they were added at a final concentration of 50 ¼M. One microliter of DSS was then added to the mixture at a final concentration of 1 mM, and subsequently incubated for 30 min at room temperature. Untreated control reactions were performed with addition of 1 ¼l of DMSO and incubated as described for the DSS-treated samples. Reactions were stopped by quenching of DSS with 2 ¼l Tris-HCl (1 M, pH 7.4) for at least 15 min at room temperature. Mixtures were then boiled in SDS-PAGE loading buffer for 4 min and separated on denaturing SDS-polyacrylamide Tris-glycine gels. Proteins were detected by staining with GelCode Blue reagent (Thermo Fisher Scientific) or were transferred on nitrocellulose membrane and detected with a HRP-conjugated anti-histidine antibody (INDIA-HisProbe-HRP antibody; Thermo Fisher Scientific).
Protein samples were made from HEK293 cells grown in 12-well plates. For each sample, three wells were cotransfected using the TurboFect reagent (Thermo Scientific) with the pair HA-ACBD6/GFP-NMT2 or HA-NMT2/GFP-ACBD6 for 48 h. Cells were lysed with lysis immunoprecipitation buffer (Thermo Scientific) and protease cocktail inhibitor (Thermo Scientific) in a final volume of 400 ¼l. Cleared lysates were stored at −80°C. For co-immunoprecipitation, 200 ¼g of each protein sample was precleared with 25 ¼l protein G-magnetic beads (NEBiolabs) for 1 h at 4°C. Following removal of the beads, 5 ¼g of mouse monoclonal anti-HA antibody (Sigma-Aldrich, H3663) was added and incubated for 1 hr at 4°C. Then, 25 ¼l of fresh protein G-magnetic beads was added and incubated overnight at 4°C. Beads were then washed three times with ice-cold PBS and with lysis IP buffer. Complexes were eluted by suspending the beads in 60 ¼l SDS-PAGE loading buffer and heating at 70°C for 10 min. After removal of the beads, 10 ¼l of each sample was analyzed by Western blot with rabbit polyclonal anti-GFP (Sigma-Aldrich, G1544) and with HRP-conjugated mouse anti-HA antibodies (Sigma-Aldrich, H6533)
ITC measurements of the binding of acyl-CoAs and fatty acids were performed on a VP-ITC instrument (MicroCal, LLC) (
). All experiments were performed in ammonium acetate (25 mM, pH 7.4), supplemented with 0.1% Triton X-100 to prevent precipitation of ecNMT2, at 30°C. The proteins were dialyzed in the same buffer and fresh 10 mM stocks of the ligands were prepared from powder with the dialyzing buffer. For NMT2 (Fig. 6B), measurements were performed with 28 injections of 10 ¼l of 165 ¼M ligand every 150 s. The chamber contained the protein at an initial concentration of 5.5 ¼M. For ACBD6ΔC measurements (Fig. 6A), protein was at a concentration of 30 ¼M and was injected with 600 ¼M of C16-CoA and 300 ¼M of C18:2-CoA. For data presented in Fig. 6C, proteins were at a concentration of 10 ¼M and C16-CoA at 100 ¼M. Control experiments were run by injecting buffer into the cell containing the protein and by injecting the ligand into the cell containing buffer. Heat generated from control runs was subtracted from the data of the experimental set performed under the same conditions
NMT activity measurements
Real-time measurements of the released CoA from acyl-CoAs by purified NMT2 were performed at 30°C in a Cary Avian UV-Vis spectrophotometer (50 Bio; Varian) in the presence of DTNB. Measurements were made in dual-beam mode against blank buffer and normalized by subtracting values obtained in reactions performed in the absence of the target peptide. Reactions were performed in 500 ¼l of 20 mM sodium phosphate buffer at pH 8.0 with 1 mM EDTA and 100 ¼M Hs pp60src#2-9 peptide, 5–100 ¼M acyl-CoAs (C14-CoA, C16-CoA, C18:2-CoA), and 0.2 mM DTNB. Unless otherwise indicated, a 2:1 molar ratio of ACBD6/NMT2 was used. Titration of NMT2 was performed to determine the optimal concentration of the partially purified proteins to maintain linearity of the measurement over a period of 5 min (usually 2 ¼M ACBD6/1 ¼M NMT2). Kinetic calculations were performed with GraphPad Prism 6.
Interaction of NMT enzymes with ACBD6
ACBD6 protein is a modular protein with an N-terminal conserved ACBD and two ankyrin-repeat motifs present in the nonconserved C-terminal domain (Fig. 1A). Affinity purification MS was performed using a C-terminally Strep-tagged ACBD6 construct, transiently expressed in HEK293 cells, in four biological replicates. From these experiments, the most specific interacting proteins were NMT1 and NMT2, identified with a total of 24 and 14 unique peptides, respectively (supplementary Table 1). NMT1 and NMT2 share high sequence identity (73%, ClustalW alignment), thus unique and shared peptide sequences were identified to each of these proteins in the MS data (supplementary Table 2). Sufficient unique peptide counts were obtained to both NMT1 and NMT2 in each experiment to confirm the presence of both proteins at comparable levels in this semi-quantitative analysis (supplementary Table 3). Two proteins, CAMK2D and UGGT1, were also identified at a lower specificity threshold, but NMT1 and NMT2 were the only proteins that reproducibly interacted with ACBD6 in all four experiments. The specificity of the association with ACBD6 was further confirmed with a mammalian two-hybrid interaction assay, which established a stronger binding of ACBD6 with NMT2 than with NMT1, as compared with the related ACBD5 protein (33% identical in the ACBD), which showed no interaction (Fig. 1B).
Association of NMT2 with ACBD6 was further investigated by co-immunoprecipitation of wild-type and mutant constructs of GFP-tagged ACBD6 by HA-NMT2, and of the inversely tagged GFP-NMT2 by HA-ACBD6 (Fig. 2A, B). A C-terminally truncated form of ACBD6 lacking the two ankyrin-repeat motifs (ACBD6ΔC, Fig. 1A) did not co-immunoprecipitate with NMT2 (Fig. 2A, B). The C terminus of NMT-2 includes residues that bind the target peptide substrate (Fig. 1A), and the C terminus itself may assist in catalysis by deprotonating the ammonium of the acceptor glycine residue (
). An NMT2 form lacking the last 58 residues, including the signature motif PS00976 (NMT2ΔC, Fig. 1A), did not interact with full-length ACBD6 either by co-immunoprecipitation or mammalian two-hybrid assay (Fig. 2C; supplementary Fig. 1A). Deletion mutants of conserved motifs of the ACBD of ACBD6 were also designed (Fig. 1, regions H2–4), and these did not prevent interaction with NMT2 (Fig. 2D). Thus, ACBD6 interacts via its C-terminal domain containing the ankyrin-repeat motifs with NMT2, and the NMT2 interaction with ACBD6 is dependent upon its C-terminal region.
ACBD6 and NMT2 form hetero-dimeric complexes
Association of the two purified proteins was investigated using covalent cross-linking with the reagent DSS, followed by analysis of the trapped complexes by SDS-PAGE. As previously reported (
), ACBD6 (32 kDa) behaved as a monomeric protein, and no high molecular mass species were detected after DSS treatment (Fig. 3A). To produce and purify NMT2, a recombinant form was expressed in E. coli without the N-terminal membrane localization sequence (also known as ecNMT2 protein) (
). Purified ecNMT2 (supplementary Fig. 1B) was primarily monomeric (48 kDa), and few bands of very high molecular mass were formed after DSS treatment (>100 kDa, not shown). When ACBD6 and ecNMT2 were mixed at a 1:1 molar ratio, most of the ecNMT2 monomeric form disappeared, and a new band was detected at approximately the calculated mass of a heterodimeric ACBD6/ecNMT2 complex (80 kDa). Addition of the NMT2 substrate, C14-CoA, and of the ACBD6 ligand, C18:1-CoA, did not prevent formation of the complex (Fig. 3A). In contrast, the truncated recombinant ACBD6ΔC mutant (21 kDa) did not form a complex with ecNMT2 (Fig. 3B).
Ligand binding to ACBD6 stimulates the NMT reaction
Activity of purified ecNMT2 enzyme was measured by the real-time detection of the release of CoA from C14-CoA in the presence of a synthetic peptide matching the amino terminal myristoylation sequence of c-Src protein (
) (supplementary Fig. 1C, D). Addition of increasing concentrations of ACBD6 protein resulted in the stimulation of activity of ecNMT2 (Fig. 4). No inhibitory effect was observed, even with a 20:1 ACBD6/ecNMT2 molar ratio. Under similar conditions, addition of purified ACBD1 had only a weak stimulatory effect. Compared with ACBD6, two ACBD6 mutant forms, Y114A and FKKY-AAAA (dubbed FKKYmut), with defects in acyl-CoA binding (
) failed to stimulate the reaction in the presence of C14-CoA, thus acyl-CoA binding of ACBD6 is required for the stimulatory effect on NMT2 activity (Fig. 5). The molecule C18:2-CoA, a ligand of ACBD6, was not an acyl donor for the NMT reaction (Figs. 4, 5). In the absence of ACBD6, addition of C18:2-CoA to C14-CoA had a weak inhibitory effect on the reaction (Fig. 5). However, in the presence of ACBD6, addition of C18:2-CoA resulted in increased stimulation of ecNMT2 activity compared with the activity obtained with only ACBD6. This stimulatory effect was not detected with the two mutant ACBD6 forms, Y114A and FKKYmut. Thus, compared with apo-ACBD6, acyl-CoA-bound ACBD6 appeared to be the form enhancing the processing of C14-CoA by NMT2, independent of its binding to C14-CoA.
ACBD6 interaction is required for NMT2 activity stimulation
The requirement for protein interaction in the stimulatory effect of ACBD6 in the presence of its ligand on the NMT reaction was investigated. The carboxy-terminal domain of ACBD6 is dispensable for ligand binding (
) and we confirmed that the truncated ACBD6ΔC form binds C18:2-CoA (Fig. 6A). ACBD6ΔC did not interact with ecNMT2 (Fig. 2), suggesting that the ankyrin-repeat motifs were involved in the contact to the C-terminal motif of NMT2 (Fig. 2C; supplementary Fig. 1A). Compared with full-length ACBD6, ACBD6ΔC could not stimulate the NMT reaction and the ecNMT2 activity rate in the presence of the truncated form was similar to those obtained with the Y114A and FKKYmut mutants (Fig. 5). Thus, interaction of ABCD6 with NMT2 was required for the ligand-mediated increase in NMT2 activity.
N-terminal ACBD6 domain prevents binding of C16-CoA to NMT2
Purified ecNMT2 binds both C14-CoA and C16-CoA with similar affinity (Fig. 6B). Binding of C16-CoA to ecNMT2 resulted in cleavage of the ester bond and release of CoA (Fig. 7A). Confirmation of the strong binding affinity of NMT2 for this acyl-CoA competitor was obtained by the observation that 20 ¼M of C16-CoA produced a stronger signal than 15 ¼M of C14-CoA (Fig. 7B). Addition of ACBD6 resulted in the stimulation of the reaction with C14-CoA, but it decreased the processing of C16-CoA by ecNMT2 (Fig. 7A, B). The mutant S106A lacks one of the two phosphorylated serine residues of ACBD6 (
), and binds to C16-CoA with stronger affinity than ACBD6. This mutant stimulated ecNMT2 activity with C14-CoA as ACBD6 (Fig. 5), but it could prevent the processing of C16-CoA at much higher concentration than ACBD6 (Fig. 7B). In contrast, the ACBD6 mutant, Y114A, which did not bind C16-CoA (
), could not prevent the processing of C16-CoA by NMT2, even at very low concentration (Fig. 7B).
NMT2 interaction to the C-terminal ACBD6 domain is required for competitor protection
As established above, binding of C16-CoA to ACBD6 prevented processing of the acyl-CoA competitor by ecNMT2. However, the effect of ACBD6 could not be accounted for by a model requiring exhaustion of free-competitor in the reaction mixture because as little as 1 ¼M of ACBD6 was enough to reduce the processing of 100 ¼M C16-CoA to half (Fig. 7B). As seen for the C18:2-CoA stimulation of C14-CoA processing by ecNMT2, ACBD6/NMT2 complex formation was also necessary to prevent usage of C16-CoA by ecNMT2. The truncated ACBD6ΔC form, which did not interact with ecNMT2 (Fig. 2A and 2B), but bound C16-CoA (Fig. 6A, inset), could not prevent processing of C16-CoA by ecNMT2. In fact, that form was even less efficient in protecting ecNMT2 than the mutant Y114A (Fig. 7B). These results suggested that in the hetero-dimeric complex, ACBD6-NMT2 formed through the interaction of their C-terminal domains, C16-CoA was prevented from accessing the NMT2 binding site through sequestration to the N-terminal ACBD of ACBD6.
Displacement of C16-CoA by linoleoyl-CoA
To confirm that protection of NMT2 from C16-CoA by ACBD6 was the result of C16-CoA binding to ACBD6, the ligand, C18:2-CoA, was added to the reaction. ACBD6 binds C18:2-CoA with greater affinity than C16-CoA (
). At an equal concentration of C18:2-CoA and C16-CoA (5 ¼M), ACBD6 could no longer prevent processing of C16-CoA by ecNMT2 and activity rate doubled (Fig. 7C). The mutant Y114A, unable to bind either acyl-CoA species (
), had no detectable effect on ecNMT2 activity, irrespective of the C18:2-CoA concentrations. The mutant S106A, which binds C16-CoA with stronger affinity than ACBD6, limited use of C16-CoA by ecNMT2, even when a 10-fold excess of C18:2-CoA was added to the reaction. Thus, selectivity of binding of the ACB motif of the acyl-CoA carrier ACBD6 modulates the NMT reaction by controlling availability of acyl-CoA to NMT2 and stimulates its activity via protein contact with their respective C-terminal domains.
The acyl-transferase activity of the NMT proteins is highly specific toward C14-CoA. On the other hand, a variety of glycine-containing nascent peptide chains can bind to the C-terminal domain of NMT (
), indicating that NMT is less selective toward its protein substrate. The catalytic reaction is a multistep process initiated by binding of C14-CoA to the N-terminal domain of apo-NMT, which triggers a conformational change exposing the peptide binding site (
). C14-CoA is then hydrolyzed and CoA is released. The aliphatic tail is transferred and covalently linked to the exposed glycine residue of the peptide substrate, releasing the acylpeptide. Whereas the transfer rates of acyl chains other than C14 are extremely slow compared with myristate (
). Thus, in the absence of a process to modulate binding or release of the acyl-CoA competitor from the C14-CoA binding site, compounds such as C16-CoA would inhibit the action of NMT enzymes in vivo. This is illustrated in Fig. 8, which provides a summary of data presented in this study, indicating an important role of acyl-CoA binding proteins in this process. Several other reports provide evidence that other cellular components modulate the NMT reaction to ensure that the correct acyl chain is linked to the protein substrate. Whereas NMT enzymes are highly specific in transferring a 14-carbon tail on nascent peptides, C14:1 and C14:2, rather than C14:0, are the fatty acids used to modify proteins such as the α-subunit of the G-protein photoreceptor in retinal photoreceptor cells (
). It can be hypothesized that acyl-CoA carriers with stronger affinity to unsaturated myristate control the acylation profile observed in this tissue.
In our study, the stimulation of the NMT activity of NMT2 in the presence of apo-ACBD6 and C14-CoA could be explained by the formation of the ACBD6/NMT2 complex (Fig. 8B). We speculate that in the ACBD6/NMT2 complex, the entry of the C16:0 acyl-chain to the binding site of the acyltransferase subunit is reduced in favor of the C14:0 acyl-chain. Mutant forms of ACBD6, which are unable to bind C16-CoA or are unable to interact with the enzyme, could not prevent NMT2 from using this acyl donor (Fig. 7). These findings suggested that protection of the NMT reaction was also the result of the local sequestration of C16-CoA by the acyl-CoA carrier subunit of the ACBD6/NMT2 complex. The highly improved preference for C14:0-CoA relative to C16:0-CoA in this complex is essential, given that these two compounds are at the low and high end of the concentration range, respectively (
). Without this shift in specificity triggered by ACBD6, it is unlikely that the minor C14:0-CoA molecule could ever access the NMT2 binding site. Because ACBD6 can also bind C14-CoA, the stimulatory effect could also indicate the channeling of C14-CoA from the binding site of ACBD6 to NMT2 in the complex. In addition, the formation of the ACBD6/NMT2 complex could also alter the binding affinity of NMT2 toward C16-CoA and render its binding to NMT2 less favorable. A particular role should be considered for the serine 106 residue of ACBD6. This site is phosphorylated in vivo (
) and, as shown in Fig. 7C, this residue is essential in controlling access of C16-CoA to NMT2. The binding of C16-CoA to the ACBD motif of phosphoACBD6 might trigger a substantial conformational change in the complex leading to blocking the entry or channeling of C16-CoA to the NMT2 binding site. In vivo, ACBD6 and NMT2 are exposed to a variety of acyl-CoAs at different (local) concentrations (
). These acyl-CoAs that are bound to the ACBD6/NMT2 complex may further stimulate the NMT reaction toward C14:0-CoA (Fig. 8C). In support of this model, we established that activity of NMT2 toward C14:0-CoA was stimulated by binding of C18:2-CoA to ACBD6 (Fig. 5). The role of serine 106 is highlighted, as the ACBD6 serine mutant prevented C16-CoA access to NMT2 independently of C18:2-CoA (Fig. 7). Together, this suggests that even under condition of excess of C16-CoA, ACBD6 would protect the specificity of the NMT reaction as long as the formation of the complex with NMT2 or phosphorylation of ACBD6 was not prevented.
A variety of pathogens rely on protein acylation processes. Because binding of the peptides does not affect acyl-CoA binding to the N-terminal motif (
), compounds that can recognize the C-terminal binding domain of the NMT proteins of invading pathogens seem to be good candidates to specifically disrupt myristoylation without impacting acylation of the proteins of the host (
), the host ACBD6 and the parasite ACBD6 homolog could cross the vacuole membrane in association with lipid droplets. Disruption of such a process may offer another strategy to affect the activity of the parasite NMT and its association to ACBD6 (
The NMT process in vivo is more complex than explored in this study. For example, the generation of the acyl-donor and acyl-acceptor of the reaction are localized processes that require movement of the fatty acyl molecules through membranes and binding to carriers such as FABP, their activation to acyl-CoAs by acyl-CoA synthetases, and their transport by ACBD proteins to the NMT enzyme. ACBD proteins are involved in a variety of cellular mechanisms through association with a multiplicity of proteins. However, to our knowledge, the role of the binding of the acyl-CoA ligand to the ACBD function has not been established in these processes. The modulation of the NMT reaction by ligand binding to ACBD6 in the enzymatic complex formed with NMT2 provides one of the first examples of a physiological role for the acyl-CoA binding property of a member of the human ACBD family.
A complex comprising phosphatidylinositol 4-kinase IIIbeta, ACBD3, and Aichi virus proteins enhances phosphatidylinositol 4-phosphate synthesis and is critical for formation of the viral replication complex.
MS for this work was provided through the University of California at San Francisco Mass Spectrometry Facility (A.L. Burlingame, director), supported by National Institutes of Health Grant 8P41GM103481. J.L.D. is supported by the Howard Hughes Medical Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
acyl-CoA binding domain
5,5′-dithio-bis-(2-nitrobenzoic acid) or Ellman's reagent