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N-terminal transmembrane domain of lipase LipA from Pseudomonas protegens Pf-5: A must for its efficient folding into an active conformation
Biochimie 105 (2014) 165e171
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Research paper
N-terminal transmembrane domain of lipase LipA from Pseudomonas protegens Pf-5: A must for its efficient folding into an active conformation Daiming Zha, Huaidong Zhang, Houjin Zhang, Li Xu, Yunjun Yan* Key Laboratory of Molecular Biophysics, Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 May 2014 Accepted 8 July 2014 Available online 16 July 2014
LipA from Pseudomonas protegens Pf-5 has been proven not to be secreted into the extracytoplasmic space, proposing that it is a membrane protein in virtue of its N-terminal transmembrane domain predicted by the TMHMM 2.0. However, LipA was confirmed to be an intracellular protein through determining the effects of lipA deletion or overexpression on the lipase activities in the whole-cell, lysis supernatant and lysis pellet, even through its transmembrane domain being able to make heterologous LacZ locate on the cytoplasmic membrane via construction of b-galactosidase reporter strains. Subsequently, lipase activity assays showed that the transmembrane domain played an indispensable role for the catalytic function of LipA through construction of the markerless deletion mutant of transmembrane domain sequence of lipA and the expression and purification of LipA and LipADTMD. To further investigate why the transmembrane domain lost its membrane localization function and significantly affected the catalytic function of LipA, the 3D structures of LipA and LipADTMD were constructed. The results indicated that the transmembrane domain, located in the interior of LipA, helped the a-helical lid to form an open conformation by the mediation of a5 helix. It seems to act as a kind of intramolecular chaperone like the b-roll motif of subfamily I.3 lipases, which is novel and is the first to notify the intramolecular chaperone of a subfamily I.1 lipase. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Pseudomonas protegens lipase Transmembrane domain Intramolecular chaperone 3D structure
1. Introduction The rhizosphere inhabitant Pseudomonas protegens Pf-5 (previously called Pseudomonas fluorescens Pf-5), isolated from the soil in College Station, Texas in the 1979, is a well-known biocontrol bacterium that can suppress plant diseases by producing secondary metabolites to inhibit various pathogenic bacteria, fungi and oomycetes [1,2]. Though Pf-5 has been widely studied thanks to the biocontrol properties, very little literature is available on its lipases. LipA (KEGG entry number PFL_0617, GenBank accession number YP_257759) is a member of subfamily I.1 lipases secreted through the type II secretion pathway and its recombinant protein (i.e., rPFL from P. fluorescens JCM5963) has been reported [3,4]. Most of the known subfamily I.1 lipases contain an N-terminal signal peptide, which directs their secretion into the extracytoplasmic space through the Sec machinery. For lipases from Gram-positive
* Corresponding author. Tel.: þ86 (0)27 87792214 12; fax: þ86 (0)27 87792213. E-mail addresses: [email protected], [email protected] (Y. Yan). http://dx.doi.org/10.1016/j.biochi.2014.07.007 0300-9084/© 2014 Elsevier Masson SAS. All rights reserved.
bacteria, this mechanism is sufficient, whereas these secreted by Gram-negative bacteria must cross a second barrier constituted by the outer membrane [4,5]. After being secreted through the inner membrane, lipases from Gram-negative bacteria fold in the periplasm into an enzymatically active conformation by means of their cognate lipase-specific foldases called Lif proteins. Subsequently, they are transported through the outer membrane via the secreton including up to 14 different proteins [4,6]. However, LipA lacks a typical N-terminal signal peptide but has a transmembrane domain (TMD) (aa 9 to 31 relative to the starting amino acid) predicted by the TMHMM Server v. 2.0 program (http://www.cbs.dtu.dk/ services/TMHMM/) and is not secreted into the extracytoplasmic space [7]. In addition, its cognate lipase-specific foldase is not found in the Pf-5 genome. These different features suggest that LipA is not a typical subfamily I.1 lipase and its N-terminal TMD may play an important role for its enzymatic function. In this study, we confirmed the role of TMD in LipA by measuring the lipase activities of various strains in the whole-cell, lysis supernatant and lysis pellet and detecting the lipase activities of LipA and LipADTMD towards tributyrin and p-nitrophenyl esters,
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and further proposed the mechanism of TMD inactivating LipA by modeling the 3D structures of LipA and LipADTMD. In addition, we also investigated the effect of TMD on the subcellular localization of heterologous LacZ. 2. Materials and methods 2.1. Strains, plasmids, culture conditions and general methods The bacterial strains and plasmids used were described in Table 1. P. protegens (28 C) and Escherichia coli (37 C) strains were propagated in liquid or solid (1.5% w/v agar) LB medium. Antibiotics were used at the following concentrations: ampicillin (Ap) (100 mg/ mL), gentamicin (Gm) (50 mg/mL), and kanamycin (Km) (50 mg/mL) for P. protegens, and spectinomycin (Sp) (100 mg/mL), gentamicin (50 mg/mL), and kanamycin (50 mg/mL) for E. coli. PrimeSTAR HS DNA Polymerase, restriction enzymes, and DNA ligation kit ver. 2.0 were purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. (Dalian, China). Genomic DNA extraction, plasmid preparation, and DNA gel extraction were carried out with commercial kits (Omega Bio-Tek, Doraville, GA) according to the manufacturer's protocols. Genomic DNA of P. protegens Pf-5 was used as the template in all PCR amplification reactions. Oligonucleotide primers were synthesized from Wuhan Anygene Biological Technology Co., Ltd. (Wuhan, China). DNA sequencing was performed by Shanghai Sunny Biotechnology Co., Ltd. (Shanghai, China). All other routine manipulations were performed by standard methods [8].
SphI/TMDD-L-XbaI (Table S1). SpeI-SphI digested TMDU, SphI-XbaI digested TMDD, and SpeI-XbaI digested pJQ200SK were ligated, and then followed by E. coli Top10 transformation. Transformants were screened on LB plates containing 50 mg/mL gentamicin and further verified by colony PCR. Then, plasmid DNA was isolated and sequenced. The recombinant plasmid was named as pJQlipADTMD (Table 1). In order to construct the lipADTMD expression plasmids, PCR amplicon encoding the lipADTMD (810-bp) was amplified using primers lipADTMDF-BamHI/lipAR-HindIII (Table S1). Then, BamHIHindIII digested amplicon was cloned into the same digested pBBRKm, creating the recombinant plasmid pBBRKlipADTMD (Table 1). The recombinant plasmids pET-lipA and pET-lipADTMD (Table 1) were constructed by PCR amplification using primers lipAF-NdeI/lipAR-HindIII and lipADTMDF-NdeI/lipAR-HindIII (Table S1) and cloning 903-bp and 810-bp DNA fragments into the plasmid pET-28a digested with NdeI and HindIII, respectively. b-galactosidase reporter plasmid pBBR003 (Table 1) was generated by PCR amplification using primers lipA-PF-KpnI/lipA0 PR-HindIII (Table S1) and cloning a 631-bp DNA fragment encompassing the promoter and the first six codons of lipA into the plasmid pBBR001 (Table 1) digested with KpnI and HindIII. Similarly, b-galactosidase reporter plasmid pBBR003TMD (Table 1) was constructed by PCR amplification using primers lipA-PF-KpnI/lipATMDR-HindIII (Table S1) and cloning a 712-bp DNA fragment encompassing the promoter and TMD sequence of lipA into the plasmid pBBR001 digested with KpnI and HindIII.
2.2. Construction of plasmids 2.3. Construction of strains To construct the markerless deletion mutation cassette of TMD sequence of lipA, homology arms of 618-bp upstream (bp 615 to 2 relative to the translational start site) and 410-bp downstream (bp 92 to 501 relative to the translational start site) were respectively amplified using primers TMDU-U-SpeI/TMDU-L-SphI and TMDD-U-
The markerless deletion mutant of TMD sequence of lipA was constructed via a double-crossover recombination event by means of a gene replacement system with the plasmid pJQ200SK [10,11]. Single- and double-crossover recombination events were verified
Table 1 Strains and plasmids used in this study. Strain or plasmid E. coli Top10 BL21(DE3) P. protegens Pf-5 Pf0617 Pf0617TMD Pf-5K Pf0617K Pf0617lipA Pf0617lipADTMD Pf003 Pf003TMD Plasmid pRK2073 pJQ200SK pJQlipADTMD pBBRKm pBBRKlipA pBBRKlipADTMD pET-lipA pET-lipADTMD pBBR001 pBBR003 pBBR003TMD
Description
Reference or source
mcrA D(mrr-hsdRMS-mcrBC)F80lacZDM15DlacX74 recA1 araD139D(ara-leu)7697galU galK rpsL (StrR) endA1 nupG F-, ompT hsdSB(rB-, mB-) dcm gal(DE3)
Invitrogen
Rhizosphere isolate; Pltþ, Apr DlipA derivative of Pf-5 Deletion mutant of transmembrane domain sequence of lipA Pf-5 with pBBRKm; Kmr Pf0617 with pBBRKm; Kmr Pf0617 with pBBRKlipA; Kmr Pf0617 with pBBRKlipADTMD; Kmr Pf0617 with pBBR003; Gmr Pf0617 with pBBR003TMD; Gmr
ATCC BAA-477 [7] This study [7] This study [7] This study This study This study
Helper plasmid for triparental mating; Spr Suicide vector with sacB counter-selectable marker used for homologous recombination; Gmr pJQ200SK carrying a 1.028 kb BamHI-XbaI insert with a deletion in the transmembrane domain sequence of lipA; Gmr Broad-host-range vector derivative of pBBR1MCS-5; lacIq-Plac, Kmr pBBRKm with a 904 bp BamHI-HindIII fragment harboring the coding region of lipA; Kmr pBBRKm carrying a 810 bp BamHI-HindIII insert with a deletion in transmembrane domain sequence of lipA; Kmr pET-28a with a 903 bp BamHI-HindIII fragment harboring the coding region of lipA; Kmr pET-28a carrying a 810 bp BamHI-HindIII insert with a deletion in transmembrane domain sequence of lipA; Kmr pBBR1MCS-5 derivative with a translational ‘lacZ fusion; Gmr pBBR001 derivative with a translational lipA’-‘lacZ fusion; Gmr pBBR001 derivative with a translational lipA-TMD-‘lacZ fusion; Gmr
[9] [10] This study
Novagen
[7] [7] This study This study This study This study This study This study
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by colony PCR using primers lipA-22U/lipA223L (Table S1). Furthermore, sequencing and gentamicin resistance assay for double-crossover colonies were carried out to determine the loss of TMD sequence and plasmid pJQ200SK. The verified doublecrossover colony, i.e., markerless deletion mutant of TMD sequence of lipA, was named as Pf0617TMD (Table 1). To further evaluate the function of LipADTMD, the strain Pf0617lipADTMD (Table 1) was constructed via introducing pBBRKlipADTMD into the mutant strain Pf0617 (Table 1) by means of triparental mating with pRK2073 as a helper plasmid [9]. To study the effect of TMD on the protein localization, the triparental mating with pRK2073 as a helper plasmid was used to respectively introduce pBBR003 and pBBR003TMD into the mutant strain Pf0617 to construct b-galactosidase reporter strains Pf003 and Pf003TMD (Table 1). 2.4. Preparation of whole-cell, lysis supernatant and lysis pellet samples Bacterial cultures of P. protegens were grown in LB medium overnight. After that, 50 mL cultures were colleted by centrifugation at 5000 rpm (rotor model: F15-6100y) for 5 min at 4 C (Thermo Scientific Sorvall ST 16R). The cell pellets were washed three times with 0.9% (w/v) NaCl solution, subsequently resuspended in 5 mL 0.9% NaCl solution, and the resuspended cells were the whole-cell samples. After sonicating for 10 min, 5 mL resuspended cells were centrifuged at 4000 rpm for 10 min at 4 C to remove the unbroken cells, subsequently, lysis supernatants and lysis pellets were separated by centrifugation at 12,000 rpm for 30 min at 4 C. The lysis supernatant samples (soluble fraction) were transferred to clean tubes, and the lysis pellet samples (membrane fraction) were washed three times with 0.9% NaCl solution and resuspended in 5 mL 0.9% NaCl solution. 2.5. Lipase activity assay Lipase activity of the whole-cell, lysis supernatant and lysis pellet samples towards the p-nitrophenyl caprylate was performed by a spectrophotometric method as described previously [7]. Lipase activity of the recombinant LipA and LipADTMD on the p-nitrophenyl esters, including p-nitrophenyl acetate, p-nitrophenyl butyrate, p-nitrophenyl caprylate, p-nitrophenyl decanoate, was measured with some modifications. The reaction system consisted of 1.48 mL TriseHCl buffer (50 mM, pH 9.0) and 10 mL pNP ester. The mixtures were pre-incubated at 55 C for 5 min, and 10 mL protein samples were subsequently added. Protein samples were substituted with equivalent amounts of NTA-200 buffer (20 mM TriseHCl, 0.3 M NaCl, 200 mM imidazole, pH 8.0) when the blank reaction was performed. One unit of lipase activity was defined as the amount of enzyme needed to release 1 mmol of p-nitrophenol per minute. Furthermore, the recombinant LipA and LipADTMD were spotted on the tributyrin plate (water, 1% (v/v) tributyrin, 1.5% (w/v) agar) to qualitatively determine the lipase activity. 2.6. Expression and purification of recombinant LipA and LipADTMD The recombinant plasmids pET-lipA and pET-lipADTMD were respectively transformed with E. coli BL21(DE3) for protein expression. The cultures were supplemented with 0.05 mM IPTG when the recombinant strains grew to OD600 of 1.0e1.2 in LB medium at 37 C, and then cultivation was continued for 12 h at 20 C. Harvested cells were suspended in lysis buffer (20 mM TriseHCl, 0.3 M NaCl, pH 8.0) and lysed by sonication. The lysate was cleared by centrifugation at 12,000 rpm for 1 h at 4 C, and the supernatant
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was purified using a Ni Sepharose™ 6 Fast Flow resin (GE healthcare, USA). The target proteins were finally eluted with the elution buffer NTA-200. The Bradford method and 12% SDS-PAGE were used to estimate the concentration and molecular mass of proteins, respectively, and the SDS-PAGE gel was stained with Coomassie Brilliant Blue R-250. 2.7. Construction of 3D structures of LipA and LipADTMD The 3D structures of LipA and LipADTMD were constructed by Rosetta 3.5 [12]. Rosetta provided both ab initio and comparative models of protein domains using the Rosetta fragment insertion method. Domains without a detectable PDB homolog were modeled with the Rosetta de novo protocol. Comparative models were built from parent PDBs detected by PSI-BLAST or HHSEARCH and aligned by HHSEARCH and SPARKS. Loop regions were assembled from fragments and optimized to fit the aligned template structure. The qualities of each modeled 3D structures were checked using MolProbity [13]. 2.8. b-galactosidase activity assay
b-galactosidase activity from P. protegens cells carrying the different reporter plasmids (Table 1) on ONPG was determined by a spectrophotometric method as described previously [14], normalized to the OD600 of the bacterial culture and expressed in Miller units. 3. Results 3.1. TMD loses its membrane localization function in LipA We had previously shown that LipA was not secreted into the extracytoplasmic space [7], suggesting that it was a membrane protein thanks to its N-terminal TMD. To verify this proposal, the lipase activities of strains Pf0617 and Pf0617lipA in the whole-cell, lysis supernatant and lysis pellet were measured and respectively compared with strains Pf-5 and Pf0617K. Compared with Pf-5, the lipase activities of Pf0617 in whole-cell and lysis supernatant notably decreased by 23% and 17%, respectively, while the lipase activity in lysis pellet lowered by 10% (Fig. 1). As shown in Fig. 1, the lipase activities of Pf0617lipA in the whole-cell, lysis supernatant and lysis pellet were respectively 187%, 176% and 108% of that of Pf0617K, and no significant difference in the lysis pellet was detected between Pf0617lipA and Pf0617K. In addition, lipA expressed in lipase-free host E. coli Top10 showed that the lipase activities of whole-cell and lysis supernatant were respectively 11.9 ± 1.2 and 12.4 ± 0.4 U/mL$OD600 and the difference was not significant, while lysis pellet did not have lipase activity. 3.2. TMD can restore its membrane localization function in heterologous LacZ Although TMD was deprived of its membrane localization function in LipA, might it restore this function in heterologous proteins? To investigate the role of TMD in membrane localization of heterologous proteins, b-galactosidase reporter strains Pf003 and Pf003TMD were constructed. As shown in Fig. 2A, the bgalactosidase activities of Pf003 in lysis supernatant and lysis pellet were respectively 98% and 7% of that of Pf003 in whole-cell, and no significant difference was detected between lysis supernatant and whole-cell. For Pf003TMD, the b-galactosidase activities in lysis supernatant and lysis pellet were respectively 12% and 83% of that in whole-cell, and their differences were significant.
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Fig. 1. Influence of TMD on the membrane localization of LipA. Pf-5, wild-type; Pf0617, markerless deletion mutant of coding region of lipA; Pf0617K, Pf0617 with pBBRKm; Pf0617lipA, Pf0617 with pBBRKlipA. The height of each bar and the error bars show the mean and standard deviation, respectively, from three independent experiments. *P < 0.05, compared with control.
3.3. TMD plays an indispensable role for the catalytic function of LipA Although TMD has not the membrane localization function in LipA, we guess it might have another role for the function of LipA. To examine the specific role of TMD in LipA, the markerless deletion mutant of TMD sequence of lipA was constructed. After successfully constructing the mutant Pf0617TMD, its lipase activities in the whole-cell, lysis supernatant and lysis pellet were determined. The lipase activity of Pf0617TMD remarkably decreased by 20% in whole-cell, significantly reduced by 16% in lysis supernatant, lowered by 6% in lysis pellet, respectively, compared with that of Pf-5 (Fig. 3A). Subsequently, the lipADTMD expressed in the Pf0617 (Pf0617lipADTMD) was investigated along with Pf-5K as the control. As shown in Fig. 3A, the lipase activities of Pf0617lipADTMD in the whole-cell, lysis supernatant and lysis pellet like those of Pf0617TMD, showed 77%, 82% and 90% of that of Pf-5K, respectively, but the lipase activity between Pf0617lipADTMD and Pf-5K in lysis pellet had no significant difference. LipA and LipADTMD were expressed in E. coli BL21(DE3) and purified by affinity chromatography on a NieNTA column to separately study the role of TMD in LipA. The molecular mass of the recombinant LipA and LipADTMD was respectively estimated to be 34 and 32 kDa by SDS-PAGE gel (Fig. 3B), which was consistent with the theoretically calculated molecular mass. After that, the lipase activity of the two proteins was measured by the tributyrin plate assay and the spectrophotometric method. The results showed that LipADTMD could not catalyze the hydrolysis of tributyrin and pnitrophenyl decanoate but was able to hydrolyze 28% p-nitrophenyl acetate, 11% p-nitrophenyl butyrate and 8% p-nitrophenyl caprylate compared with LipA (Fig. 3C and D). 3.4. TMD affects the conformation of a-helical lid of LipA To determine how TMD affects the catalytic function of LipA, the 3D structures of LipA and LipADTMD were constructed. The 3D structures of LipA and LipADTMD most resembled 1EX9 and 4GW3, respectively, and their remark scores were 0.83 and 0.95. Taken as a whole, the 3D structure of LipA was similar to that of LipADTMD
Fig. 2. Effect of TMD on the subcellular localization of heterologous LacZ. (A) Pf003, Pf0617 with b-galactosidase reporter plasmid pBBR003; Pf003TMD, Pf0617 with bgalactosidase reporter plasmid pBBR003TMD. The height of each bar and the error bars show the mean and standard deviation, respectively, from three independent experiments. *P < 0.05, **P < 0.01, compared with control. (B) Schematic representation of the pBBR003 (i) and pBBR003TMD (ii) used in this study.
except for the a-helical lid (Fig. 4). It was worth noting that TMD was embedded in the interior of 3D structure of LipA and showed the structure of b-strand (Fig. 4A). As shown in Fig. 4B, the deletion of TMD mainly changed the position of a5 helix, and hence led to the a-helical lid to be in a closed conformation, blocking substrates assess into the catalytic pocket. 4. Discussion Generally, subfamily I.1 lipases secreted by the type II secretion pathway have an N-terminal signal peptide [4,5]. However, P. protegens Pf-5 LipA lacks a signal peptide but has a TMD at its Nterminal, and it is not secreted into the extracytoplasmic space [7]. These suggest that LipA is a special member of subfamily I.1 lipases, its N-terminal TMD may have a specific role for its catalytic function. LipA has been proven not to be an extracellular lipase [7], which suggests that it is a membrane protein as a result of its N-terminal TMD. To verify this suggestion, lipase activities of Pf-5, Pf0617, Pf0617K and Pf0617lipA in the whole-cell, lysis supernatant and lysis pellet were determined. However, the results showed that the deletion or overexpression of lipA significantly changed the lipase activities in the whole-cell and lysis supernatant while in no
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Fig. 3. Impact of TMD on the catalytic function of LipA. (A) Effect of TMD sequence deletion on the lipase activity of whole-cell, lysis supernatant and lysis pellet. Pf-5, wild-type; Pf0617TMD, markerless deletion mutant of TMD sequence of lipA; Pf-5K, Pf-5 with pBBRKm; Pf0617lipADTMD, Pf0617 with pBBRKlipADTMD. (B) SDS-PAGE analysis of the recombinant LipA and LipADTMD. M, protein marker; Lane 1, the supernatant of E. coli BL21(DE3) harboring pET-28a eluted by NTA-200; Lane 2, the recombinant LipA eluted by NTA-200; Lane 3, the recombinant LipADTMD eluted by NTA-200. (C) Tributyrin plate assay of the recombinant LipA and LipADTMD. (D) Activities of the recombinant LipA and LipADTMD towards p-nitrophenyl esters. C2, acetate; C4, butyrate; C8, caprylate; C10, decanoate. The height of each bar and the error bars show the mean and standard deviation, respectively, from three independent experiments. *P < 0.05, compared with control.
significant way altered the lipase activity in the lysis pellet (Fig. 1), which denied this proposal. On the contrary, LipA was observed to be distributed in the cytoplasm, manifesting that TMD lost its membrane localization function in LipA. Moreover, lipA expressed in lipase-free host E. coli further confirmed this phenomenon.
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Although TMD loses its membrane localization function in its cognate LipA, it can restore this function in heterologous proteins. The b-galactosidase reporter assay showed that LacZ with TMD was mostly localized at the cytoplasmic membrane while LacZ without TMD was almost located in the cytoplasm (Fig. 2A). These confirmed that TMD did affect the subcellular localization of LacZ, verifying the prediction by the TMHMM 2.0, which might be due to its hydrophobicity and its heterology to LacZ leading to TMD located in the surface of the heterologous protein. Considering its specificity in LipA, TMD may play an important role for the catalytic function of LipA. To explore this specific role, the markerless deletion mutant of TMD sequence of lipA was constructed by a gene replacement system with the plasmid pJQ200SK, which utilized a double-crossover recombination event to knockout target sequence and clear the plasmid pJQ200SK by means of the Bacillus subtilis sacB (levansucrase) for counterselection [16]. Subsequently, lipase activities of Pf-5 and Pf0617TMD in the wholecell, lysis supernatant and lysis pellet were detected. The deletion of TMD sequence changed the lipase activities in the whole-cell, lysis supernatant and lysis pellet in a manner similar to the deletion of lipA (Figs. 1 and 3A), which indicated that the deletion of TMD sequence made LipA inactivate. In addition, pBBRKlipADTMD expressed in Pf0617 further confirmed the above conclusion (Fig. 3A). However, both Pf0617 and Pf0617TMD still remained 80% or so of the total lipase activity, which was due to Pf-5 containing another intracellular lipase (KEGG entry number PFL_2722, GenBank accession number YP_259829) and other intracellular enzymes associated with the lipase activity. To separately study the role of TMD in LipA, LipA and LipADTMD were expressed and purified. Lipase activity assays showed that LipADTMD was not able to hydrolyze tributyrin or p-nitrophenyl decanoate (Fig. 3C and D), these further proved the role of the TMD in LipA. However, LipADTMD could partly catalyze the hydrolysis of p-nitrophenyl ester whose carbon chain length was less than ten compared with LipA (Fig. 3D), which may be because of the deletion of TMD sequence changing the conformation of LipADTMD in a fine-tuning but important manner. Although other studies had shown that the N-terminal domain could also affect the function of lipases, its deletion did not inactivate the lipases [17e19]. Therefore, the 3D structures of LipA and LipADTMD were modeled by Rosetta 3.5 to further investigate how TMD influences the catalytic function of LipA. Rosetta 3.5 has been successfully used to model the 3D structures of other proteins [20,21], and it produced the closest pose to the target crystal structure [20]. The results of 3D structures showed that the deletion of TMD mainly affected the conformation of a-helical lid through the displacement of a5 helix that made the lid be in a closed conformation to block substrates access into the catalytic pocket (Fig. 4A and B), which was consistent with LipADTMD having partial activity toward pnitrophenyl esters with short chain fatty acids (C2eC8) (Fig. 3D). This indicates that the flexibility of the lid mainly results from motions of a5 helix, as was found in the case of other lipases from crystal structure or molecular dynamics simulations [22,23], further confirming the authenticity and reasonability of the modeling by Rosetta 3.5. Thanks to the fact that LipA lacks its cognate lipase-specific foldase, TMD might act as a kind of intramolecular chaperone like the b-roll motif of subfamily I.3 lipases contributing to the folding of the catalytic domain [24-26]. In addition, the deletion of the b-roll motif also inactivated subfamily I.3 lipases [24]. Surprisingly, TMD was embedded inside LipA and was modeled as a b-strand (Fig. 4A), being inconsistent with the prediction of the TMHMM 2.0, which further confirmed its specificity in LipA (Fig. 1). Many studies have demonstrated that identical short peptide sequences in unrelated proteins could have different conformations and a-helices could also be converted into b-strands
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Fig. 4. 3D structures of LipA (A) and LipADTMD (B). The residues forming the catalytic triad Ser-His-Asp were shown as cylinders; the position of the a-helical lid was highlighted with the label LID and numbered as the lipase from P. aeruginosa PAO1 (PDB ID：1EX9) [15]; LipA was shown as ribbons and colored in green, and TMD was colored in magenta; LipADTMD was shown as ribbons and colored in light blue.
[27e29]. Thus, it is possible that TMD was b-strand in LipA but ahelix in LacZ. In conclusion, this study demonstrated that in LipA its N-terminal TMD lost membrane localization function but played an indispensable role for its catalytic function, and probably acted as a kind of intramolecular chaperone helping the a-helical lid to form an open conformation mediated by a5 helix. This is the first to notify the intramolecular chaperone of a subfamily I.1 lipase. We found that such N-terminal TMD also exists in many other lipases by BLAST search in NCBI, therefore, similar studies should also be done to verify whether their TMD has the function of intramolecular chaperone in these lipases in further. Conflict of interest None. Acknowledgments The authors acknowledge the financial support of the National Natural Science Foundation of China (NSFC) (Nos. 31070089, 31170078 and J1103514), the National High Technology Research and Development Program of P. R. China (863 Program) (No. 2011AA02A204), and the Innovation Foundation of Shenzhen Government (JCYJ20120831111657864). Many thanks are due to Dr. Jingyuan Chen (Hubei Academy of Forestry, Wuhan, China) for kindly presenting P. protegens Pf-5. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biochi.2014.07.007. References [1] A. Ramette, M. Frapolli, M. Fischer-Le Saux, C. Gruffaz, J.M. Meyer, G. Defago, L. Sutra, Y. Moenne-Loccoz, Pseudomonas protegens sp. nov., widespread plantprotecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin, Syst. Appl. Microbiol. 34 (2011) 180e188.
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Research paper
N-terminal transmembrane domain of lipase LipA from Pseudomonas protegens Pf-5: A must for its efficient folding into an active conformation Daiming Zha, Huaidong Zhang, Houjin Zhang, Li Xu, Yunjun Yan* Key Laboratory of Molecular Biophysics, Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 May 2014 Accepted 8 July 2014 Available online 16 July 2014
LipA from Pseudomonas protegens Pf-5 has been proven not to be secreted into the extracytoplasmic space, proposing that it is a membrane protein in virtue of its N-terminal transmembrane domain predicted by the TMHMM 2.0. However, LipA was confirmed to be an intracellular protein through determining the effects of lipA deletion or overexpression on the lipase activities in the whole-cell, lysis supernatant and lysis pellet, even through its transmembrane domain being able to make heterologous LacZ locate on the cytoplasmic membrane via construction of b-galactosidase reporter strains. Subsequently, lipase activity assays showed that the transmembrane domain played an indispensable role for the catalytic function of LipA through construction of the markerless deletion mutant of transmembrane domain sequence of lipA and the expression and purification of LipA and LipADTMD. To further investigate why the transmembrane domain lost its membrane localization function and significantly affected the catalytic function of LipA, the 3D structures of LipA and LipADTMD were constructed. The results indicated that the transmembrane domain, located in the interior of LipA, helped the a-helical lid to form an open conformation by the mediation of a5 helix. It seems to act as a kind of intramolecular chaperone like the b-roll motif of subfamily I.3 lipases, which is novel and is the first to notify the intramolecular chaperone of a subfamily I.1 lipase. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Pseudomonas protegens lipase Transmembrane domain Intramolecular chaperone 3D structure
1. Introduction The rhizosphere inhabitant Pseudomonas protegens Pf-5 (previously called Pseudomonas fluorescens Pf-5), isolated from the soil in College Station, Texas in the 1979, is a well-known biocontrol bacterium that can suppress plant diseases by producing secondary metabolites to inhibit various pathogenic bacteria, fungi and oomycetes [1,2]. Though Pf-5 has been widely studied thanks to the biocontrol properties, very little literature is available on its lipases. LipA (KEGG entry number PFL_0617, GenBank accession number YP_257759) is a member of subfamily I.1 lipases secreted through the type II secretion pathway and its recombinant protein (i.e., rPFL from P. fluorescens JCM5963) has been reported [3,4]. Most of the known subfamily I.1 lipases contain an N-terminal signal peptide, which directs their secretion into the extracytoplasmic space through the Sec machinery. For lipases from Gram-positive
* Corresponding author. Tel.: þ86 (0)27 87792214 12; fax: þ86 (0)27 87792213. E-mail addresses: [email protected], [email protected] (Y. Yan). http://dx.doi.org/10.1016/j.biochi.2014.07.007 0300-9084/© 2014 Elsevier Masson SAS. All rights reserved.
bacteria, this mechanism is sufficient, whereas these secreted by Gram-negative bacteria must cross a second barrier constituted by the outer membrane [4,5]. After being secreted through the inner membrane, lipases from Gram-negative bacteria fold in the periplasm into an enzymatically active conformation by means of their cognate lipase-specific foldases called Lif proteins. Subsequently, they are transported through the outer membrane via the secreton including up to 14 different proteins [4,6]. However, LipA lacks a typical N-terminal signal peptide but has a transmembrane domain (TMD) (aa 9 to 31 relative to the starting amino acid) predicted by the TMHMM Server v. 2.0 program (http://www.cbs.dtu.dk/ services/TMHMM/) and is not secreted into the extracytoplasmic space [7]. In addition, its cognate lipase-specific foldase is not found in the Pf-5 genome. These different features suggest that LipA is not a typical subfamily I.1 lipase and its N-terminal TMD may play an important role for its enzymatic function. In this study, we confirmed the role of TMD in LipA by measuring the lipase activities of various strains in the whole-cell, lysis supernatant and lysis pellet and detecting the lipase activities of LipA and LipADTMD towards tributyrin and p-nitrophenyl esters,
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and further proposed the mechanism of TMD inactivating LipA by modeling the 3D structures of LipA and LipADTMD. In addition, we also investigated the effect of TMD on the subcellular localization of heterologous LacZ. 2. Materials and methods 2.1. Strains, plasmids, culture conditions and general methods The bacterial strains and plasmids used were described in Table 1. P. protegens (28 C) and Escherichia coli (37 C) strains were propagated in liquid or solid (1.5% w/v agar) LB medium. Antibiotics were used at the following concentrations: ampicillin (Ap) (100 mg/ mL), gentamicin (Gm) (50 mg/mL), and kanamycin (Km) (50 mg/mL) for P. protegens, and spectinomycin (Sp) (100 mg/mL), gentamicin (50 mg/mL), and kanamycin (50 mg/mL) for E. coli. PrimeSTAR HS DNA Polymerase, restriction enzymes, and DNA ligation kit ver. 2.0 were purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. (Dalian, China). Genomic DNA extraction, plasmid preparation, and DNA gel extraction were carried out with commercial kits (Omega Bio-Tek, Doraville, GA) according to the manufacturer's protocols. Genomic DNA of P. protegens Pf-5 was used as the template in all PCR amplification reactions. Oligonucleotide primers were synthesized from Wuhan Anygene Biological Technology Co., Ltd. (Wuhan, China). DNA sequencing was performed by Shanghai Sunny Biotechnology Co., Ltd. (Shanghai, China). All other routine manipulations were performed by standard methods [8].
SphI/TMDD-L-XbaI (Table S1). SpeI-SphI digested TMDU, SphI-XbaI digested TMDD, and SpeI-XbaI digested pJQ200SK were ligated, and then followed by E. coli Top10 transformation. Transformants were screened on LB plates containing 50 mg/mL gentamicin and further verified by colony PCR. Then, plasmid DNA was isolated and sequenced. The recombinant plasmid was named as pJQlipADTMD (Table 1). In order to construct the lipADTMD expression plasmids, PCR amplicon encoding the lipADTMD (810-bp) was amplified using primers lipADTMDF-BamHI/lipAR-HindIII (Table S1). Then, BamHIHindIII digested amplicon was cloned into the same digested pBBRKm, creating the recombinant plasmid pBBRKlipADTMD (Table 1). The recombinant plasmids pET-lipA and pET-lipADTMD (Table 1) were constructed by PCR amplification using primers lipAF-NdeI/lipAR-HindIII and lipADTMDF-NdeI/lipAR-HindIII (Table S1) and cloning 903-bp and 810-bp DNA fragments into the plasmid pET-28a digested with NdeI and HindIII, respectively. b-galactosidase reporter plasmid pBBR003 (Table 1) was generated by PCR amplification using primers lipA-PF-KpnI/lipA0 PR-HindIII (Table S1) and cloning a 631-bp DNA fragment encompassing the promoter and the first six codons of lipA into the plasmid pBBR001 (Table 1) digested with KpnI and HindIII. Similarly, b-galactosidase reporter plasmid pBBR003TMD (Table 1) was constructed by PCR amplification using primers lipA-PF-KpnI/lipATMDR-HindIII (Table S1) and cloning a 712-bp DNA fragment encompassing the promoter and TMD sequence of lipA into the plasmid pBBR001 digested with KpnI and HindIII.
2.2. Construction of plasmids 2.3. Construction of strains To construct the markerless deletion mutation cassette of TMD sequence of lipA, homology arms of 618-bp upstream (bp 615 to 2 relative to the translational start site) and 410-bp downstream (bp 92 to 501 relative to the translational start site) were respectively amplified using primers TMDU-U-SpeI/TMDU-L-SphI and TMDD-U-
The markerless deletion mutant of TMD sequence of lipA was constructed via a double-crossover recombination event by means of a gene replacement system with the plasmid pJQ200SK [10,11]. Single- and double-crossover recombination events were verified
Table 1 Strains and plasmids used in this study. Strain or plasmid E. coli Top10 BL21(DE3) P. protegens Pf-5 Pf0617 Pf0617TMD Pf-5K Pf0617K Pf0617lipA Pf0617lipADTMD Pf003 Pf003TMD Plasmid pRK2073 pJQ200SK pJQlipADTMD pBBRKm pBBRKlipA pBBRKlipADTMD pET-lipA pET-lipADTMD pBBR001 pBBR003 pBBR003TMD
Description
Reference or source
mcrA D(mrr-hsdRMS-mcrBC)F80lacZDM15DlacX74 recA1 araD139D(ara-leu)7697galU galK rpsL (StrR) endA1 nupG F-, ompT hsdSB(rB-, mB-) dcm gal(DE3)
Invitrogen
Rhizosphere isolate; Pltþ, Apr DlipA derivative of Pf-5 Deletion mutant of transmembrane domain sequence of lipA Pf-5 with pBBRKm; Kmr Pf0617 with pBBRKm; Kmr Pf0617 with pBBRKlipA; Kmr Pf0617 with pBBRKlipADTMD; Kmr Pf0617 with pBBR003; Gmr Pf0617 with pBBR003TMD; Gmr
ATCC BAA-477 [7] This study [7] This study [7] This study This study This study
Helper plasmid for triparental mating; Spr Suicide vector with sacB counter-selectable marker used for homologous recombination; Gmr pJQ200SK carrying a 1.028 kb BamHI-XbaI insert with a deletion in the transmembrane domain sequence of lipA; Gmr Broad-host-range vector derivative of pBBR1MCS-5; lacIq-Plac, Kmr pBBRKm with a 904 bp BamHI-HindIII fragment harboring the coding region of lipA; Kmr pBBRKm carrying a 810 bp BamHI-HindIII insert with a deletion in transmembrane domain sequence of lipA; Kmr pET-28a with a 903 bp BamHI-HindIII fragment harboring the coding region of lipA; Kmr pET-28a carrying a 810 bp BamHI-HindIII insert with a deletion in transmembrane domain sequence of lipA; Kmr pBBR1MCS-5 derivative with a translational ‘lacZ fusion; Gmr pBBR001 derivative with a translational lipA’-‘lacZ fusion; Gmr pBBR001 derivative with a translational lipA-TMD-‘lacZ fusion; Gmr
[9] [10] This study
Novagen
[7] [7] This study This study This study This study This study This study
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by colony PCR using primers lipA-22U/lipA223L (Table S1). Furthermore, sequencing and gentamicin resistance assay for double-crossover colonies were carried out to determine the loss of TMD sequence and plasmid pJQ200SK. The verified doublecrossover colony, i.e., markerless deletion mutant of TMD sequence of lipA, was named as Pf0617TMD (Table 1). To further evaluate the function of LipADTMD, the strain Pf0617lipADTMD (Table 1) was constructed via introducing pBBRKlipADTMD into the mutant strain Pf0617 (Table 1) by means of triparental mating with pRK2073 as a helper plasmid [9]. To study the effect of TMD on the protein localization, the triparental mating with pRK2073 as a helper plasmid was used to respectively introduce pBBR003 and pBBR003TMD into the mutant strain Pf0617 to construct b-galactosidase reporter strains Pf003 and Pf003TMD (Table 1). 2.4. Preparation of whole-cell, lysis supernatant and lysis pellet samples Bacterial cultures of P. protegens were grown in LB medium overnight. After that, 50 mL cultures were colleted by centrifugation at 5000 rpm (rotor model: F15-6100y) for 5 min at 4 C (Thermo Scientific Sorvall ST 16R). The cell pellets were washed three times with 0.9% (w/v) NaCl solution, subsequently resuspended in 5 mL 0.9% NaCl solution, and the resuspended cells were the whole-cell samples. After sonicating for 10 min, 5 mL resuspended cells were centrifuged at 4000 rpm for 10 min at 4 C to remove the unbroken cells, subsequently, lysis supernatants and lysis pellets were separated by centrifugation at 12,000 rpm for 30 min at 4 C. The lysis supernatant samples (soluble fraction) were transferred to clean tubes, and the lysis pellet samples (membrane fraction) were washed three times with 0.9% NaCl solution and resuspended in 5 mL 0.9% NaCl solution. 2.5. Lipase activity assay Lipase activity of the whole-cell, lysis supernatant and lysis pellet samples towards the p-nitrophenyl caprylate was performed by a spectrophotometric method as described previously [7]. Lipase activity of the recombinant LipA and LipADTMD on the p-nitrophenyl esters, including p-nitrophenyl acetate, p-nitrophenyl butyrate, p-nitrophenyl caprylate, p-nitrophenyl decanoate, was measured with some modifications. The reaction system consisted of 1.48 mL TriseHCl buffer (50 mM, pH 9.0) and 10 mL pNP ester. The mixtures were pre-incubated at 55 C for 5 min, and 10 mL protein samples were subsequently added. Protein samples were substituted with equivalent amounts of NTA-200 buffer (20 mM TriseHCl, 0.3 M NaCl, 200 mM imidazole, pH 8.0) when the blank reaction was performed. One unit of lipase activity was defined as the amount of enzyme needed to release 1 mmol of p-nitrophenol per minute. Furthermore, the recombinant LipA and LipADTMD were spotted on the tributyrin plate (water, 1% (v/v) tributyrin, 1.5% (w/v) agar) to qualitatively determine the lipase activity. 2.6. Expression and purification of recombinant LipA and LipADTMD The recombinant plasmids pET-lipA and pET-lipADTMD were respectively transformed with E. coli BL21(DE3) for protein expression. The cultures were supplemented with 0.05 mM IPTG when the recombinant strains grew to OD600 of 1.0e1.2 in LB medium at 37 C, and then cultivation was continued for 12 h at 20 C. Harvested cells were suspended in lysis buffer (20 mM TriseHCl, 0.3 M NaCl, pH 8.0) and lysed by sonication. The lysate was cleared by centrifugation at 12,000 rpm for 1 h at 4 C, and the supernatant
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was purified using a Ni Sepharose™ 6 Fast Flow resin (GE healthcare, USA). The target proteins were finally eluted with the elution buffer NTA-200. The Bradford method and 12% SDS-PAGE were used to estimate the concentration and molecular mass of proteins, respectively, and the SDS-PAGE gel was stained with Coomassie Brilliant Blue R-250. 2.7. Construction of 3D structures of LipA and LipADTMD The 3D structures of LipA and LipADTMD were constructed by Rosetta 3.5 [12]. Rosetta provided both ab initio and comparative models of protein domains using the Rosetta fragment insertion method. Domains without a detectable PDB homolog were modeled with the Rosetta de novo protocol. Comparative models were built from parent PDBs detected by PSI-BLAST or HHSEARCH and aligned by HHSEARCH and SPARKS. Loop regions were assembled from fragments and optimized to fit the aligned template structure. The qualities of each modeled 3D structures were checked using MolProbity [13]. 2.8. b-galactosidase activity assay
b-galactosidase activity from P. protegens cells carrying the different reporter plasmids (Table 1) on ONPG was determined by a spectrophotometric method as described previously [14], normalized to the OD600 of the bacterial culture and expressed in Miller units. 3. Results 3.1. TMD loses its membrane localization function in LipA We had previously shown that LipA was not secreted into the extracytoplasmic space [7], suggesting that it was a membrane protein thanks to its N-terminal TMD. To verify this proposal, the lipase activities of strains Pf0617 and Pf0617lipA in the whole-cell, lysis supernatant and lysis pellet were measured and respectively compared with strains Pf-5 and Pf0617K. Compared with Pf-5, the lipase activities of Pf0617 in whole-cell and lysis supernatant notably decreased by 23% and 17%, respectively, while the lipase activity in lysis pellet lowered by 10% (Fig. 1). As shown in Fig. 1, the lipase activities of Pf0617lipA in the whole-cell, lysis supernatant and lysis pellet were respectively 187%, 176% and 108% of that of Pf0617K, and no significant difference in the lysis pellet was detected between Pf0617lipA and Pf0617K. In addition, lipA expressed in lipase-free host E. coli Top10 showed that the lipase activities of whole-cell and lysis supernatant were respectively 11.9 ± 1.2 and 12.4 ± 0.4 U/mL$OD600 and the difference was not significant, while lysis pellet did not have lipase activity. 3.2. TMD can restore its membrane localization function in heterologous LacZ Although TMD was deprived of its membrane localization function in LipA, might it restore this function in heterologous proteins? To investigate the role of TMD in membrane localization of heterologous proteins, b-galactosidase reporter strains Pf003 and Pf003TMD were constructed. As shown in Fig. 2A, the bgalactosidase activities of Pf003 in lysis supernatant and lysis pellet were respectively 98% and 7% of that of Pf003 in whole-cell, and no significant difference was detected between lysis supernatant and whole-cell. For Pf003TMD, the b-galactosidase activities in lysis supernatant and lysis pellet were respectively 12% and 83% of that in whole-cell, and their differences were significant.
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Fig. 1. Influence of TMD on the membrane localization of LipA. Pf-5, wild-type; Pf0617, markerless deletion mutant of coding region of lipA; Pf0617K, Pf0617 with pBBRKm; Pf0617lipA, Pf0617 with pBBRKlipA. The height of each bar and the error bars show the mean and standard deviation, respectively, from three independent experiments. *P < 0.05, compared with control.
3.3. TMD plays an indispensable role for the catalytic function of LipA Although TMD has not the membrane localization function in LipA, we guess it might have another role for the function of LipA. To examine the specific role of TMD in LipA, the markerless deletion mutant of TMD sequence of lipA was constructed. After successfully constructing the mutant Pf0617TMD, its lipase activities in the whole-cell, lysis supernatant and lysis pellet were determined. The lipase activity of Pf0617TMD remarkably decreased by 20% in whole-cell, significantly reduced by 16% in lysis supernatant, lowered by 6% in lysis pellet, respectively, compared with that of Pf-5 (Fig. 3A). Subsequently, the lipADTMD expressed in the Pf0617 (Pf0617lipADTMD) was investigated along with Pf-5K as the control. As shown in Fig. 3A, the lipase activities of Pf0617lipADTMD in the whole-cell, lysis supernatant and lysis pellet like those of Pf0617TMD, showed 77%, 82% and 90% of that of Pf-5K, respectively, but the lipase activity between Pf0617lipADTMD and Pf-5K in lysis pellet had no significant difference. LipA and LipADTMD were expressed in E. coli BL21(DE3) and purified by affinity chromatography on a NieNTA column to separately study the role of TMD in LipA. The molecular mass of the recombinant LipA and LipADTMD was respectively estimated to be 34 and 32 kDa by SDS-PAGE gel (Fig. 3B), which was consistent with the theoretically calculated molecular mass. After that, the lipase activity of the two proteins was measured by the tributyrin plate assay and the spectrophotometric method. The results showed that LipADTMD could not catalyze the hydrolysis of tributyrin and pnitrophenyl decanoate but was able to hydrolyze 28% p-nitrophenyl acetate, 11% p-nitrophenyl butyrate and 8% p-nitrophenyl caprylate compared with LipA (Fig. 3C and D). 3.4. TMD affects the conformation of a-helical lid of LipA To determine how TMD affects the catalytic function of LipA, the 3D structures of LipA and LipADTMD were constructed. The 3D structures of LipA and LipADTMD most resembled 1EX9 and 4GW3, respectively, and their remark scores were 0.83 and 0.95. Taken as a whole, the 3D structure of LipA was similar to that of LipADTMD
Fig. 2. Effect of TMD on the subcellular localization of heterologous LacZ. (A) Pf003, Pf0617 with b-galactosidase reporter plasmid pBBR003; Pf003TMD, Pf0617 with bgalactosidase reporter plasmid pBBR003TMD. The height of each bar and the error bars show the mean and standard deviation, respectively, from three independent experiments. *P < 0.05, **P < 0.01, compared with control. (B) Schematic representation of the pBBR003 (i) and pBBR003TMD (ii) used in this study.
except for the a-helical lid (Fig. 4). It was worth noting that TMD was embedded in the interior of 3D structure of LipA and showed the structure of b-strand (Fig. 4A). As shown in Fig. 4B, the deletion of TMD mainly changed the position of a5 helix, and hence led to the a-helical lid to be in a closed conformation, blocking substrates assess into the catalytic pocket. 4. Discussion Generally, subfamily I.1 lipases secreted by the type II secretion pathway have an N-terminal signal peptide [4,5]. However, P. protegens Pf-5 LipA lacks a signal peptide but has a TMD at its Nterminal, and it is not secreted into the extracytoplasmic space [7]. These suggest that LipA is a special member of subfamily I.1 lipases, its N-terminal TMD may have a specific role for its catalytic function. LipA has been proven not to be an extracellular lipase [7], which suggests that it is a membrane protein as a result of its N-terminal TMD. To verify this suggestion, lipase activities of Pf-5, Pf0617, Pf0617K and Pf0617lipA in the whole-cell, lysis supernatant and lysis pellet were determined. However, the results showed that the deletion or overexpression of lipA significantly changed the lipase activities in the whole-cell and lysis supernatant while in no
D. Zha et al. / Biochimie 105 (2014) 165e171
Fig. 3. Impact of TMD on the catalytic function of LipA. (A) Effect of TMD sequence deletion on the lipase activity of whole-cell, lysis supernatant and lysis pellet. Pf-5, wild-type; Pf0617TMD, markerless deletion mutant of TMD sequence of lipA; Pf-5K, Pf-5 with pBBRKm; Pf0617lipADTMD, Pf0617 with pBBRKlipADTMD. (B) SDS-PAGE analysis of the recombinant LipA and LipADTMD. M, protein marker; Lane 1, the supernatant of E. coli BL21(DE3) harboring pET-28a eluted by NTA-200; Lane 2, the recombinant LipA eluted by NTA-200; Lane 3, the recombinant LipADTMD eluted by NTA-200. (C) Tributyrin plate assay of the recombinant LipA and LipADTMD. (D) Activities of the recombinant LipA and LipADTMD towards p-nitrophenyl esters. C2, acetate; C4, butyrate; C8, caprylate; C10, decanoate. The height of each bar and the error bars show the mean and standard deviation, respectively, from three independent experiments. *P < 0.05, compared with control.
significant way altered the lipase activity in the lysis pellet (Fig. 1), which denied this proposal. On the contrary, LipA was observed to be distributed in the cytoplasm, manifesting that TMD lost its membrane localization function in LipA. Moreover, lipA expressed in lipase-free host E. coli further confirmed this phenomenon.
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Although TMD loses its membrane localization function in its cognate LipA, it can restore this function in heterologous proteins. The b-galactosidase reporter assay showed that LacZ with TMD was mostly localized at the cytoplasmic membrane while LacZ without TMD was almost located in the cytoplasm (Fig. 2A). These confirmed that TMD did affect the subcellular localization of LacZ, verifying the prediction by the TMHMM 2.0, which might be due to its hydrophobicity and its heterology to LacZ leading to TMD located in the surface of the heterologous protein. Considering its specificity in LipA, TMD may play an important role for the catalytic function of LipA. To explore this specific role, the markerless deletion mutant of TMD sequence of lipA was constructed by a gene replacement system with the plasmid pJQ200SK, which utilized a double-crossover recombination event to knockout target sequence and clear the plasmid pJQ200SK by means of the Bacillus subtilis sacB (levansucrase) for counterselection [16]. Subsequently, lipase activities of Pf-5 and Pf0617TMD in the wholecell, lysis supernatant and lysis pellet were detected. The deletion of TMD sequence changed the lipase activities in the whole-cell, lysis supernatant and lysis pellet in a manner similar to the deletion of lipA (Figs. 1 and 3A), which indicated that the deletion of TMD sequence made LipA inactivate. In addition, pBBRKlipADTMD expressed in Pf0617 further confirmed the above conclusion (Fig. 3A). However, both Pf0617 and Pf0617TMD still remained 80% or so of the total lipase activity, which was due to Pf-5 containing another intracellular lipase (KEGG entry number PFL_2722, GenBank accession number YP_259829) and other intracellular enzymes associated with the lipase activity. To separately study the role of TMD in LipA, LipA and LipADTMD were expressed and purified. Lipase activity assays showed that LipADTMD was not able to hydrolyze tributyrin or p-nitrophenyl decanoate (Fig. 3C and D), these further proved the role of the TMD in LipA. However, LipADTMD could partly catalyze the hydrolysis of p-nitrophenyl ester whose carbon chain length was less than ten compared with LipA (Fig. 3D), which may be because of the deletion of TMD sequence changing the conformation of LipADTMD in a fine-tuning but important manner. Although other studies had shown that the N-terminal domain could also affect the function of lipases, its deletion did not inactivate the lipases [17e19]. Therefore, the 3D structures of LipA and LipADTMD were modeled by Rosetta 3.5 to further investigate how TMD influences the catalytic function of LipA. Rosetta 3.5 has been successfully used to model the 3D structures of other proteins [20,21], and it produced the closest pose to the target crystal structure [20]. The results of 3D structures showed that the deletion of TMD mainly affected the conformation of a-helical lid through the displacement of a5 helix that made the lid be in a closed conformation to block substrates access into the catalytic pocket (Fig. 4A and B), which was consistent with LipADTMD having partial activity toward pnitrophenyl esters with short chain fatty acids (C2eC8) (Fig. 3D). This indicates that the flexibility of the lid mainly results from motions of a5 helix, as was found in the case of other lipases from crystal structure or molecular dynamics simulations [22,23], further confirming the authenticity and reasonability of the modeling by Rosetta 3.5. Thanks to the fact that LipA lacks its cognate lipase-specific foldase, TMD might act as a kind of intramolecular chaperone like the b-roll motif of subfamily I.3 lipases contributing to the folding of the catalytic domain [24-26]. In addition, the deletion of the b-roll motif also inactivated subfamily I.3 lipases [24]. Surprisingly, TMD was embedded inside LipA and was modeled as a b-strand (Fig. 4A), being inconsistent with the prediction of the TMHMM 2.0, which further confirmed its specificity in LipA (Fig. 1). Many studies have demonstrated that identical short peptide sequences in unrelated proteins could have different conformations and a-helices could also be converted into b-strands
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Fig. 4. 3D structures of LipA (A) and LipADTMD (B). The residues forming the catalytic triad Ser-His-Asp were shown as cylinders; the position of the a-helical lid was highlighted with the label LID and numbered as the lipase from P. aeruginosa PAO1 (PDB ID：1EX9) [15]; LipA was shown as ribbons and colored in green, and TMD was colored in magenta; LipADTMD was shown as ribbons and colored in light blue.
[27e29]. Thus, it is possible that TMD was b-strand in LipA but ahelix in LacZ. In conclusion, this study demonstrated that in LipA its N-terminal TMD lost membrane localization function but played an indispensable role for its catalytic function, and probably acted as a kind of intramolecular chaperone helping the a-helical lid to form an open conformation mediated by a5 helix. This is the first to notify the intramolecular chaperone of a subfamily I.1 lipase. We found that such N-terminal TMD also exists in many other lipases by BLAST search in NCBI, therefore, similar studies should also be done to verify whether their TMD has the function of intramolecular chaperone in these lipases in further. Conflict of interest None. Acknowledgments The authors acknowledge the financial support of the National Natural Science Foundation of China (NSFC) (Nos. 31070089, 31170078 and J1103514), the National High Technology Research and Development Program of P. R. China (863 Program) (No. 2011AA02A204), and the Innovation Foundation of Shenzhen Government (JCYJ20120831111657864). Many thanks are due to Dr. Jingyuan Chen (Hubei Academy of Forestry, Wuhan, China) for kindly presenting P. protegens Pf-5. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biochi.2014.07.007. References [1] A. Ramette, M. Frapolli, M. Fischer-Le Saux, C. Gruffaz, J.M. Meyer, G. Defago, L. Sutra, Y. Moenne-Loccoz, Pseudomonas protegens sp. nov., widespread plantprotecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin, Syst. Appl. Microbiol. 34 (2011) 180e188.
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