By substituting Ala426 and Lys438 with Phe and His, respectively, the inactive mutant showed a much stronger interaction with phosphatidylcholine and a weaker interaction with phosphatid
Trang 1Yoshiko Uesugi, Jiro Arima, Masaki Iwabuchi and Tadashi Hatanaka
Research Institute for Biological Sciences (RIBS), Okayama, Japan
Phospholipase D (PLD; EC 3.1.4.4) catalyzes
phos-pholipid hydrolysis and phosphatidyl transfer
(Fig 1A) This is a ubiquitous and important enzyme
involved in signal transduction in mammals [1,2]
StreptomycesPLDs can be categorized into two types:
one is an iron-containing enzyme, such as that from
Streptomyces chromofuscus (chromofuscus PLD) for
which tightly bound iron is necessary for its catalytic
activity [3]; and the other is a member of the PLD
superfamily whose hallmark is the possession of two
catalytic HxKxxxxD (HKD) motifs [4–6] Because
enzymes of the latter type have a simple structure
containing two HKD motifs, they are useful as a
suitable model of mammalian PLDs
A study of the chemical modification of PLD from
Streptomycessp PMF (PMFPLD) suggested that Lys,
not His, is essential for PLD activity [7] Iwasaki et al
[8] revealed that two HKD motifs are essential for the
activity, using the N- and C-terminal halves of
Strep-tomyces PLD Furthermore, Leiros et al [9] showed
that His170 in the N-terminal HKD motif of PMFPLD
acts as the initial nucleophile that attacks the phospho-rus atom of the substrate, on the basis of the crystal structures of PMFPLD Previously, using two Strep-tomyces PLDs in repeat-length independent and broad spectrum (RIBS) in vivo DNA shuffling, we constructed
a random chimera library to investigate the recognition
of phospholipids by Streptomyces PLD We revealed that the N-terminal HKD motif contains the nucleo-phile, using an inactive chimera and surface plasmon resonance (SPR) analysis [10]
To date, the functions of the HKD motifs in cata-lytic mechanisms have been extensively studied [11–13] At present, PLD-catalyzed reactions are con-sidered to consist of two steps: first, the formation
of a covalently linked phosphatidyl enzyme interme-diate via the His residue of the N-terminus HKD motif; and second, the hydrolysis or transphosphati-dylation of the intermediate by a water or alcohol molecule (Fig 1A)
As mentioned above, previous experimental studies have focused on the relationship between HKD motifs
Keywords
phospholipase D; phospholipid; substrate
recognition; SPR; Streptomyces
Correspondence
T Hatanaka, Research Institute for
Biological Sciences (RIBS), Okayama,
7549-1 Kibichuo-cho, Kaga-gun, Okayama
716-1241, Japan
Fax: +81 866 56 9454
Tel: +81 866 56 9452
E-mail: hatanaka@bio-ribs.com
(Received 12 January 2007, revised 14
March 2007, accepted 22 March 2007)
doi:10.1111/j.1742-4658.2007.05802.x
Recently, we identified Ala426 and Lys438 of phospholipase D from Strep-tomyces septatus TH-2 (TH-2PLD) as important residues for activity, sta-bility and selectivity in transphosphatidylation These residues are located
in a C-terminal flexible loop separate from two catalytic HxKxxxxD motifs To study the role of these residues in substrate recognition, we eval-uated the affinities of inactive mutants, in which these residues were substi-tuted with Phe and His, toward several phospholipids by SPR analysis By substituting Ala426 and Lys438 with Phe and His, respectively, the inactive mutant showed a much stronger interaction with phosphatidylcholine and
a weaker interaction with phosphatidylglycerol than the inactive TH-2PLD mutant We demonstrated that Ala426 and Lys438 of TH-2PLD play a role in sensing the head group of phospholipids
Abbreviations
PA, phosphatidic acid; PC, phosphatidylcholine; PG, phosphatidylglycerol; PLD, phospholipase D; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho- L -serine]; PpNP, phosphatidyl-p-nitrophenol; RU, resonance unit; SPR, surface plasmon resonance; SUV, small unilamellar vesicle.
Trang 2and activity Recently, we demonstrated that four
amino acid residues, Gly188, Asp191, Ala426 and
Lys438, of PLD from Streptomyces septatus TH-2
(TH-2PLD) are associated with PLD activity and
sub-strate recognition [10,14] (Fig 2A) Substituting Ala426
and Lys438 with Phe and His, respectively, led to
improvements in PLD activity, thermostability, and
organic solvent tolerance and to a change in the
selectiv-ity of transphosphatidylation activselectiv-ity compared with
that in the original chimera [14] This suggests that
Ala426 and Lys438 are involved in substrate recognition
Hughes et al [15] demonstrated that human
PLD1b interacts with fluorescence-labeled
phospha-tidylcholine (PC), whereas PLD1 does not interact
with fluorescence-labeled phosphatidylethanolamine
This finding shows that the PLD–phospholipid
inter-action correlates with PLD activity, because PLD1
has no catalytic activity toward
phosphatidylethanol-amine Recently, SPR analysis has been used to
investigate the effects of the rat PLD1 Phox
homo-logy (PX) domain on membrane binding properties
[16], and the specific association of PLD1b with its
regulator proteins, PKCa, Rac1 and ARF6 [17] In
addition, because of the interaction of inactive PLDs
and PC retaining a covalent phosphatidyl-enzyme
intermediate determined by SPR analysis, the
N-ter-minal HKD motif was found to act as a catalytic
nucleophile [10]
In this study, to investigate the roles of Ala426
and Lys438 of TH-2PLD in substrate recognition in
more detail, we analyzed the association of inactive
mutants of TH-2PLD, in which these residues were
substituted with Phe and Ala, respectively,
conco-mitantly with the substitution of His443 of the
C-terminal HKD motif with Ala, with three phos-pholipid substrates (Fig 1) by SPR analysis
Results
Preparation of inactive mutants of TH-2PLD
In a previous study, we used two homologous Streptomyces PLDs, TH-2PLD and PLD from Strep-tomyces sp (PLDP), as parental enzymes by RIBS shuffling [10] PLDP had Phe and His corresponding
to Ala426 and Lys438 of TH-2PLD, respectively Thus, we substituted these residues of TH-2PLD with Phe and His in this study In addition, to evaluate the effect of the residues on phospholipid recognition by SPR analysis, we constructed inactive mutants of TH-2PLD, in which His443 of an HKD motif was substituted with Ala, as shown in Fig 2B
We then expressed the resultant genes and purified the proteins they encode All of the purified mutants mostly showed a single band with the same molecu-lar mass ( 57 kDa) as that of wild-type TH-2PLD
on SDS⁄ PAGE (Fig 2C) Furthermore, using west-ern blot analysis with anti-(wild-type TH-2PLD) serum, these mutants were found to have similar uniformities and purities (Fig 2D) All the mutants had low activities toward phosphatidyl-p-nitrophenol (PpNP) (< 0.7 lmolÆmin)1Æmg)1), whereas wild-type TH-2PLD had high activity (59 lmolÆmin)1Æmg)1)
To confirm the folding of the inactive mutants
of TH-2PLD, their CD spectra were measured
As shown in Fig 3, the CD spectra showed that the inactive mutants folded with a secondary struc-ture similar to wild-type TH-2PLD These results
A
B
Fig 1 Reactions catalyzed by PLD (A) and
structure of phospholipid head groups (B).
Trang 3suggest that we successfully prepared inactive mutant
enzymes
Association of inactive mutants with
phospholipid vesicles
To investigate the association of key C-terminal
resi-dues with the head group of phospholipid substrates,
we analyzed the binding profiles of TH-2(H443A),
TH-2-F(H443A) and TH-2-FH(H443A) for several
phospholipid vesicles with a covalent
phosphatidyl-PLD intermediate using SPR analysis Sensorgrams
obtained by SPR analysis showed real-time
biomole-cular interaction Overlaid sensorgrams were obtained
when TH-2(H443A), TH-2-F(H443A) and
TH-2-FH(H443A) were passed at different concentrations
over immobilized oleoyl-sn-glycero-3-phosphocholine (POPC; Fig 4A–C), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (POPS; Fig 4D–F)
or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG; Fig 4G–I) vesicles As shown in Fig 4A–F, TH-2-F(H443A) and TH-2-FH(H443A) exhibited significantly higher binding abilities for POPC and POPS vesicles than TH-2(H443A) By con-trast, sensorgrams of TH-2-F(H443A) were similar to those of TH-2(H443A) for POPG vesicles, and their interactions were stronger than those of TH-2-FH(H443A) (Fig 4G–I) These differences in interac-tion were not caused by the heterogeneity of the mutants If they were, each mutant would have shown the same association and dissociation curves for all the phospholipids; however, the results did not show such
A
B
Fig 2 (A) 3D structure around identified key residues (i.e residues 188, 191, 426 and 438 of TH-2PLD) associated with activ-ity The overall structure of TH-2PLD is
sho-wn using the Swiss-PDB viewer and is based on the crystal structure of PMFPLD The identified key residues are indicated in red The N-terminal and C-terminal HKD motifs are shown in light blue and purple, respectively (B) Primary structures of wild-type TH-2PLD and its inactive mutants The gray box indicates the His residue of the C-terminal HKD motif mutated to Ala The identified residues related to the PLD reaction are shown in black boxes.
(C) SDS ⁄ PAGE results of purified PLDs Lanes 1–4 contained 2 lg of 2PLD, TH-2(H443A), TH-2-F(H443A) and TH-2-FH-(H443A), respectively Lane M indicates SDS ⁄ PAGE standard proteins (molecular masses: 100 000, 80 000, 60 000, 50 000,
40 000, 30 000 and 20 000 Da) Samples were loaded on a 10% acrylamide gel (D) Western blot analysis of purified PLDs using anti-(wild-type TH-2PLD) serum Lanes 1–3 contained 2 lg of TH-2(H443A), TH-2-F(H443A) and TH-2-FH(H443A), respectively Lane M indicates prestained SDS ⁄ PAGE standards (molecular masses: 111 000,
93 000, 53 500, 36 100 and 29 500 Da) The samples were loaded on a 10% acrylamide gel The arrowhead indicates the position of the purified PLDs.
Trang 4curves Therefore, these results suggest that
substitu-tion of Ala426 with Phe led to considerably stronger
interactions with POPC and POPS It should be noted
that the double mutant showed a decrease in the
strength of its interaction with POPG vesicles,
although the interactions of the mutant with POPC
and POPS vesicles remained strong
The kinetic constant was calculated from each
sensor-gram using bia evaluation 4.1 analysis software
according to the global fitting of 1:1 binding with a mass
transfer model Affinity constants (KD) for POPC
vesi-cles were 5.3 ± 0.9, 5.5 ± 1.4 and 4.8 ± 0.7 nm for
TH-2(H443A), TH-2-F(H443A) and TH-2-FH(H443A),
respectively There was no significant difference in KD
value between these proteins; however, the maximal
responses of these proteins differ markedly In the case
of POPG and POPS vesicles, KDcould not be calculated
because their response curves did not fit the evaluation
curves In particular, sensorgrams toward POPG
showed an increase and a decrease in interactions during
the association process at a low mutant concentration
(Fig 4G–I) The results suggest that the mutants have
more than two binding sites, with different affinities for
POPG vesicles Unfortunately, there are no evaluation
models for determining the interaction between a ligand
and an analyte involving more than two binding sites
Thus, the sensorgrams of mutants toward POPG could
not be analyzed appropriately
To compare the differences in affinity for
phospho-lipids among TH-2(H443A), TH-2-F(H443A) and
TH-2-FH(H443A), the maximal responses measured
for these inactive mutant associations with POPC,
POPS and POPG vesicles are shown in Fig 5 Injection
of 532 nm TH-2(H443A) resulted in a binding signal of
1409 resonance units (RU) for POPC vesicles, which was similar to that of 1204 RU for POPG vesicles (t-test; P > 0.05), whereas the association with POPS vesicles was significantly weaker (477 RU; P < 0.001)
As shown in Fig 4A,G, the sensorgram of TH-2(H443A) increased more sharply in the association phase when POPG vesicles rather than POPC vesicles were used In contrast, TH-2-FH(H443A) bound to POPG vesicles slowly (Fig 4I), and the degree of bind-ing response for POPG vesicles was 5.1-fold lower than that for POPC vesicles TH-2-F(H443A) also showed a degree of binding response for POPG vesicles 2.6-fold lower than that for POPC vesicles Interestingly, the interactions of each inactive mutant with POPS vesicles were similar and low in degree compared with those with POPC vesicles, although each mutant exhibited different degrees of interaction with POPG vesicles From these results, it is suggested that residues 426 and
438 of TH-2PLD play a role in sensing the head group
of phospholipid vesicles
Conformational change of inactive mutants induced by phospholipids
To analyze changes in the tertiary and secondary struc-tures of inactive mutants induced by phospholipids, we further measured the fluorescence and CD spectra of inactive mutants in the absence and presence of POPC and POPG vesicles [18] TH-2PLD has 11 Trp residues that contribute to its fluorescence emission spectrum
As shown in Fig 6A–C, the emission maxima, around
340 nm, were the same for all the inactive mutants With inactive mutant alone, TH-2-F(H443A) showed a similar fluorescence emission spectrum to TH-2(H443A), and TH-2-FH(H443A) had a higher fluores-cence emission intensity than TH-2(H443A) For the mutant TH-2-FH(H443A), the local environment around Trp434 is probably changed by substituting His for Lys438, because Lys438 is located adjacent to Trp434 ( 4 A˚) The fluorescence emission intensities
of TH-2-F(H443A) and TH-2-FH(H443A) increased at
340 nm with the addition of POPC vesicles, and the degrees of increase were 0.186 and 0.083 relative to those without POPC, respectively Interestingly, the fluorescence emission intensity of TH-2-FH(H443A) with POPG vesicles decreased to that without POPG
at 0.11 degrees However, the fluorescence emission intensity of TH-2(H443A) did not change with or without phospholipids In contrast, the CD spectra of all the inactive mutants were similar with or without phospholipids (Fig 6D–F) From these results, it sug-gests that TH-2-F(H443A) and TH-2-FH(H443A) were
Fig 3 CD spectra of PLDs The spectrum of each PLD
(0.1 mgÆmL)1) in 10 m M potassium phosphate buffer (pH 7.0) was
measured at 25 C.
Trang 5induced conformational changes in their tertiary
struc-tures with phospholipid introduction, although their
secondary structures remain unchanged
Discussion
Recently, Sato et al [19] reported that the
phosphati-dic acid (PA) contents produced by a side reaction of
several Streptomyces PLDs differ markedly during
transphosphatidylation from PC to
phosphatidylgly-cerol (PG) Among the PLDs used, TH-2PLD showed the lowest selectivity in transphosphatidylation, and the amount of hydrolyzed PA increased with reaction time This phenomenon was considered to be the result
of synthesized PG being hydrolyzed to PA during transphosphatidylation We speculated that TH-2PLD recognizes synthesized PG as well as PC; therefore, the amount of hydrolyzed PA increases Recently, we showed that the C-terminal flexible loop in Strepto-myces PLD (residues 425–442) is separate from the
Fig 4 Sensorgrams at different concentrations of inactive mutants of TH-2PLD As substrate, POPC (A–C), POPS (D–F) and POPG (G–I) vesicles were immobilized on an L1 sensor chip, as described in Experimental procedures The SPR sensorgrams were obtained when TH-2(H443A) (A,D,G), TH-2-F(H443A) (B,E,H) and TH-2-FH(H443A) (C,F,I) were passed over the phospholipid vesicles at 532, 355, 236 and
158 n M , respectively (from top to bottom), at a flow rate of 20 lLÆmin)1for 5 min at 25 C, followed by a buffer at the same flow rate for 10 min The affinities of the mutants to the phospholipid vesicles were determined by fitting these SPR sensorgrams using BIA EVALUATION 4.1 analysis software.
Trang 6two highly conserved catalytic HKD motifs, and that
it is formed at the entrance of the active-site well and
has multiple functional roles A mutant PLD with
Ala426 and Lys438 substituted with Phe and His,
respectively, improved its selectivity in
transphosphati-dylation from PC to PG [14] Thus, we plan to
investi-gate the relationship between the recognition of several
phospholipids, such as PC, PG and phosphatidylserine
(PS), and the residues identified by SPR analysis
These results agree well with the present findings
that TH-2(H443A) has comparable interactions with
POPC and POPG, and that TH-2-FH(H443A) has a
much stronger interaction with POPC and a weaker
interaction with POPG than TH-2(H443A) (Fig 5) In
addition, by determining the corresponding active
mutants in terms of their activity toward POPC
vesi-cles by the method using choline oxidase and
peroxi-dase [14], the activities of TH-2-F (3.3 lmolÆmin)1Æ
mg)1) and TH-2-FH (3.3 lmolÆmin)1Æmg)1) were
found to be twofold higher than that of TH-2PLD
(1.7 lmolÆmin)1Æmg)1) toward POPC (data not shown)
These results suggest that the activities of active
mutants correlate well with the binding data of
inac-tive mutants These findings suggest that TH-2PLD
recognizes PC and PG similarly, and that residues
426 and 438 of TH-2PLD play an important role in
phospholipid recognition To date, nine sequences of
Streptomyces PLDs have been determined All
Strep-tomyces PLDs except TH-2PLD have a Phe residue
corresponding to Ala426 of TH-2PLD By contrast, at Lys438 of TH-2PLD, four of them have a Lys residue and the rest have a His residue Thus, the main cause
of the low selectivity of TH-2PLD in transphosphati-dylation seems to be Ala426 In this study, we con-firmed that these residues are associated with the interaction between TH-2PLD and its substrate SPR analysis revealed that Streptomyces PLD inter-acts to a much higher degree with zwitterionic phos-pholipid vesicles (i.e PC) than with anionic phospholipid vesicles (i.e PG and PS) (Fig 5) PLDs from mammals and poppy seedlings hydrolyze PC most efficiently among several phospholipids [20,21] That is, Streptomyces PLD containing HKD motifs seems to prefer zwitterionic phospholipid vesicles, such
as POPC, to anionic phospholipid vesicles POPS and POPG, similarly to other PLDs
Previous SPR analysis indicated that the PLD1 PX domain has a high phosphoinositide specificity, that is, the KD value for POPC⁄ POPE ⁄ phosphatidylinositol 3,4,5-trisphosphate (77 : 20 : 3) is 18 ± 4 nm [16] Powner et al [17] showed KDvalues between PLD1b and regulator proteins, PKCa, Rac1 and ARF6 (i.e
42 ± 15, 143 ± 28 and 660 ± 63 nm, respectively) [17]
In this study, KDvalues for POPC vesicles were found
to be 5.3 ± 0.9, 5.5 ± 1.4 and 4.8 ± 0.7 nm for TH-2(H443A), TH-2-F(H443A) and TH-2-FH(H443A), respectively These results suggest that the affinities of these mutants for POPC vesicles are stronger than those of the specific association between the PLD1 PX domain and phosphatidylinositol 3,4,5-trisphosphate,
or PLD1b and its regulators
We speculate that the difference in the degree of interaction with POPC among TH-2PLD mutants is a result of the influence of two flexible loops, i.e resi-dues 188–203 and 425–442, of TH-2PLD on each other Iwasaki et al [8] and Xie et al [22] showed that PLD activity is restored when the N- and C-terminal fragments of Streptomyces PLD and PLD1 coexist, although these fragments have only negligible activities
in isolation From these findings, we speculate that PLD changes its conformation markedly before and after binding to the substrate In fact, SPR analysis (Figs 4,5) and fluorescence spectroscopy (Fig 6) showed that the inactive mutants, in which Ala426 and Lys438 were substituted with Phe and Ala, showed ter-tiary structural changes with phospholipid binding Combined with the results of SPR analysis and fluores-cence spectroscopy, it seems that the interaction between Streptomyces PLD and POPG differs from that between the PLD and POPC, although this phe-nomenon cannot be explained experimentally at pre-sent These two flexible loops may play a role as a
Fig 5 Interaction of PLDs and phospholipid vesicles The maximal
responses of TH-2(H443A), TH-2-F(H443A) and TH-2-FH(H443A)
were measured for their specific associations with phospholipids
vesicles Each PLD (532 n M ) was injected at a flow rate of 20
lLÆmin)1 Each value represents the mean ± SD from three
inde-pendent experiments.
Trang 7trigger of conformational change when PLDs bind to
the substrate Therefore, residues 426 and 438 located
in the C-terminal loop could affect the interaction of
PLDs with substrate
TH-2(H443A) and TH-2-F(H443A) exhibited
stron-ger interactions with POPG vesicles than
TH-2-FH(H443A) Sensorgrams of these mutants showed
an increase and a decrease in the degree of interactions
during the association process at a low concentration
of mutants (Fig 4G–I) These results suggest that
there are more than two binding sites that have
differ-ent affinities to PG vesicles in the mutants Using
com-puter analysis with the automated docking program
autodock, Aikens et al [23] showed that the glycerol
group of PG is bound to a region composed of
Ser453, Lys454, Asn455, Tyr457, Ser459 and Leu461
of PMFPLD These residues correspond to Ser458, Lys459, Asn460, Tyr462, Ser464 and Leu466, respect-ively, in the C-terminal region of TH-2PLD PMFPLD and TH-2PLD are 85% homologous in primary struc-ture Hence, we surmise that another PG-recognizing site is present in the C-terminal region
Ala426 and Lys438 of TH-2PLD are located in a C-terminal flexible loop separate from two catalytic HKD motifs The loop, in coordination with the N-terminal loop, forms the entrance of the active well comprising two HKD motifs (Fig 2A) It is reasonable
to consider that these residues play a role in sensing the head group of phospholipids from a geometrical point of view It might be possible to change the sub-strate preference of Streptomyces PLD by substituting these two residues with other amino acid residues
Fig 6 Fluorescence emission spectra of TH-2(H443A) (A),TH-2-F(H443A) (B) and TH-2-FH(H443A) (C) in the absence and presence of phosp-holipids vesicles The inactive mutants were excited at 290 nm and emission spectra were recorded between 300 and 380 nm Fluores-cence measurements were carried out at 25 C with 1.2 l M PLDs in 10 m M Tris ⁄ HCl (pH 7.0) containing 4 m M CaCl2 CD spectra of TH-2(H443A) (D), TH-2-F(H443A) (E) and TH-2-FH(H443A) (F) in the absence and presence of phospholipids vesicles The spectrum was meas-ured at 25 C with 1.7 l M PLDs in 10 m M Tris ⁄ HCl (pH 7.0) containing 4 m M CaCl 2 POPC or POPG of SUVs was added PLD solution at a final concentration of 1 m M , and incubated for over 1 h All spectra were corrected by subtracting the spectra of the corresponding back-ground media without PLD.
Trang 8Experimental procedures
Materials
The plasmid pETKmS2 [24] was kindly provided by
T Yamane (Nagoya University, Japan) PpNP was
pre-pared from soybean phosphatidic acid and p-nitrophenol
according to the procedure of D’Arrigo et al [25] POPC,
POPS and POPG were obtained from Avanti Polar Lipids
(Alabaster, AL, USA) and used without further purification
All the other chemicals were of the highest purity available
Preparation of PLDs
To construct the mutant TH-2(H443A), the mutagenic gene
was amplified by PCR using the following primers:
5¢-CCCTGCGCGCGCTCGTCGGCA-3¢ (corresponding to
the gene th-2pld, nucleotides 962–982) and 5¢-ACCAG
[corresponding to nucleotides 1316–1340 from th-2pld,
cloned, sequenced and digested with BglII and Van91I
Next, the plasmid pUC19(TH-2) [26] was digested with
BglII and Van91I, and the product was substituted for the
corresponding region in the subcloned th-2pld The
resul-tant muresul-tant gene was digested with NcoI and BamHI
and ligated into the NcoI–BamHI gap of the vector
pETKmS2(TH-2(H443A))
To prepare the mutants 2-F(H443A) and
TH-2-FH(H443A), a partial th-2pld was amplified by PCR using
the following primers: 5¢-ACTACGTCGACACCTCCCA
CC-3¢ (corresponding to nucleotides 575–595 from th-2pld)
silent mutation of the NheI site (underlined) (corresponding
to nucleotides 1257–1278 from th-2pld) Then the amplified
DNA fragments were cloned, sequenced and digested with
PstI and NheI Next, the plasmids pETKmS2(G-F) and
pETKmS2(G-FH) [14] were digested with NheI and BsiWI
The two resulting fragments were ligated into the PstI–
BsiWI gap of the vector pETKmS2(TH-2(H443A)) to
con-struct the expression vector The expression vectors obtained
were confirmed by DNA sequencing
The recombinant TH-2PLD and inactive mutant enzymes
were expressed as secreted proteins with a C-terminal His6
tag, and purified with TALON metal affinity resin (Clontech,
Palo Alto, CA, USA) according to standard protocols The
purities of proteins obtained were confirmed by SDS⁄ PAGE
[10] and western blot analysis using an anti-(wild-type
TH-2PLD) serum
Assay for PLD activity using PpNP
Hydrolytic activity was determined on the basis of the
hydrolytic activity of PpNP The procedure was similar
one described previously [27] One unit of PLD was defined as the amount of the enzyme that releases 1 lmol
of p-nitrophenol per minute under the assay conditions The reactions were carried out in 1.5-mL cuvettes The 1-mL reaction mixture consisted of 0.07–0.2 lg of purified PLDs and 2 mm PpNP in 0.1 m sodium acetate buffer (pH 5.5) at 37C
CD spectroscopy
The folding of PLDs was confirmed by CD spectroscopy using a J-720WI spectrometer (Jasco, Tokyo, Japan) Pro-teins were dissolved to a final concentration of 0.1 mgÆmL)1
in 10 mm potassium phosphate buffer (pH 7.0) Spectra were acquired at 25C using a 2-mm path-length cuvette The spectra of PLDs that averaged 10 scans were corrected
by subtracting the spectra of the corresponding background media without PLD
Preparation of vesicles
An aliquot of phospholipids dissolved in chloroform was evaporated and further dried in vacuum for at least 3 h The lipids were hydrated to a concentration of 10 mm in phosphate-buffered saline for SPR analysis or in 10 mm Tris⁄ HCl (pH 7.0) for fluorescence and CD measurements The lipid suspension was vortexed vigorously, and frozen and thawed 10 times in liquid nitrogen To obtain small uni-lamellar vesicles (SUVs), the suspension was passed 30 times through polycarbonate membranes (50 nm pore diameter) using a Lipofast extruder (Avestin, Ottawa, Canada) [28]
SPR analysis
Real-time interactions between PLD molecules and phos-pholipids were measured using a Biacore instrument (Bia-core 2000, Bia(Bia-core AB, Uppsala, Sweden) Liposomes were captured on the surface of a Sensor Chip L1 (Biacore AB) as
‘ligand’ The surface of the L1 sensor chip was first cleaned with 20 mm 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS) at a flow rate of 5 lLÆmin)1 followed by the injection of SUVs (60 lL, 0.5 mm phospho-lipids) at a flow rate of 2 lLÆmin)1 in buffer A (10 mm sodium acetate, pH 5.5, 4 mm CaCl2) This resulted in the deposition of 5000–7000 RU To measure the association
of PLDs with phospholipids, a purified inactive mutant enzyme (105–532 nm diluted in buffer A) as ‘analyte’ was applied to the captured SUVs at a flow rate of 20 lLÆmin)1
at 25C After the binding of PLDs to phospholipids, disso-ciation was observed at the same flow rate for 10 min The evaluation of the kinetic parameters of PLD binding to phospholipids was performed by the nonlinear fitting of binding data using bia evaluation 4.1 analysis software The apparent association (ka) and dissociation (kd) rate
Trang 9constants were evaluated from the differential binding curves
(sample–control) shown in Fig 4, assuming an A + B¼ AB
association type for protein–lipid interaction The affinity
constant KDwas calculated from the equation KD¼ kd⁄ ka
Fluorescence spectroscopy
Fluorescence spectra were obtained with an F-4500
spectro-fluorometer (Hitachi, Tokyo, Japan) All measurements
were carried out at 25C with 1.2 lm PLDs in 10 mm
Tris⁄ HCl (pH 7.0) containing 4 mm CaCl2 using 2-mm
path-length quartz cuvette The excitation wavelength was
290 nm, and excitation and emission slits were 5 nm
Emis-sion was scanned from 300 to 380 nm PLDs were mixed
with 1 mm SUVs and incubated over 1 h The spectra of
PLDs that averaged four scans were corrected by
subtract-ing the spectra of the correspondsubtract-ing background media
without PLD The degree of change in the fluorescence
intensity was calculated as (I) I0)⁄ I0, where I0is the
maxi-mum intensity of PLD alone, and I is the maximaxi-mum
inten-sity in the presence of phospholipids [29]
Statistical analysis
All statistical evaluations were performed using an unpaired
Student’s t test All data are presented as mean ± SD of at
least three determinations
Acknowledgements
We thank Ms M Taniai (Hayashibara Biochemical
Laboratories) for technical advice on SPR analysis This
research was financially supported by the Sasakawa
Sci-entific Research Grant from The Japan Science Society
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