From the Department of Biochemistry and the
** Department of Odontology, Umeå University, 90187 Umeå, Sweden and the
Department of Biochemistry and
Biophysics, Stockholm University,
10691 Stockholm, Sweden
Received for publication, November 11, 2002, and in revised form, November 29, 2002
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ABSTRACT |
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In membranes of Acholeplasma
laidlawii two consecutively acting glucosyltransferases, the (i)
Lipid bilayer properties, important for membrane
barrier and protein function, are regulated at several levels in
cells. Acholeplasma laidlawii and
Escherichia coli membrane lipids are metabolically designed
to yield a bilayer "window" with certain features between the gel
and nonbilayer phases (1, 2). In A. laidlawii this aims at
maintaining (i) a certain lipid bilayer surface charge density and (ii)
a constant radius of spontaneous curvature (elastic packing stress),
including similar bilayer/nonbilayer transition temperatures (1, 3).
Regulation of (i) and (ii) resides mainly at the polar headgroup level
(4), whereas in E. coli curvature is regulated at the acyl
chain level (2), with more or less constant headgroup composition.
The bilayer-forming glucolipid -monoglucosyldiacylglycerol (MGlcDAG) synthase (alMGS) (EC
2.4.1.157) and the (ii)
-diglucosyl-DAG (DGlcDAG) synthase (alDGS)
(EC 2.4.1.208), are involved in maintaining (i) a certain anionic lipid
surface charge density and (ii) constant nonbilayer/bilayer conditions
(curvature packing stress), respectively. Cloning of the alDGS gene
revealed related uncharacterized sequence analogs especially in several
Gram-positive pathogens, thermophiles and archaea, where the encoded
enzyme function of a potential Streptococcus pneumoniae DGS
gene (cpoA) was verified. A strong stimulation of alDGS by
phosphatidylglycerol (PG), cardiolipin, or nonbilayer-prone 1,3-DAG was
observed, while only PG stimulated CpoA. Several secondary structure
prediction and fold recognition methods were used together with
SWISS-MODEL to build three-dimensional model structures for three MGS
and two DGS lipid glycosyltransferases. Two Escherichia
coli proteins with known structures were identified as the best
templates, the membrane surface-associated two-domain glycosyltransferase MurG and the soluble GlcNAc epimerase. Differences in electrostatic surface potential between the different models and
their individual domains suggest that electrostatic interactions play a
role for the association to membranes. Further support for this was
obtained when hybrids of the N- and C-domain, and full size alMGS with
green fluorescent protein were localized to different regions of the
E. coli inner membrane and cytoplasm in vivo.
In conclusion, it is proposed that the varying abilities to bind, and
sense lipid charge and curvature stress, are governed by typical
differences in charge (pI values), amphiphilicity, and hydrophobicity
for the N- and (catalytic) C-domains of these structurally similar
membrane-associated enzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-diglucosyldiacylglycerol
(DGlcDAG)1 is one of the
major lipids in the small cell wall-less A. laidlawii, the
other is the nonbilayer-prone
-monoglucosyldiacylglycerol (MGlcDAG),
cf. pathway below. Another pathway leads from phosphatidic acid to phosphatidylglycerol (PG). Under
certain circumstances more acylated, and more nonbilayer-prone,
variants of MGlcDAG and DGlcDAG, i.e. MAMGlcDAG and
MADGlcDAG, respectively, are synthesized (5). A. laidlawii
can only synthesize saturated acyl chains, and to still be able to
adapt to membrane stress and new environments, the amounts of the two
major glucolipids are strongly adjusted to maintain a functional
membrane bilayer. The MGlcDAG-synthesizing enzyme alMGS (EC 2.4.1.157)
((i) above) was recently cloned (6) and found to be a
surface-associating protein with no transmembrane segments, where
bilayer binding was dependent on phosphatidylglycerol and stimulated by
small amounts of nonbilayer
lipids.2 This seemed logical
given the strong involvement of this enzyme in the lipid surface charge
regulation, i.e. (i) above (8).
View larger version (8K):
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Scheme 1.
Glucolipid
pathway.
The 1,2-diacylglycerol-3--glucose (1
2)-
-glucosyl
transferase (DGS) (EC 2.4.1.208) catalyzes the consecutive transfer of
glucose from UDP-Glc to MGlcDAG to yield DGlcDAG (cf. (ii) above). This reaction is activated in an essential manner by certain anionic lipids and stimulated by other additives promoting nonbilayer tendencies both in vivo (1, 9) and with crude or pure
enzymes in vitro (10, 11), keeping or restoring bilayer
packing conditions. Furthermore, an additional modulation of the
activity is achieved by certain phosphorylated metabolites,
double-stranded DNA (12), and by a low redox potential (7). Due to
their small polar headgroups, nonbilayer-prone lipids of the reversed
type, like MGlcDAG, make the two monolayers in a bilayer each want to
curl concavely toward the water phase. This induces a curvature elastic stress (increased spontaneous curvature), with an increased chain order
and a closer approach to a bilayer-nonbilayer phase transition. Similar
packing features can be inferred for the major galactolipids in the
membranes of chloroplasts and photosynthetic bacteria (13). A number of
peripheral and integral membrane proteins are functionally affected by
this lateral stress, but for none the exact mechanisms are known (14),
although correlation with the lipid properties is very evident
(e.g. Refs. 15-17).
Hence, a question of large importance is how the A. laidlawii DGS is able to sense the curvature stress and respond by
increased or decreased synthesis of DGlcDAG. The cloned gene (this
work) yielded an active enzyme from E. coli, and the deduced
amino acid sequence and the predicted secondary and three-dimensional
structure models strongly indicate this enzyme to be, unexpectedly,
very similar in structure and surface association as the preceding MGlcDAG synthase (glucosyltransferase) and several other
glycosyltransferases. Hence, the bilayer/nonbilayer lipid balance, and
the connected bilayer physical forces, are sensed at the bilayer
surface by the DGlcDAG synthase. Data base searches revealed a new
group of related lipid glycosyltransferases in Gram-positive bacteria and archaea. Functional cloning of CpoA from Streptococcus
pneumoniae suggests this group to be responsible for synthesis of
-diglycosyldiacylglycerols in a number of species. However, CpoA
responds differently to the properties of the surrounding bilayer, and
the latter is substantiated by certain differences in the enzyme
structure models.
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MATERIALS AND METHODS |
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Genomic Preparation-- A. laidlawii strain A-EF22 was grown in 28 °C in tryptose/bovine serum albumin medium (11), supplemented with 0.12 mM oleic acid (18:1c). The cells were harvested, and the DNA was prepared with the GenomicPrepTM kit (Amersham Biosciences). The S. pneumoniae strain 19FCCUG 3030 was grown in Todd-Hewitt medium, pH 7.8 at 37 °C overnight. The DNA was prepared by heating the resuspended cell pellet at 95 °C for 10 min. After the subsequent centrifugation, the DNA was present in the supernatant.
Cloning of DGS Genes--
Degenerated DNA primers were made
based on the N-terminal amino acid sequence of the purified A. laidlawii DGlcDAG synthase (alDGS) determined through Edman
degradation (10). An internal sequence was also determined after
proteolytic cleavage and fragment purification by reverse phase-high
performance liquid chromatography. The primers 5'-GGT CGT GCT
TTT TAT CA(C/T) CA(A/G) AAA-3' and 5'-AAT AAC AGC ACC (A/G)TC IAC
(A/G)TG-3' were used in a PCR amplification procedure that resulted in
a ~600-bp DNA fragment. The PCR products were purified and ligated
into the pCR-Script SK(+) cloning vector (Stratagene) linearized with
SrfI and then sequenced. The purified PCR product was also
radioactively labeled by -35S-dATP and used as a probe
in a Southern blot procedure with completely HindIII/EcoRI digested DNA from A. laidlawii. The hybridization results were visualized by electronic
autoradiography (Packard Instant ImagerTM). The DNA
fragments with corresponding size to the hybridization band were
purified and ligated into the pCR-Script SK(+) cloning vector and
transformed into E. coli TOP 10F heat-shock competent cells.
Positive clones were found by colony replicating to nitrocellulose filter and hybridization (18) with the radioactively labeled probe. The
nucleotide sequence of the inserted DNA fragment was determined using
both gene-specific and vector-specific primers with ABI PRISM® Big Dye
terminator cycle sequencing ready reaction kit (PE Applied Biosystems).
Using these results, a construct was made where the gene
ALdgs from A. laidlawii A-EF22 strain was cloned
into a pET15b-vector and transformed into E. coli BL21(DE3). This recombinant was used for the enzymatic assays.
The gene coding for the spDGS in S. pneumoniae (cpoA), identified with the alDGS aa sequence in a computer search, was isolated by PCR with the primers 5'-TAG TTA TGG AGA AAA AGA AAT TAC G-3' and 5'-TAC CTC ACT TTT TAC TTT CTC CC-3', which bind to the Shine-Dalgarno and stop codon sequence regions, respectively. The PCR product was purified, ligated into a SmaI-digested pCR-script vector, and transformed into TOP10F.
Lipids and Amphiphiles Used--
Synthetic
rac-1,3-dioleoylglycerol (1,3-DOG) was purchased from
Larodan (Malmö, Sweden). The MGlcDAG and DGlcDAG were
prepared as described (8). Synthetic -MGalDAG was obtained from Dr. D. Mannock (cf. Dahlqvist et al. (11)).
Synthetic DOPA and DOPG were purchased from Avanti Polar Lipids
(Alabaster, AL) and CHAPS detergent from Roche Molecular Biochemicals.
Growth of Recombinants--
Protein expression in the
recombinant strains was performed in 1× LB medium supplemented with
100 µg of carbenicillin/ml. The strains were grown at 37 °C, and
0.3 mM
isopropyl-1-thio--D-galactopyranoside was added
at OD600 = 0.75. Cells were harvested by centrifugation after another 16 h at 23 °C. Harvested recombinant E. coli cells were solubilized in assay buffer (110 mM
Tris-HCl, pH 8.0, 22 mM MgCl2, and 22 mM CHAPS) to a total protein concentration of ~1.3 mg/ml,
by 4 × 30 s in a sonication bath and three times extensive vortexing during incubation on ice for 3 h, before enzymatic assays.
Enzymatic Assays--
In the standard assay for glycolipid
synthesis 25 µl of protein solution (cf. above) was added
to 20 µl of lipid micellar solution and incubated on ice for 30 min.
The enzymatic reaction was started by addition of 5 µl of
UDP-[14C]galactose or UDP-[14C]glucose to a
concentration of 1 mM (30 GBq mol1) in a
total volume of 50 µl. Standard lipid concentration was 10 mM (1 mM MGlcDAG substrate in addition to the
activator DOPG (or CL) and the nonbilayer-prone 1,3-DOG, with DGlcDAG
as balance). After 30 min of incubation at 28 °C, the reaction was
stopped with 375 µl of methanol/chloroform 2:1 (v/v), and the lipids
were extracted and separated by TLC (10, 19). The lipid products on the
TLC plates were visualized and quantified by electronic autoradiography
(Packard Instant ImagerTM). All assays were done in
duplicate. A variety of glycolipids was used as TLC references
(20).
Localization of alMGS-GFP Fusion Variants--
Full-size (398 aa), N- (aa 1-226), and C-domain (aa 227-398) alMGS, respectively,
were cloned upstream and in-frame with the gene for green fluorescent
protein (GFP) using a PCR primer approach based on the alMGS sequence
(6) and the vector pGFPuv (Clontech), in E. coli JM109. Ligation, transformation, and selection followed standard procedures, and the obtained constructs were verified by DNA
sequencing. Enzymatic activities of the hybrid proteins were analyzed
after induction with
isopropyl-1-thio--D-galactopyranoside by labeling
of E. coli lipids in vivo with radioactive
acetate (6) and by assay for MGlcDAG synthesis after detergent
solubilization in vitro (6). Localization of the MGS-GFP
variants in E. coli was analyzed by fluorescence microscopy
at various growth and induction conditions, using a Zeiss Axioplan2
microscope, fluorescein isothiocyanate filters, a CCD camera (Sony),
and Image Access 3.0 software (Imagic Bildverarbeitung AG).
Sequence Analysis and Structure Prediction-- To find homologs to the alDGS and to predict a function for the genes in flanking regions, PSI-BLAST (21) at NCBI in the nonredundant data base and the data base for finished and unfinished microbial genomes, was used. Preliminary genomic sequence data were obtained from The Institute for Genomic Research. Features of the primary structure of the alDGS sequence were further analyzed with tools available at the ExPASy Molecular Biology Server (www.expasy.ch) (Swiss Institute of Bioinformatics) and with the Wisconsin Package Version 9.1 (Genetics Computer Group, Madison, WI).
Potential secondary and three-dimensional structure models were predicted from aa sequences by several local structure (PsiPred, Target99, and Jpred2) and fold recognition methods (FFAS, 3D-PSSM, GenTHREADER, mGenTHREADER, INBGU, Sam-T99, and FUGUE) and jointly evaluated using the Pcons consensus method (22) at the MetaServer (www.bioinfo.pl) (23). The scores stating the similarity between the obtained models are derived from Levitt and Gerstein (cf. Ref. 22). The aa sequences from several known glycosyltransferase structures were analyzed ("benchmarked") in a similar manner (with PDB Test, www.bioinfo.pl) and the suggested PDB structures compared with the DALI method (24).
Structural models of the glycosyltransferases were built, based on the sequence alignments and recognized folds given by the MetaServer (above), using the Swiss-PdbViewer program for alignment (Version 3.7; www.expasy.ch/spdbv/) (25) and the optimize-mode of the SWISS-MODEL program server (Version 3.5; www.expasy.ch/swissmod/SWISS-MODEL.html) for automatic model building. For the MGS sequences the three protein structures of ecMurG (PDB-ID 1F0K), ecEpim (PDB-ID 1F6D), and aoGtfB (PDB-ID 1IIR) were used as templates, while the DGS sequences were modeled with the first two (ecMurG and ecEpim) as templates. All five proteins were successfully modeled. Although the models obtained from the SWISS-MODEL server are already energy minimized, they were subjected to an additional global energy minimization. Prior to the final minimization a few side chains were adjusted as they were trapped within the ring of aromatic side chains, a situation an energy minimization would not be able to correct. For the global energy minimization the Swiss-PdbViewer implementation of the GROMOS96 43B1 force field was used, together with the SWISS-MODEL server minimization protocol, namely 200 steps of steepest descent, followed by 300 steps of conjugate gradient minimization. The quality of the models was accessed using the WhatCheck program (26). Identification of secondary structure elements, calculation of the electrostatic surface potential, and visualization were made using the program MOLMOL (27). The electrostatic potentials were calculated assuming point charges on heavy atoms, dielectric constants of 2 and 80 for protein and solvent, respectively, an ionic strength of 0.3 M, a 2-Å salt radius, and a boundary condition of zero potential at 15 Å. A solvent radius of 1.4 Å was used when the accessible van der Waals surfaces of the heavy atoms of each model were identified. The relative solvent accessibility of residues was calculated from their coordinates and divided into three groups; buried (0-5%), intermediate (5-25%) and exposed (25-100%) (28).
Nucleotide Sequence Accession Number--
Nucleotide sequence
data for A. laidlawii DGlcDAG synthase have been deposited
at GenBankTM with accession number AY078412.
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RESULTS AND DISCUSSION |
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Gene Cloning and Organization--
The N-terminal and an internal
amino acid sequence of the purified DGlcDAG synthase from A. laidlawii (alDGS) were determined by Edman degradation (10).
Designed degenerated PCR primers yielded a gene-specific probe, which
was used in a Southern blot procedure identifying a 2.2-kbp DNA
fragment that was cloned. The sequence of this contig (Table
I) revealed an open reading frame (ORF)
of 999 bp coding for the alDGS sequence named ALdgs (Fig.
1) with a potential Shine-Dalgarno
sequence starting at position 16. The G+C content was 33%, which is
typical for A. laidlawii DNA (31.7-35.7% (29)). The
translated ORF found just downstream the ALdgs gene was
related to proteins from the aldo/keto-reductase family. The sequence
further downstream show similarity to trigger factor, a conserved
chaperone found in all prokaryotes. No ORFs in the opposite direction
were found. The A. laidlawii contigs containing the
preceding glucosyltransferase gene ALmgs cloned earlier (6)
and the ALdgs determined here (Table I) have no nucleotide
sequence in common and were not localized to the same DNA fragment
according to hybridization. Hence, it was concluded that these two
glycosyltransferase (GT) genes are not adjacent to each other on the
chromosome and do not belong to the same operon.
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Homologs and Analogs to alDGS--
BLAST searches in the
finished/unfinished and the nonredundant data bases at NCBI with the
alDGS amino acid sequence gave several significant hits in eubacteria
and archaea (Table II). All the top hits
were Gram-positive sequences, and most were from pathogenic
species. The best score showed a gene in Enterococcus faecium with 49% aa identity to the alDGS, and sequences from streptococcal or other closely related species (Lactococcus) had an
identity above 45%. These species do all contain an
-diglycosyldiacylglycerol identical to the A. laidlawii one (30, 31), and the sugar moieties are solely glucose.
In the corresponding glycolipid from S. pneumoniae
(cf. Table II) the outer glucose is replaced by a galactose.
For the alMGS glucosyltransferase aa identities of 31 and 29% were
recorded to similar enzymes in S. pneumoniae and Borrelia burgdorferi, and these were shown to encode
homologous functions to the A. laidlawii enzyme (6). Most
important, alDGS and all the sequences of Table II contain the
conserved EX7E motif characteristic for the
retaining (
) GTs of CAZy family 4 of glycosyltransferases (32); the
preceding enzyme alMGS (cf. Introduction) also belongs to
this family (6). They also contain a typical motif for
glycosyltransferases belonging to Pfam family 00534 (conserved domain
search). Proteins in this family transfer nucleoside
diphosphate-linked sugars, like glucose, galactose, mannose, and
X-glucose, to a variety of acceptor substrates such as glycogen and
lipopolysaccharides.
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The BLAST searches revealed no proteins with an established function,
since many of them are from recently published genome projects and so
far lack annotation. However, the S. pneumoniae gene in
Table II was first sequenced by Grebe et al. (33) and named
cpoA. This protein was proposed to be involved in resistance to -lactams and was isolated from first-step piperacillin-resistant mutants. The cpoA and the consecutive ORF5 gene, both part
of the same operon, were suggested (but not shown) to be involved in
formation of the linkage unit in lipoteichoic acid or polymerization of
teichoic acid precursors prior to translocation (33). We showed
recently that this ORF5 encoded an enzyme homologous in function to
alMGS (cf. step (i) in Introduction) and that was named
spMGS. This enzyme was of similarly high sequence identity to alMGS as
the CpoA protein is to alDGS (cf. Table II and Fig. 1). Most
intriguing, the majority of organisms with a potential alDGS homolog
(Table II) also encode a homolog to alMGS, according to
BLAST sequence analyses (data not shown) (6). The fact that the
homologs of DGS and MGS genes in S. pneumoniae were adjacent to each other (above), potentially in an operon organization, was true
also for most of the species in Table II. However, not for A. laidlawii and the related species Clostridium
acetobutylicum and not for Pyrococcus furiosus (data
not shown). Since the function of the S. pneumoniae MGS
enzyme (i.e. ORF5, see above) has been verified (6), the
adjacent gene cpoA was PCR-cloned (see "Materials and
Methods") to establish its function (cf. below).
The BLAST searches also revealed analogs in the three archaeal species
Halobacterium sp. NRC-1, Pyrococcus horikoshii
and P. furiosus (Table II); all sequences are of unknown
function. The membrane of Halobacterium spp. contains the
major glycolipid 3-HSO3--Galp-(1-6)-
-Manp-(1-2)-
-Glcp-(1-1)-diether
and several minor glycolipids. Since alDGS, and all CAZy family 4 members (32), are retaining enzymes the Halobacterium analog
(Vng0600c, Table II) can be proposed to catalyze the glycosylation with
the second mannose, and the adjacent Vng0598c (6), the first glucose. In another thermophile Thermotoga maritima, a DGlcDAG was
found with the rare
(1
4) diglucosyl structure (34), and an
analog (pir C72340) with low score to alDGS was found by BLAST (Table II). Hence, the A. laidlawii alDGS glucosyltransferase gene
has homologs in a number of especially Gram-positive pathogens, and analogs in archaea and thermophiles (cf. Table II). Most of
these homologs are encoded next to an alMGS homolog, most likely in an
operon organization.
Gene Function--
Enzymatic synthesis of A. laidlawii
DGlcDAG from MGlcDAG, with crude native cell proteins or purified
alDGS, only yielded the -DGlcDAG product; no other glucose acceptor
than
-MGlcDAG was active (like
or Gal variants) (10, 11). Here,
alDGS and S. pneumoniae CpoA were cloned and overexpressed
in E. coli and enzymatic activity assays verified the
function (see "Materials and Methods"). As expected a new lipid
product was synthesized by the cloned alDGS when the assay mixture was
supplemented with
-MGlcDAG purified from A. laidlawii and
radiolabeled UDP-Glc (Fig. 2, lane
2). This lipid, tentatively identified as the
1,2-diacyl-3-O-[
-D-glucopyranosyl-(1
2)-O-
-D-glucopyranosyl]-sn-glycerol
(DGlcDAG), showed the same TLC migration as the lipid product
obtained from corresponding syntheses using native, crude or purified,
DGS enzyme from A. laidlawii (Fig. 2, lane 1)
(10), and several diglycosyl-DAG reference lipids (20). Assays
with
-MGalDAG or UDP-Gal yielded no products.
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The sequence similarity between alDGS and CpoA (Fig. 1) suggested the
latter to possess a similar function as alDGS. All combinations of the
lipid substrates -MGalDAG and
-MGlcDAG, and the radiolabeled nucleotide sugars UDP-Gal and UDP-Glc, were tried. All assays were
supplemented with the anionic DOPG. Fig. 2 (lane 3-6) shows that the overexpressed CpoA (spDGS) was able to catalyze the
glycosylation of only
-MGlcDAG by using the sugar nucleotide
UDP-Gal. This is in agreement with earlier characterization of the
lipid composition in S. pneumoniae, where the two major
glycolipids were characterized to be monoglucosyl diacylglycerol
(MGlcDAG) and galactosyl-glucosyldiacylglycerol (GalGlcDAG) (36, 37),
and where all sugars were in
-configuration and identically
linked as in A. laidlawii (38, 39). We have recently characterized the ORF5 gene adjacent downstream to
cpoA as encoding the S. pneumoniae
-MGlcDAG glucosyltransferase (spMGS) (6). Hence, the CpoA protein is
the consecutively acting spDGS, analogous to the alDGS above.
Furthermore, synthesis of DAG, MGlcDAG, and GalGlcDAG could be achieved
in a joint CHAPS extract of three E. coli clones separately
expressing each of a phosphatidic acid phosphatase identified by us,
spMGS (6) and spDGS (this work), respectively (S. pneumoniae
genes sp0489, sp1076, and
sp1075 (40)), data not shown. Consequently, an efficient
synthesis of this lipid pathway can be achieved without the genes
in an operon, cf. biosynthetic route in introduction.
Modulation by Anionic and Nonbilayer Lipids--
The native alDGS
enzyme is very responsive toward the presence of certain anionic and
nonbilayer lipids, as well as some phosphorylated metabolites and the
redox state (7, 10, 12). These features are likely to constitute the
basis for regulation of the nonbilayer/bilayer packing conditions in
the A. laidlawii membrane. Packing properties brought by
nonbilayer-prone molecules (curvature elastic stress) can be
qualitatively analyzed also in mixed lipid-detergent bilayer-like aggregates (41-43). Assays (see "Materials and Methods") for the DGlcDAG synthase from A. laidlawii and the GalGlcDAG
synthase from S. pneumoniae (CpoA/spDGS) showed that both
enzymes respond to PG in a similar manner, but that only the A. laidlawii variant was stimulated by CL, and strongly by the
nonbilayer-prone 1,3-DOG (Fig. 3).
Activation of alDGS correlated with the amount of negatively charged
lipid phosphates in the mixed micelles; CL has two phosphates per mole
lipid compared with one in PG. 1,3-DOG behaves similarly to 1,2-DOG,
which has a well established ability to shift the phase equilibria of
membrane lipids toward nonbilayer aggregates (reversed
type) (44). The combined effect of CL and 1,3-DOG was a very strong
stimulation of alDGS (Fig. 3, top), perhaps also promoted by
the nonbilayer-prone character of the unsaturated CL used. It is very
evident that spDGS responds differently to the immediate lipid
environment (Fig. 3, bottom). In S. pneumoniae CL
is a major membrane component and larger than PG (45), as is GalGlcDAG
(37). Reasons why CL did not activate spDGS may be either that the
enzyme does not bind (unlikely) alternatively that binding gives an
unfavorable orientation at the membrane or that the larger negative
potential from the lipid surface repels the acidic C-domain
(cf. pI in Table III),
abolishing enzyme activity. The lack of response for spDGS toward the
nonbilayer-prone 1,3-DOG may indicate that this S. pneumoniae enzyme is not designed to sense such packing
properties. In A. laidlawii strains there is a correlation
between low MGlcDAG amounts, and hence more DGlcDAG, and high CL
amounts (46, 47). Yet, in A. laidlawii A-EF22 CL is lacking.
Most important, the response here for the cloned alDGS (Fig. 3) is in
agreement with the purified native enzyme (10, 12). The differences in
response toward CL between alDGS and spDGS are analogous to what was
recorded for the preceding alMGS and spMGS enzymes versus CL
amounts (6). Hence, these A. laidlawii and S. pneumoniae lipid GTs must have certain differentiating features in
their sequence properties.
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Sequence Features and Properties--
Fig. 1 shows the sequences
of some selected homologs/analogs to alDGS (Table II), including the
cloned CpoA/spDGS with an established function (above). Conserved
residues are spread along the whole sequences but concentrated to two
regions: one contains the triplet
Arg170, Lys171, Gly172 in
the aa stretch 161-192 in alDGS, and the other includes ~30 aa
around the characteristic EX7E-motif of CAZy GT
family 4 (aa 242-250 in alDGS). The most variable region is the end of
the C-terminal part. However, a Conserved domain search (cf.
"Materials and Methods") proposed the second half of the alDGS
sequence to contain a nucleotide-binding domain. Mutant studies of the
-mannosyltransferase AceA (also CAZy family 4) suggested three aa
positions (Lys211, Glu287, and
Glu295) to be involved in the catalytical mechanism (48).
We propose Lys171, Glu242, and
Glu250 to be the corresponding residues in alDGS, all
conserved in the two regions with higher identity. A pair-wise
alignment with BLAST2 (not shown) revealed a similar region between the
two A. laidlawii glucosyltransferases at position 181-346
(alMGS) and 129-294 (alDGS), with 28% identical residues
(cf. Figs. 1 and 5). The alDGS, and its homologs/analogs (in
Fig. 1), have not any predicted transmembrane segments (HMMTOP (49)),
yet DGS is associated with the membrane (10, 50). Likewise, the main
portion of the CpoA was found in the membrane fraction, suggesting that
it associates with membrane components in vivo (33).
A high pI value is typical for membrane-associated proteins (51), and
essentially all membrane lipid surfaces are negatively charged due to
their anionic lipids (low pKa values), e.g. the alMGS enzyme binds especially to phospholipids like
PG.2 alDGS has a calculated pI around 9.4 (Table III),
hence is positively charged at physiological pH, and charged residues
do frequently appear as pairs. The N-terminal half is predominantly
positively charged, with a calculated pI of 9.7. These charge features
were not conserved among the closest analogs and the pI (full size) varies from 4.7 (Halobacterium) to 9.1 (Streptococcus
pyogenes). The characteristic highly positive N-terminal half was
also the case for the proteins from S. pyogenes and
Streptococcus mutans. A common pattern for other
homologs/analogs was an N-terminal half with higher pI and a C-terminal
half with lower pI (Table III). Note the substantially lower pI values
for the two S. pneumoniae enzymes compared with the A. laidlawii ones, especially for the C-terminal domains (Table III).
This may constitute the basis for the different response toward CL of
the A. laidlawii and S. pneumoniae GTs (Fig. 3
and 4).
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Fold Recognition and Structure Modeling--
A number
of established local structure prediction and fold recognition methods
(23), with the Pcons evaluation method (22), were used to analyze the
potential structural relationships for alDGS, spDGS, and alMGS (Table
IV in the Supplemental Material) and spMGS. A substantial similarity
was proposed to the three-dimensional structures of the
glycosyltransferases MurG, GtfB, and the soluble UDP-GlcNAc
2-epimerase, which are all strongly related to phage T4
-glucosyltransferase. The predicted sequence alignments were the
basis for an alignment in Swiss-PdbViewer and model building in
SWISS-MODEL. The proteins alMGS, spMGS, and csMGS were modeled onto the
template protein structures of ecMurG, GlcNAc-epimerase, and aoGtfB,
while modeling of alDGS and spDGS were performed with only the first
two templates (see Supplement Material and Refs. 53-57). A
presentation of the structural models and their surface charge
potentials are shown in Fig. 4, and some important quality indicators
of the models are given in Table V (see the Supplemental Material). In
general the model structures have normal (fairly good) packing quality
and Ramachandran plot behavior, together with distinct energy minima.
Note the general similarity of the cucumber
-MGalDAG synthase GT to
the four acholeplasma and streptococcal
-GTs. Hence, these
glycosyltransferases, most of which are membrane-associated, seem to
conform to a similar structural design despite low sequence similarities.
Membrane Association--
The established binding properties of
alMGS to anionic phospholipid bilayers (6), the look of the potential
active-site clefts, and the bilayer localization of the DAG and MGlcDAG
acceptor lipid substrates, strongly support the positively charged
bottom faces indicated (Fig. 4) as the lipid bilayer-binding surfaces of the enzymes. These model proposals were further supported by extensive sequence segment comparisons (data not shown) with 11 proteins of known structures of which
most bind to lipid membranes3
and to the sequences of another 10 proteins established to bind to
lipids and/or membranes.4 For both A. laidlawii
enzymes sequence similarities were confined to six regions, with
multiple hits to each region. In most instances, the proposed structure
of a certain alDGS or alMGS segment (as in Fig. 4) corresponded to the
structure in the target protein, provided the sequence similarities
were sufficiently high. This was the case for certain amphiphilic
helices, some -strands, and especially several, more hydrophobic
loops at the bottom surface and active site clefts. Such loops, flanked
by or containing positively charged residues, are important
lipid-binding segments in several proteins like the C2 domains in human
Factor V and phosphatidylinositol-phospholipase C
(58, 59).
Furthermore, no sequence segment similarities were recorded for the
structures of several interface-anchored integral proteins
(prostaglandin synthase and squalene cyclase).
Potential lipid surface- or hydrophobic interface-associating segments
were predicted by the Membrane Protein
Explorer.5 Several obvious
differences were noted (Fig. 5),
e.g. alDGS has more exposed hydrophobic segments than spDGS
and notably one stronger in the C-domain. These two also differ by two
sequence gaps in their in the N-domain. The soluble epimerase reveals
no hydrophobic stretch, and MurG has one in the N-domain (Fig. 4), also
suggested to be the membrane-binding (amphiphilic) segment (52). The
latter seems to have counterparts at similar locations in the three MGS enzymes. These proposals indicate important differences in charge and
hydrophobicity between alDGS and spDGS, which may be related to their
different behavior toward the anionic CL activator and nonbilayer DOG
additive. Likewise, similar differences between alDGS and alMGS may be
the basis for the strong stimulation of alDGS by nonbilayer-prone
lipids.
|
Domain Charge and Membrane Binding in Vivo--
The N-
and C-domains of alMGS have a high and low calculated pI, respectively
(Table III), which should yield different membrane binding. DNA
encoding these domains, and full-size alMGS, were ligated in front (in
frame) of the gene for GFP in the pGFPuv vector and transformed into
E. coli JM109 wt (see "Materials and Methods").
Full-size, but not the N- and C-domain alMGS hybrids, displayed
enzymatic activity as revealed from synthesis of radioactively labeled
MGlcDAG by E. coli in vivo, as well as after
detergent solubilization in vitro (data not shown), similar
to the cloned enzyme (6). Localization of the GFP fusion proteins was
assayed by fluorescence microscopy at various growth and gene induction conditions (Fig. 6). A membrane
association in E. coli of the full size alMGS-GFP hybrid was
evident (Fig. 6A), very similar to a MinD-GFP hybrid (cell
division protein) of established phospholipid binding properties (60).
The low pI of the native GFP protein should prevent membrane binding,
as is indicated in Fig. 6D. Likewise, the C-domain-GFP
hybrid had a cytoplasmic distribution, similar to the native GFP, with
an occasional localization to an intracellular structure (Fig.
6C). A membrane distribution of the N-domain-GFP with a
strong enrichment to the cell poles (Fig. 6B) is similar to
the GFP fusion of the E. coli integral membrane, secretion protein YidC (61), and MinD-GFP under control by MinE (62). The alMGS
N-domain-GFP was released from the cell poles to the cytoplasm by a
slight increase in NaCl concentration (data not shown). Furthermore,
the GelK N-domain (high pI, cf. Table III), but not the
C-domain (low pI), binds to the acidic E. coli membrane (63). Similar pI values are also valid for mannosyltransferase AceA
(Table III), which is found associated with the membrane (48). Clearly,
the three GFP variants of the MGS domains have different membrane
binding properties in vivo.
|
E. coli membrane lipids contain ~25 mol % anionic lipids (PG plus CL), sufficient to yield binding of purified alMGS to defined liposomes in vitro.2 CL promotes binding and activity of alMGS, and activity of alDGS (Fig. 3), better than PG (6).2 It has been shown by different techniques that the cell poles of E. coli are enriched in the anionic CL compared with the average membrane (64, 65). The different in vivo binding properties of the alMGS domains, correlating to protein/domain charge (Table III), is analogous to the differences recorded for C1 and C2 membrane lipid targeting domains in many peripheral proteins (35). The latter yields a synergistic and/or regulatory interplay between targeting and activation (35). Substantial differences between the alDGS and alMGS N- and C-domains were revealed here, and the homologous spMGS and spDGS are both more similar to alMGS in this respect (Table III). Most likely this will affect the binding, activities, and responsiveness of the various enzymes, as is indicated in Fig. 3 and Ref. 6.
Conclusions--
From experimental data, the predicted structural
models, and the comparisons with many lipid-associating proteins, it is
evident that these lipid glucosyltransferases are associated with the membrane lipid interface. Hence, the sensing of the bilayer packing properties and the regulatory activities must be confined to the exact
mechanisms by how the N- and C-domains of these enzymes are anchored
and activated, as indicated from alDGS, spDGS, alMGS, and spMGS here.
This is analogous to the C1 and C2 membrane targeting domains in many
eukaryotic peripheral proteins. The different amounts of charged and
hydrophobic residues exposed on the enzyme bilayer-binding surfaces
(see Figs. 4 and 5) support that lipid surface charge and curvature
elastic packing stress might affect the orientation and potentially the
conformation between the N- and C-terminal domains. This can be coupled
to the interactions of the soluble and lipid substrates and to other
stimulating agents like metabolic phosphates and DNA.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Viola Tegman for excellent technical assistance, Dr. Mari Norgren for the S. pneumoniae strain, Hanna Eriksson for constructing the GFP hybrids, Petra Björk and Dr. Lars Wieslander for microscope access, and Dr. Anders Berglund and Dr. Arne Elofsson for valuable discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by the Swedish Science Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY078412.
The on-line version of this article (availabe at
http://www.jbc.org) contains supplemental text, supplemental Tables IV
and V, and supplemental Refs. 1-8.
§ Both authors have contributed equally to this work.
¶ Present address: Dept. of Microbiology, Colorado State University, Fort Collins, CO 80523-0015.
To whom correspondence should be addressed. Tel.:
46-8-16-24-63; Fax: 46-8-15-36-79; E-mail: ake@dbb.su.se.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M211492200
2 L. Li, submitted for publication.
3
E. coli SRP receptor FtsY and
D-lactate dehydrogenase, Neisseria meningitidis
glycosyltransferase LgtC, B. subtilis glycosyltransferase SpsA, cytochrome c (several species), human Factor V C2
domain, galactose oxidase A, phosphatidylinositol-phospholipase
C, synaptotagmin C2 domain, human pleckstrin, and Epsin 1.
4
E. coli proteins DnaA, SecA,
phosphatidylserine synthase, RecA and PutA, rat phosphocholine
cytidylyltransferase, A. xylium glycosyltransferase AceA,
mouse -spectrin, and human dynamin.
5 S. Jaysinghe, K. Hristova, and S. H. White (blanco.biomol.uci. edu/mpex).
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
DGlcDAG, 1,2-diacyl-3-O-[-D-glucopyranosyl-(1
2)-O-
-D-glucopyranosyl]-sn-glycerol;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate;
MGlcDAG, 1,2-diacyl-3-O-(
-D-glucopyranosyl)-sn-glycerol;
GalGlcDAG, 1,2-diacyl-3-O-[
-D-glucopyranosyl-(1
2)-O-
-D-galactopyranosyl]-sn-glycerol;
1, 3-DOG, 1,3-dioleoylglycerol;
MGalDAG, 1,2-diacyl-3-O-(
-D-galactopyranosyl)-sn-glycerol;
MAMGlcDAG, 1,2-diacyl-3-O-[6-O-acyl(
-D-glucopyranosyl)]-sn-glycerol;
MADGlcDAG, 1,2-diacyl-3-O-[
-D-glucopyranosyl-(1
2)-O-(6-O-acyl-
-D-glucopyranosyl)]-sn-glycerol;
DGS, disugar-glycolipid synthase;
MGS, monosugar-glycolipid synthase;
PG, phosphatidylglycerol;
DOPG, dioleoylphosphatidylglycerol;
DOPA, dioleoylphosphatidic acid;
CL, cardiolipin;
GT, glycosyltransferase;
GFP, green fluorescent protein;
aa, amino
acids;
ORF, open reading frame.
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