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Journal of Bacteriology, March 2000, p. 1432-1436, Vol. 182, No. 5
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Novel Missense Mutations That Affect the Transport
Function of MalK, the ATP-Binding-Cassette Subunit of the
Salmonella enterica Serovar Typhimurium Maltose
Transport System
Sabine
Hunke,
Heidi
Landmesser, and
Erwin
Schneider*
Institut für
Biologie/Bakterienphysiologie, Humboldt-Universität zu Berlin,
D-10099 Berlin, Germany
Received 11 August 1999/Accepted 8 December 1999
 |
ABSTRACT |
We report on novel mutations in the malK gene of
Salmonella enterica serovar Typhimurium, encoding the
ATPase subunit of the maltose transporter (MalFGK2).
Biochemical analysis suggests that (i) L86 might be involved in a
signaling step during substrate translocation and (ii) E306 may be
critical for the structural integrity of the protein.
| |
TEXT |
In enterobacteria, such as
Escherichia coli and Salmonella enterica serovar
Typhimurium, the uptake of maltose-maltodextrins is mediated by the
MalFGK2 transport complex, which is a member of the
superfamily of ATP-binding-cassette (ABC) transport proteins (1). MalF and MalG are hydrophobic membrane-integral
components, while MalK represents the ABC subunit of the complex. In
addition, a soluble extracellular (periplasmic) substrate-binding
protein, MalE, is also required for activity. When separated from the
hydrophobic subunits, MalK displays a spontaneous ATPase activity
(10, 19), while in the MalFGK2 complex ATP
hydrolysis is observed only concomitantly with transport (3,
7). Thus, ligand translocation and ATP hydrolysis are dependent
on a signaling mechanism initiated by the substrate-loaded binding
protein and transmitted through MalF-MalG (4). Besides being
indispensable for transport, MalK displays a regulatory function by
phenotypically acting as a repressor of genes belonging to the maltose
regulon (1). The mechanism of this activity is currently
unknown, but evidence for binding of MalK to MalT, the positive
regulator of the maltose regulon, has recently been presented
(12). These and other functions can be separated by
mutations (8, 9); this finding, together with data from the
study of half molecules and chimeras (13), has led to the
proposal of a domain structure as presented in Fig.
1. Accordingly, the protein is composed
of two structurally distinct entities, with the regulatory functions
being mostly confined to the C-terminal extension of about 100 residues
by which MalK differs in length from a prototype ABC domain
(15). However, recent results from this laboratory also
indicated that the C-terminal domain may not simply be hooked to a
consensus ABC domain to provide a regulatory activity but is an
integral part of the tertiary structure (13). To further
investigate the structure-function relationship between the proposed
domains of MalK, we have used chemical mutagenesis to isolate novel,
previously uncharacterized mutations in the malK gene of
Salmonella serovar Typhimurium.
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FIG. 1.
Proposed domain structure of MalK. The relative
positions of mutated residues described in this communication are
indicated. See text for details (modified from reference
21).
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|
Random mutagenesis, sequencing, and characterization of mutants in
vivo.
Serovar Typhimurium and E. coli strains,
plasmids, media, and culture conditions were previously described
(13, 14, 21). Chemical mutagenesis of the malK
gene was performed by treatment of plasmid pSW7 (malK under
the control of the trc promoter on plasmid vector pSE380)
(20) with hydroxylamine as described elsewhere
(21). Purified plasmid DNA was subsequently used to transform the MalK
strain ES25 (17).
Transformants were screened for mutations on MacConkey
agar-maltose-ampicillin plates. Plasmid DNA from four putative
candidates (white colonies) were isolated and subjected to nucleotide
sequence analysis using a LiCor semiautomatic sequencer (MWG Biotech,
Ebersberg, Germany). Base changes translating into the following amino
acid substitutions were found: L86F-R113C (malK819), P160L
(malK824), D165N (malK825), and E306K
(malK822). For further analysis of malK819, both
changes were separately introduced into the wild-type allele (on pSW7)
by means of site-directed mutagenesis (QuikChange kit; Stratagene) and
the resulting plasmids were used to transform ES25. While plasmid
pSH17-N42, carrying the malK821 allele (R113C), gave rise to
red colonies on MacConkey agar-maltose plates, expression of
malK820 (L86F) from plasmid pSH17-N41 resulted in white
colonies. Thus, the growth defect of malK819 was due solely
to the L86F mutation (malK820), and consequently, the
malK821 allele was not further considered.
The failure of the remaining MalK mutants to support growth of strain
ES25 on maltose was confirmed by transport assays that were performed
as described in reference 17. The initial rates of
[14C]maltose uptake never exceeded 4% of the rate
measured with control cells that harbored the plasmid-borne wild-type
allele (pSW7) (Table 1). All mutant
proteins were stably produced as verified by immunoblot analysis of
whole-cell extracts. Interestingly, the E306K mutation caused a
mobility shift of the protein (not shown, but see Fig. 2).
None of the substitutions eliminated the repressing activity of the
MalK variants on MalT-dependent gene expression, as analyzed by
assaying 
-galactosidase activity of strain SK1280 transformed with
the plasmid-borne mutant alleles. Strain SK1280 carries a chromosomal
malK-lacZ fusion that results in a maltose-negative phenotype but places the lacZ gene under the control of the
pmalK promoter (8). Similar to the
wild type, the mutant alleles substantially reduced expression of the
lacZ fusion (Table 1).
Analysis of nucleotide-binding activity in membrane vesicles.
To further elucidate the functional defects of the mutant proteins, we
first tested for their capability to bind nucleotides. To this end,
inside-out membrane vesicles were prepared from cells of strain ES25
transformed with the respective derivatives of pSE380 and subjected to
photo-cross-linking with the ATP analog [
-32P]N3-ATP as described in reference
19. Results from such an experiment demonstrated
that all variants reacted with the probe (not shown). Quantitation by
densitometric scanning of the autoradiographs revealed that the mutant
proteins incorporated between 48 and 92% of the label relative to the
wild type. Preincubation of the samples with excess unlabeled ATP
resulted in a substantial reduction of photo-cross-linking, indicating
the specificity of the reaction. Thus, it is concluded that
nucleotide-binding activity was basically retained in each mutant protein.
Biochemical properties of purified MalK variants.
Next, the
effects of the mutations on ATP-induced conformational changes and on
ATPase activity were investigated for the purified proteins (Fig.
2). The L86F, P160L, and D165N mutants were purified by published methods (16, 19) from E. coli strain BL21(DE3) carrying derivatives of plasmid pES67
(16). In the case of the P160L mutant, the strain
additionally harbored plasmid pOFX-T7-SL1, which encodes the E. coli GroESL chaperones (2), to increase the amount of
soluble protein. The E306K mutant was purified as an N-terminal
hexahistidine fusion from strain BL21(DE3)(pOFX-T7-SL1) carrying the
respective derivative of plasmid pGS91-1 (14) by red agarose
chromatography (16), followed by nickel-nitrilotriacetic acid chromatography according to the manufacturer's instructions (Qiagen). Wild-type MalK was isolated by any of these methods, using
plasmids pES67 (16) and pGS91-1 (14).
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FIG. 2.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis of purified MalK wild-type and mutant proteins. A total
of 5 µg of protein was loaded in each well, electrophoresed, and
stained as described in reference 17. Lanes: 1, molecular mass standards; 2, wild type, prepared as described in
reference 19; 3, L86F mutant; 4, wild type, prepared
as described in reference 16; 5, P160L mutant; 6, D165N mutant; 7, wild type (His6-MalK); 8, His6-E306K mutant.
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|
The purified proteins were subjected to limited proteolysis with
trypsin to monitor a conformational change upon interaction with MgATP
(18). Representative results from such an experiment are
shown in Fig. 3. Typically, MalK is
rapidly cleaved at R66, R146, R153, and R185 into four major,
transiently occurring peptide fragments (T1 to T4) (18).
Increasing concentrations of MgATP stabilize mature MalK and fragment
T1 (Fig. 3A). Thus, binding of the nucleotide renders potential
cleavage sites less accessible to the protease, indicating a change in
protein tertiary structure. The L86F, P160L, and D165N variants
displayed similar fragment patterns except that resistance to trypsin
required the presence of 2 mM MgATP (Fig. 3B; data shown for L86F
only), suggesting that the mutations somewhat lowered the affinity of
the proteins for the nucleotide. In contrast, the peptide fragments
obtained from the E306K mutant exhibited intrinsic resistance against
further degradation that was unaffected by 0.2 mM MgATP. However, 2 mM MgATP substantially protected the mature form of the protein
against the protease (Fig. 3C). Although the mutation created a
potential new tryptic cleavage site, no change in the apparent
molecular masses of the accumulated fragments was observed, indicating
that the residue is rather inaccessible to the protease. Thus, the data
suggest that the mutation might cause a structural alteration of the
protein.
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FIG. 3.
Limited proteolysis of purified MalK variants. Mutant
proteins were digested with trypsin at a concentration of 48:1 (by
weight) in the absence or presence of ATP. Samples were withdrawn at
the indicated times and subsequently analyzed by immunoblotting as
described previously (18). (A) Wild type, prepared according
to reference 16; (B) L86F mutant; (C) E306K mutant.
The relative positions of the previously recognized major cleavage
products (T1 to T4) (18) are indicated. Fragments T2 and T3
were not separated under the conditions used here. Lane 1, undigested
control. Results from representative experiments are shown.
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|
Analysis of the ATPase activities of the purified variants, carried out
as described elsewhere (10), revealed reduced rates of
hydrolysis for the P160L, D165N, and E306K variants relative to the
corresponding wild-type preparations (Fig.
4). Thus, the data provide a reasonable
explanation for the failure of these mutant proteins to support maltose
transport in vivo. In contrast, the ATPase activity of the L86F protein
compared favorably to that obtained with the wild type (Fig. 4), giving
rise to the hypothesis that the effect of the mutation may become
evident only in the assembled transport complex.
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FIG. 4.
ATPase activity of purified MalK variants. Assays were
performed as described previously (10). Rates of hydrolysis
are compared to those recorded with wild-type (WT) proteins that were
prepared by the same protocol. WT proteins were purified from the
soluble fraction of strain BL21(DE3)(pLysS, pES67) (16) (a),
from the insoluble fraction of the same strain by a
denaturation-renaturation protocol (19) (b), and from the
soluble fraction of strain BL21(DE3)(pGS91-1, pOFX-T7-SL1) by
nickel-nitrilotriacetic acid-agarose chromatography (c). The data are
the averages of at least two independent assays.
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ATPase activity of mutant transport complexes in
proteoliposomes.
To test the above notion, we analyzed the
maltose-binding protein (MalE)-maltose-stimulated ATPase activity of
the mutant transport complex incorporated into liposomes. To this end,
wild-type and mutant transport complexes were solubilized with
octylglucoside (1.5%) from membranes of strain ES54
(
atpB-D), harboring pES62-97 (malF malG under
the control of the trc promoter) (7) and pGS91-1 (malK+) (14) or pSH60-N41
(malK820). Subsequently, solubilized proteins were
mixed with sonicated soybean phospholipids in the presence or absence
of maltose and purified MalE (7) and proteoliposomes were
formed by detergent dilution according to reference
3. ATPase activity of proteoliposomes was monitored
as described previously (19). As shown in Fig.
5A, the mutant transport complex containing the L86F variant exhibited <10% of the enzymatic activity monitored with a wild-type complex. By immunoblot analysis using polyclonal antisera raised against MalK, MalF, and MalG, it was confirmed that comparable amounts of complex proteins were solubilized (Fig. 5B) and recovered in the proteoliposomes (not shown). These data
are consistent with the above hypothesis.
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FIG. 5.
(A) ATPase activity of the complex containing the MalK
L86F variant incorporated into liposomes. Proteoliposomes containing
wild-type or mutant transport complexes were formed in the presence
(filled symbols) or absence (open symbols) of maltose and
maltose-binding protein (MalE) and assayed for ATPase activity as
described in reference 7. Symbols:  and  ,
wild-type complex;  and  , MalFG(KL86F)2. (B)
Immunochemical detection of the individual subunits in the
octylglucoside-solubilized fractions from membranes containing
wild-type or mutant complexes. The reactions corresponding to MalK (K),
MalF (F), and MalG (G) are indicated by arrows.
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In addition, mutant transport complexes containing the MalK P160L or
E306K variant were analyzed by the same protocol to serve as additional
controls. As expected from the results obtained with the purified
subunits, the proteoliposomes displayed ATPase activities of 4%
(P160L) and 17% (E306K) relative to the wild type.
Potential implications on function and domain structure of
MalK.
P160 and D165 immediately succeed the Walker B motif of the
nucleotide binding site encompassing the invariant aspartate 158, which
is essential for ATP hydrolysis (14) (Fig. 1). While P160 is
conserved only in some bacterial import systems and in both nucleotide
binding domains of the mammalian CFTR protein, D165 is almost invariant
in the ABC family overall (15). The crystal structure of the
bacterial ABC protein HisP revealed that the corresponding residues
(P180 and D185) are not involved in ATP ligation (6), which
is consistent with our observation that the mutations did not eliminate
nucleotide binding. Nonetheless, our data suggest that both residues
are crucial for catalysis.
From the biochemical analysis of the L86F protein, it is tempting to
speculate that the mutation might prevent the variant from becoming
activated as a consequence of conformational changes initiated by
maltose-loaded MalE. If so, this would imply that L86 and possibly
neighboring residues participate in subunit-subunit interactions in the
transport complex. The recent finding that A85 of MalK, when mutated to
methionine, acted as a suppressor of mutations in a conserved peptide
loop ("EAA loop") in the E. coli MalF and MalG proteins
is at least not contradictory to this notion (11).
E306 is localized to the C-terminal extension of the protein that is
confined to the MalK subfamily of bacterial and archaeal ABC proteins
(1, 5, 21). E306 is embedded within a conserved short
sequence motif (consensus, vxvVExxG) of unknown function. Substitution
by lysine resulted in a protein with a substantially reduced ATPase
activity. Since a direct role of the residue in ATP hydrolysis is
unlikely, the catalytic site might rather be affected by an altered
protein structure. The tryptic cleavage pattern that differs from the
wild type provides some evidence in favor of this notion. In this
respect, it is also interesting that mutations (S295N and V305I)
affecting residues in the same region of LacK, the MalK homolog of the
ABC transporter for lactose in Agrobacterium radiobacter,
improved the capability of certain LacK variants to substitute for
MalK in maltose transport (21; M. Brinkmann,
F. Scheffel, C. Brunkhorst, S. Hunke, and E. Schneider, unpublished results).
The finding that the E306K variant has retained the repressor-like
activity of MalK on other maltose-regulated genes is somewhat unexpected, because in E. coli MalK aspartate substituting
for G302 (corresponding to G300 in the serovar Typhimurium protein) abolished this function (8). Thus, the regulatory activity appears to be dependent on the presence of particular amino acid residues, while even nearby substitutions with structural implications are tolerated.
In summary, the mutations described in this report add to our knowledge
of the structure-function relationship of the proposed domains of MalK
and related proteins. In particular, the phenotype of a mutation,
E306K, supports the notion that in the native protein the N- and
C-terminal domains both affect the proper folding of the polypeptide
chain (13).
| |
ACKNOWLEDGMENTS |
We thank Birgit Sattler for excellent technical assistence, Oliver
Fayet (Toulouse, France), for providing plasmid pOFXT7-SL1, and Beth
Traxler (Seattle, Wash.) and Elie Dassa (Paris, France) for generous
gifts of MalF and MalG antisera, respectively.
This work was supported by the Deutsche Forschungsgemeinschaft (SCHN
271/6-1,6-2) and by the Fonds der Chemischen Industrie.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Humboldt-Universität zu Berlin, Institut für
Biologie/Bakterienphysiologie, Chausseestr. 117, D-10115 Berlin,
Germany. Phone: 49 (0)30-2093-8121. Fax: 49 (0)30-2093-8126. E-mail:
erwin.schneider{at}rz.hu-berlin.de.
| |
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Journal of Bacteriology, March 2000, p. 1432-1436, Vol. 182, No. 5
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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