Identification of the proteolytically labile sites in the C-domain of AcrA.
Since the structural role of the N-terminal lipid modification of AcrA remains unclear, for this study we purified the native AcrA, which is lipid modified in the periplasm. For purification purposes, the protein was modified with the C-terminal six-histidine tag (AcrAhis). MALDI-TOF analysis of the purified AcrAhis showed a single peak with a molecular mass of 41,696 Da. This value is in good agreement with the expected 41,624 Da for AcrA modified with N-acyl-S-diacylglycerol containing two palmitoyl residues and one oleoyl residue (Table ). No additional peaks were detected in this preparation, suggesting that most of the purified AcrAhis is lipid modified.
Peptide masses of whole-length AcrAhis and its major tryptic fragments
We next used limited proteolysis to evaluate the structure of the purified AcrAhis
. After 60 min of tryptic digestion, AcrAhis
was completely cleaved into five major fragments (Fig. ). The molecular masses of these AcrAhis
-derived peptides were determined to be 40.4, 36.9, 35.1, 32.1, and 28.9 kDa (Table and Fig. ). In addition, several minor fragments are also seen in the tryptic profile of AcrAhis
. Based on the sequence analysis and extant literature, we identified the 36.9-kDa fragment as a product of cleavage of the distal N- and C-terminal ends of AcrA (28 to 374 aa) (Table and Fig. ) (14
). The 32.1- and 35.1-kDa fragments are generated by further digestion from the N and C termini of AcrA. Finally, the 28.9-kDa band is the previously characterized 46- to 315-aa proteolytically stable core of AcrA (14
Proteolytic profile of the overproduced AcrAhis in intact cells is similar to that of the purified protein.
We next used tryptic digestion to compare conformations of the C-domain of AcrAhis in vitro and in intact cells. For this purpose, AG100AX cells lacking both AcrAB and AcrEF multidrug efflux pumps were transformed with the pAhis plasmid. We took advantage of the fact that AcrA is located in the periplasm and made it accessible to trypsin by means of osmotic shock. Even before treatment with trypsin, the overproduced AcrAhis was notably degraded by periplasmic proteases (Fig. ). Incubation with increasing concentrations of trypsin produced a limited number of the specific bands. By comparison to the immune- and silver-stained proteolytic profiles of the in vitro-digested AcrAhis, these bands were identified as 40.4-, 37.4-, 36.9-, 32.1-, 28.9-, and 26.5-kDa fragments. Overall, immunoblots of the proteolytic profile of the in vivo-overproduced AcrAhis were surprisingly similar to those of the purified protein. The 26.5-kDa fragment (this mass is estimated from the mobility in SDS-PAGE), which is a minor band in the proteolytic profile of the purified AcrAhis (Fig. ), could be clearly detected by immunoblotting in the AcrAhis digests in vivo. The proteolytic profile of AcrAhis overproduced in ECM2112 cells lacking all three genes, acrA, acrB, and tolC, was similar to that in AG100AX cells (data not shown). We conclude that in intact cells, the overall conformation of the overproduced AcrAhis closely resembles that of the purified protein.
The coexpression of AcrAhis with AcrB under the native acrAB promoter (pAhisB plasmid) did not significantly affect the proteolytic profile of AcrAhis. However, we noticed that at high trypsin concentrations, amounts of the 26.5-kDa fragment were higher when AcrB was overproduced together with AcrA, suggesting that the interaction with AcrB protects this fragment from trypsin degradation (see also below).
Assembly of the tripartite AcrAB-TolC complex protects the C-domain of AcrA from trypsin.
Overproduction of AcrAhis from pAhis and pAhisB plasmids results in a significant excess of AcrA compared to levels of the two other components of the multidrug efflux complex, AcrB and TolC. Thus, the tryptic digestion profile of the overproduced AcrAhis reflects mainly the conformation of the free AcrAhis. To investigate whether association with AcrB, TolC, or both brings any changes into AcrA conformation, we compared proteolytic profiles of AcrA in wild-type E. coli cells producing all three components of the complex from the chromosome and in mutant cells lacking either AcrB or TolC.
Surprisingly, we found that the tryptic digestion profiles of the chromosomally produced AcrA differ significantly from those of the purified/overproduced protein (Fig. ). In the wild-type E. coli cells producing all three proteins, AcrA, AcrB, and TolC, only three tryptic fragments of AcrA are accumulated; by comparison to the tryptic digestion of the purified AcrAhis, these were identified as 37.4-, 36.9-, and 26.5-kDa fragments (Fig. ). Not even traces of the core 28.9-kDa fragment of AcrA could be detected in the wild-type E. coli cells. Thus, the 28.9-kDa fragment, which is invariably present in tryptic digests of free AcrA, is not accumulated when AcrA is assembled into a complex. In contrast, large amounts of the 26.5-kDa fragment of AcrA could be detected already after a 5-min digestion with trypsin, and this fragment resists further cleavage even after 60 min of incubation with large amounts of trypsin (Fig. ).
The proteolytic profile of AcrA changed dramatically in cells lacking either AcrB or TolC. Only traces of the 26.5-kDa fragment of AcrA could be seen on immunoblots of cells treated with trypsin for 5 min, and none could be seen after 60-min digests (Fig. ). When ΔacrB and ΔtolC strains were transformed with plasmids producing AcrB or TolC, respectively, the 26.5-kDa fragment of AcrA became highly abundant again (Fig. ). We conclude that the increase in the amounts of the 26.5-kDa fragment is specific for the assembly of the functional tripartite AcrAB-TolC complex.
Previous genetic and biochemical studies demonstrated that AcrA forms bipartite complexes with AcrB and TolC even in the absence of the respective third component (22
). Thus, in ΔacrB
strains, AcrA is engaged in interactions with TolC and AcrB, respectively. Yet the proteolytic profiles of AcrA are very similar in these two genetic backgrounds, with small amounts of the 26.5-kDa fragment and the 28.9-kDa core present at low concentrations of trypsin and short digestion times (Fig. ). To determine whether such small amounts of fragments reflect the increased stability of the whole-length AcrA or, alternatively, indicate the rapid degradation of fragments, we quantified relative amounts of the whole-length AcrA on immunoblots of mutants and wild-type cells. We found that the decreases in the amounts of AcrA with increasing concentrations of trypsin and times of digestion were similar for all three strains (data not shown). Thus, the C-domain of AcrA is accessible to trypsin and rapidly degraded when one of the components of the AcrAB-TolC complex is missing.
The C-domain of AcrA is important for multidrug efflux function.
The experiments described above showed that the C-domain of AcrA is protected from tryptic digestion in AcrAB-TolC, suggesting that this domain might be involved in the assembly of the complex. To test the functional significance of the C-domain, we identified 12 highly conserved amino acid residues of AcrA, P309, V313, V332, R335, G352, L353, G356, D357, V359, V360, G363, and V373, and replaced them with cysteines by site-directed mutagenesis. In addition, cysteine substitutions were introduced into the variable S362 and K366 positions. Plasmids were transformed into E. coli cells deficient in acrAB and acrEF, and the expression of AcrAhis and its derivatives was determined by immunoblotting (Fig. ). The expression levels of all AcrAhis mutants were similar to that of wild-type AcrAhis, with the exception of that of the G352C mutant, which was reproducibly expressed at levels two- to threefold below that of the wild-type protein.
FIG. 3. Expression of AcrAhis and AcrB is not affected by mutations in the C-domain of AcrAhis. (A) Total E. coli W4680AE cells harboring plasmids producing AcrB and either wild-type or mutant AcrAhis were boiled in the SDS sample buffer for 5 min, separated (more ...)
To determine the functional competence of the AcrAhis mutants, we measured MICs of the selected known substrates of AcrAB-TolC, including erythromycin, norfloxacin, novobiocin, ethidium bromide, and SDS (Table ). Most of the AcrAhis mutants fully complemented the function of the wild-type AcrAhis. However, E. coli W4680AE cells carrying plasmids with AcrAhis G352C, G356C, and G363C mutants were more susceptible to multiple drugs. The effect of G356C substitution was modest, with only a twofold decrease in the MICs of the tested compounds. The G352C mutation in AcrAhis resulted in a two- to eightfold decrease in MICs of the tested drugs, with the exception of SDS. Surprisingly, cells carrying the AcrAhis G363C mutant were highly susceptible (up to 32-fold) to all tested compounds.
Antibiotic susceptibility of E. coli W4680AE cells carrying plasmids that produce the wild-type and mutant derivatives of AcrAhis
As shown in Fig. , all three antibiotic-susceptible mutants were produced at the levels similar to those of the wild-type AcrAhis. Thus, changes in protein expression cannot account for the decrease in multidrug efflux activity. In agreement, we found no undesired mutations in the sequences of the upstream noncoding region of plasmids producing these three AcrAhis mutants (data not shown). Also, no mutations were found in the acrB sequence of the pAhisG363CB plasmid. Furthermore, the immunoblotting analysis showed that the amounts of AcrB transporter were similar in E. coli W4680AE cells carrying pAhisG363CB and pAhisB plasmids (Fig. ). We conclude that the decreased antibiotic resistance is caused by defects in the C-domain of AcrA. Cells carrying pAhisG352CB and pAhisG356CB plasmids produced notably smaller amounts of AcrB (Fig. ). Thus, in these cells, the low expression levels of AcrB could contribute to the decreased resistance to antibiotics.
The G363C mutant fails to properly assemble into the functional AcrAB-TolC complex.
One possible reason why G352C and G363C substitutions inactivate AcrAhis is that these mutants have lost the ability to interact properly with AcrB, TolC, or both. Therefore, we next compared AcrAhis and its G352C and G363C variants in their ability to copurify with AcrB and TolC. For this purpose, all E. coli cells carrying pAHis (produces AcrAhis alone), pAhisB, pAhisG352CB, or pAhisG363CB plasmids were treated with the amine-reactive cross-linker DSP (spacer arm, 12 Å), and protein complexes were purified using metal affinity chromatography. AcrB and TolC copurified with AcrAhis, and its G352C and G363C mutants were detected by immunoblotting. As shown in Fig. , in all three complexes, the amounts of AcrB and TolC were similar. Thus, G352C and G363C substitutions do not disrupt formation of the tripartite AcrAB-TolC complex.
FIG. 4. The AcrAhis G363C mutant interacts with AcrB and TolC but fails to assemble into the functional AcrAB-TolC complex. (A) AcrB (top panel) and TolC (bottom panel) are copurified with the AcrAhis G363C mutant. E. coli W4680AE cells carrying pAhis, pAhis (more ...)
In vivo tryptic digestion was used to compare the structural features of the overproduced AcrAhis and its G363C derivative when these two proteins are coexpressed with AcrB. Overall, the proteolytic profile of G363C was similar to that of AcrAhis, with all major fragments in place (Fig. ). This result is consistent with the above finding that G363C substitution does not alter the structure of AcrA and that this mutant can assemble into the AcrAB-TolC complex. However, the G363C profile was more similar to that of the AcrAhis protein overproduced in the absence of AcrB (Fig. ). The 26.5-kDa fragment of G363C was notably more susceptible to trypsin than the corresponding fragment of AcrAhis (Fig. ). This result suggested that despite interactions with AcrB and TolC, the G363C mutant fails to properly assemble into the AcrAB-TolC complex.