Qian Chai

Insights into the Function and Structural Flexibility of the Periplasmic Molecular Chaperone SurA

SurA is the primary periplasmic molecular chaperone that facilitates the folding and assembling of outer membrane proteins (OMPs) in Gram-negative bacteria. Deletion of the surA gene in Escherichia coli leads to a decrease in outer membrane density and an increase in bacterial drug susceptibility. Here, we conducted mutational studies on SurA to identify residues that are critical for function. One mutant, SurA(V37G), significantly reduced the activity of SurA. Further characterization indicated that SurA(V37G) was structurally similar to, but less stable than, the wild-type protein. The loss of activity in SurA(V37G) could be restored through the introduction of a pair of Cys residues and the subsequent formation of a disulfide bond. Inspired by this success, we created three additional SurA constructs, each containing a disulfide bond at different regions of the protein between two rigid secondary structural elements. The formation of disulfide bond in these mutants has no observable detrimental effect on protein activity, indicating that SurA does not undergo large-scale conformational change while performing its function.

Effect of crowding by Ficolls on OmpA and OmpT refolding and membrane insertion

Folding of outer membrane proteins (OMPs) has been studied extensively in vitro. However, most of these studies have been conducted in dilute buffer solution, which is different from the crowded environment in the cell periplasm, where the folding and membrane insertion of OMPs actually occur. Using OmpA and OmpT as model proteins and Ficoll 70 as the crowding agent, here we investigated the effect of the macromolecular crowding condition on OMP membrane insertion. We found that the presence of Ficoll 70 significantly slowed down the rate of membrane insertion of OmpA while had little effect on those of OmpT. To investigate if the soluble domain of OmpA slowed down membrane insertion in the presence of the crowding agent, we created a truncated OmpA construct that contains only the transmembrane domain (OmpA171). In the absence of crowding agent, OmpA171 refolded at a similar rate as OmpA, although with decreased efficiency. However, under the crowding condition, OmpA171 refolded significantly faster than OmpA. Our results suggest that the periplasmic domain slows down the rate, while improves the efficiency, of OmpA folding and membrane insertion under the crowding condition. Such an effect was not obvious when refolding was studied in buffer solution in the absence of crowding.

Functional Relevance of AcrB Trimerization in Pump Assembly and Substrate Binding

AcrB is a multidrug transporter in the inner membrane of Escherichia coli. It is an obligate homotrimer and forms a tripartite efflux complex with AcrA and TolC. AcrB is the engine of the efflux machinery and determines substrate specificity. Active efflux depends on several functional features including proton translocation across the inner membrane through a proton relay pathway in the transmembrane domain of AcrB; substrate binding and migration through the substrate translocation pathway; the interaction of AcrB with AcrA and TolC; and the formation of AcrB homotrimer. Here we investigated two aspects of the inter-correlation between these functional features, the dependence of AcrA-AcrB interaction on AcrB trimerization, and the reliance of substrate binding and penetration on protein-protein interaction. Interaction between AcrA and AcrB was investigated through chemical crosslinking, and a previously established in vivo fluorescent labeling method was used to probe substrate binding. Our data suggested that dissociation of the AcrB trimer drastically decreased its interaction with AcrA. In addition, while substrate binding with AcrB seemed to be irrelevant to the presence or absence of AcrA and TolC, the capability of trimerization and conduction of proton influx did affect substrate binding at selected sites along the substrate translocation pathway in AcrB.

Role of a Conserved Residue R780 in Escherichia coli Multidrug Transporter AcrB

Multidrug efflux pumps play important roles in bacteria drug resistance. A major multidrug efflux system in Gram-negative bacteria is composed of the inner membrane transporter AcrB, outer membrane protein channel TolC, and membrane fusion protein AcrA. These three proteins form a large complex that spans both layers of cell membranes and the periplasmic space. AcrB exists and functions as a homotrimer. To identify residues at the trimer interface that play important roles in AcrB function, we conducted site directed mutagenesis and discovered a key residue, R780. Although R780K was partially functional, all other R780 mutants tested were completely nonfunctional. Replacement of R780 by other residues disrupted trimer association. However, a decrease of trimer stability was not the lone cause for the observed loss of activity, because the activity loss could not be restored by strengthening trimer interaction. Using both heat and chemical denaturation methods, we found that the mutation decreased protein stability. Finally, we identified a repressor mutation, M774K, through random mutagenesis. It restored the activity of AcrBR780A to a level close to that of the wild-type protein. To examine the mechanism of activity restoration, we monitored denaturation of AcrBR780A/M774K and found that the repressor mutation improved protein stability. These results suggest that R780 is critical for AcrB stability. When R780 was replaced by Ala, the protein retained the overall structure, still trimerized in the cell membrane, and interacted with AcrA. However, local structural rearrangement might have occurred and lead to the decrease of protein stability and loss of substrate efflux activity.

Repressive mutations restore function-loss caused by the disruption of trimerization in Escherichia coli multidrug transporter AcrB

AcrAB-TolC and their homologs are major multidrug efflux systems in Gram-negative bacteria. The inner membrane component AcrB functions as a trimer. Replacement of Pro223 by Gly in AcrB decreases the trimer stability and drastically reduces the drug efflux activity. The goal of this study is to identify suppressor mutations that restore function to mutant AcrBP223G and explore the mechanism of function recovery. Two methods were used to introduce random mutations into the plasmid of AcrBP223G. Mutants with elevated drug efflux activity were identified, purified, and characterized to examine their expression level, trimer stability, interaction with AcrA, and substrate binding. Nine single-site repressor mutations were identified, including T199M, D256N, A209V, G257V, M662I, Q737L, D788K, P800S, and E810K. Except for M662I, all other mutations located in the docking region of the periplasmic domain. While three mutations, T199M, A209V, and D256N, significantly increased the trimer stability, none of them restored the trimer affinity to the wild type level. M662, the only site of mutation that located in the porter domain, was involved in substrate binding. Our results suggest that the function loss resulted from compromised AcrB trimerization could be restored through various mechanisms involving the compensation of trimer stability and substrate binding.

Diverse sequences are functional at the C-terminus of the E. coli periplasmic chaperone SurA

SurA is a major periplasmic molecular chaperone in Escherichia coli and has been shown to assist the biogenesis of several outer membrane proteins. The C-terminal fragment of SurA folds into a short β-strand, which forms a small three-stranded anti-parallel β-sheet module with the N-terminal β-hairpin. We found that the length of the C-terminal fragment, rather than its exact amino acid composition, had a big impact on SurA function. To investigate the determinant factor of the C-terminal sequence, we created a library of SurA constructs randomized in the last 10 residues. We screened the library and randomly analyzed 19 constructs that displayed SurA activity. The C-termini of these constructs shared little sequence similarity, except that β-strand-forming residues were preferentially enriched. Three SurA constructs were expressed and purified for structural characterization. Circular dichroism and fluorescence spectroscopy analyses revealed that their structures were similar to the structure of the wild-type SurA. Our results suggest that for scaffolding purpose proteins may tolerate various sequences provided certain general requirements such as hydrophobicity and secondary structure propensity are satisfied. Furthermore, the sequence tolerance of SurA at the C-terminus indicates that this area is not likely to be involved in substrate binding.

Correlation between AcrB trimer association affinity and efflux activity.

The majority of membrane proteins function as oligomers. However, it remains largely unclear how the oligomer stability of protein complexes correlates with their function. Understanding the relationship between oligomer stability and activity is essential to protein research and to virtually all cellular processes that depend on the function of protein complexes. Proteins make lasting or transient interactions as they perform their functions. Obligate oligomeric proteins exist and function exclusively at a specific oligomeric state. Although oligomerization is clearly critical for such proteins to function, a direct correlation between oligomer affinity and biological activity has not yet been reported. Here, we used an obligate trimeric membrane transporter protein, AcrB, as a model to investigate the correlation between its relative trimer affinity and efflux activity. AcrB is a component of the major multidrug efflux system in Escherichia coli. We created six AcrB constructs with mutations at the transmembrane intersubunit interface, and we determined their activities using both a drug susceptibility assay and an ethidium bromide accumulation assay. The relative trimer affinities of these mutants in detergent micelles were obtained using blue native polyacrylamide gel electrophoresis. A correlation between the relative trimer affinity and substrate efflux activity was observed, in which a threshold trimer stability was required to maintain efflux activity. The trimer affinity of the wild-type protein was approximately 3 kcal/mol more stable than the threshold value. Once the threshold was reached, an additional increase of stability in the range observed had no observable effect on protein activity.

The ssrA-Tag Facilitated Degradation of an Integral Membrane Protein

ATP-dependent degradation plays a critical role in the quality control and recycling of proteins in cells. However, complete degradation of membrane proteins by ATP-dependent proteases in bacteria is not well-studied. We discovered that the degradation of a multidomain and multispan integral membrane protein AcrB could be facilitated by the introduction of a ssrA-tag at the C-terminus of the protein sequence and demonstrated that the cytoplasmic unfoldase-protease complex ClpXP was involved in the degradation. This is the first report to our knowledge to reveal that the ClpXP complex is capable of degrading integral membrane proteins. The chaperone SspB also played a role in the degradation. Using purified proteins, we demonstrated that the addition of the ssrA-tag did not drastically affect the structure of AcrB, and the degradation of detergent solubilized AcrB by purified ClpXP could be observed in vitro.

Accessibility from the cytoplasm is critical for ssrA tag mediated degradation of integral membrane proteins by ClpXP protease

The AAA+ protease ClpXP has long been established as the cellular rescue system that degrades ssrA-tagged proteins resulting from stalled ribosomes. Until recently, in all of these studies soluble proteins were used as model substrates, since the ClpXP complex and the related adapter SspB are all cytosolic proteins. In a previous study, we found that the introduction of an ssrA tag can facilitate complete degradation of a large and stable trimeric integral membrane protein AcrB, which is the first reported example of a membrane protein substrate. To investigate the mechanism of degradation of a membrane protein by a soluble protein complex, we experimented with the truncation of the C-terminal tail of AcrB. We found that the C-terminal tail is important for degradation, as systematic truncation of the tail diminished degradation. Thus, we hypothesize that membrane proteins need a cytosolic tail/domain for ClpXP-SspB to latch on to initiate degradation. To test this hypothesis, we introduced the ssrA tag at the C-terminal of several membrane proteins, including AqpZ, YiiP, YajR, as well as their truncation fragments, and examined their degradation. We found that the ssrA-facilitated degradation of membrane proteins by ClpXP-SspB depends on the presence of a CT tail or domain, which is critical for accessibility of the tag by ClpXP-SspB. When the ssrA tag is not well-exposed to the cytosol, FtsH can access and degrade the tagged protein, given that the substrate protein is metastable.