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Fully synthesized bacterial secretory proteins are transported across the plasma membrane post-translationally by the ATPase SecA. SecA is a member of the large AAA ATPase family and uses the energy from ATP hydrolysis to push nascent polypeptides through the SecY channel. However, the mechanism by which SecA couples its energy consumption to this translocation activity is unclear. Here, we use single molecule FRET to observe the conformational changes of SecA as it translocates a model polypeptide substrate and ensemble biochemistry experiments, including protease protection assays, to monitor the corresponding movements of the substrate itself. Upon ATP-binding, SecA’s two-helix finger inserts into the SecY channel and pushes the translocating polypeptide chain forward. Subsequently, a clamp domain closes around the substrate and holds it in place as the two-helix finger resets during ATP hydrolysis. The clamp domain re-opens after phosphate release, freeing the polypeptide to passively slide through the SecY channel until the subsequent ATP binding event. The alternating movements of these two domains ensure that translocation is directional and provide a model that may be applicable to a wide range of ATPases that work on polypeptide chains.

Due to the emergence and dissemination of multidrug resistance, bacterial pathogens have been causing a serious public health problem in recent years. To address the existing drug resistant problem, there is an urgent need to find new antimicrobials, especially those against drug-resistant bacteria. SecA is the central component of Sec-dependent secretion pathway, which is responsible for the secretion of many essential proteins as well as many toxins and virulence factors. Two SecA homologues are indentified in some important Gram-positive pathogens. SecA1 is involved in general secretion pathway and essential for viability, whereas SecA2 contribute to secretion of specific virulence factors. The high conservation among a wide range of bacteria and no human counterpart make SecA homologues attractive targets for exploring novel antimicrobials. We hypothesize that inhibition of these SecA homologues could reduce virulence, inhibit bacteria growth, and kill bacteria. SecA1 and SecA2 from four different species were cloned, purified, and characterized. All these SecA homologues show ATPase activities, thus screening ATPase inhibitors might help to develop new antimicrobials. In this study, three structurally different classes of SecA inhibitors were developed and optimized: 1) Rose Bengal (RB) and RB analogs derived from systematical dissection RB and Structure-Activity relationship (SAR) study; 2) pyrimidine analogs derived from virtual screening based on the ATP binding pocket of EcSecA and SAR study; and 3) bistriazole analogs derived from random screening and SAR study. Several potent SecA inhibitors show promising enzymatic inhibition against SecA homologues as well as bacteriostatic and bactericidal effects. Two major efflux pumps of S. aureus, NorA and MepA, have little negative effect on the antimicrobial activities of SecA inhibitors, suggesting that targeting SecA could by-pass efflux pumps. Moreover, these inhibitors impair the secretion of important toxins of S. aureus and B. anthracis, indicating the inhibition of in vivo SecA function could reduce virulence. Target identification assays confirm that these inhibitors could directly bind to SecA homologues, and specifically identify SecA from whole cell lysate of E. coli and S. aureus, suggesting that these inhibitors are really targeting on SecA. These studies validate that SecA is a good target for development antimicrobials.

SecA is a central component of the general secretion system that is essential for growth and virulence of bacteria. A series of fluorescein analogs were tested against ATPase activities of Escherichia coli SecA. Rose Bengal (RB) and Erythrosin B are potent inhibitors abolishing the activities of three forms of SecA ATPase with IC50 in µM range. Both inhibit SecA intrinsic ATPase with two mechanisms depending on ATP concentrations, indicating they influence the two non-identical nucleotide binding sites differently. RB shows different inhibitory effects against three forms of SecA ATPase activities, suggesting that the inhibition is related to the conformation of SecA. RB with IC50 at sub-µM level is the most potent inhibitor of SecA ATPases and SecA-dependent protein translocation to date. The fluorescein analogs inhibit intrinsic ATPase of Bacillus subtilis SecA similarly, and also exhibit antibacterial effects in E. coli and B. subtilis. Our findings indicate the value of fluorescein analogs as probes for mechanistic studies of SecA and the potential development of new SecA-targeted antimicrobial agents. A series of SecA derivatives with truncated C-terminus within the first long α-helix of the helix-bundle extending the ATPase catalytic domain of N68 was analyzed. These SecA variants interact with lipids, and those containing the C-terminal portion of the long α-helix starting at residues #639 form the ring-like structure in liposomes, indicating the critical domains for forming the protein-conducting channel. The presence and length of the C-domain influence the response to RB of NBDII mutants and C-terminal truncates of SecA. Thus this region may interact with the inhibitors and is involved in the structure and regulation of SecA ATPase activity. B. subtilis SecA was analyzed for interspecies comparison. Despite sharing high homology, this SecA homolog cannot complement E. coli mutants with SecA defect. Phospholipids do not stimulate ATPase activities of B. subtilis SecA, but induce its conformational changes, leading to the lipid-specific domains and ring-like structures similar to E. coli SecA. These pore-ring structures may represent part of the protein-conducting channels. Therefore, the potential structural roles of SecA in the protein translocation machinery may be universal in both Gram-negative and Gram-positive bacteria.

Sec translocon is the major machinery for protein translocation in E.coli including SecYEG, SecA and other Sec proteins. It is generally assumed that during translocation process, SecYEG serves as a protein-conducting channel and transports the protein across membranes by using SecA ATPase as driving force. However, previous work suggested that protein translocation can occur without SecYEG. In order to understand the role of SecA in this SecYEG-independent process, we use voltage clamp recording as a tool to study the ionic activity of SecA-dependent protein-conducting channel. In a major deviation from the conventional view, we found that SecA alone is sufficient to promote the channel activity with liposomes made of E.coli phospholipids in both whole cell recording in the oocytes and in the single channel recording with patch clamp. The activity is strictly dependent on the presence of functional SecA, including those from different species of bacteria. However, this SecA-alone dependent channel activity is less efficient compared to the membranes containing SecYEG. Furthermore, the channel activity loses the signal peptide specificity. Addition of purified SecYEG restores the signal peptide specificity as well as the efficiency. This channel activity is more sensitive to SecA-specific inhibitors compared with membranes containing wild-type SecYEG but is less sensitive to membranes containing suppressor proteins. This is the first time it has been shown that SecA binds to lipid low-affinity site and functions as a protein-conducting channel. To further characterize the structural roles of SecA as the core of the channel, we use several SecA variants to reconstitute with liposomes to determine the domains involved in forming functional channels. Using deletion truncated domains of 901 residues SecA and liposomes in the oocytes recordings, we identify two critical SecA domains for the formation of pore channel activity: with phospholipids alone, and for interacting with SecYEG to gain higher activity. These data provide fundamental understanding for the SecA-dependent protein –conducting channels. Our findings also suggest the possible evolution process on the protein translocation pathways from prokaryotes through eukaryotes.

We have developed a novel, sensitive and less time-consuming method to detect activity of the SecA-dependent protein-conducting channels. Nanogram levels of E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. The observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein, proOmpA, or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5’-(β, γ-methylene)-diphosphonate, a non-hydrolyzable ATP analog, both of which are known to inhibit SecA protein activity. Channel activity was also stimulated by oocyte endogenous precursor proteins, which could be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides, but not defective signal peptides, stimulated the ionic currents. We also measured SecA-dependent currents with membranes depleted of SecYEG. Wild-type LamB signal peptides, or precursor proteins stimulated ionic currents following a co-injection of SecYEG¯ membranes with puromycin. Excess exogenous SecA stimulated ionic currents through SecYEG¯ membranes. Similar activities of added SecA were observed with reconstituted membranes depleted of SecYEG. Currents through such SecYEG-depleted membranes were also stimulated by addition of defective LamB signal peptides and unfolded mature PhoA protein. In contrast, currents produced by the membranes containing wild-type SecYEG were not so stimulated, but ionic currents were stimulated through mutant strains, similar to PrlA (SecY) suppressors, e.g. PrlA4, or PrlA665 membranes, suggesting that the proofreading function of SecY was bypassed in these membranes. We have observed that azide can inhibit ionic currents when E. coli wild-type MC4100 membranes were injected with proOmpA or LamB signal peptides into Xenopus oocytes. However, such inhibition was lost when observed with oocyte-endogenous signal peptides in the absence of bacterial signal peptides. Moreover, azide did not show complete inhibition upon using SecYEG¯ membranes or SecYEG¯ reconstituted membranes plus excess SecA in the presence or absence of LamB signal peptides. Such conformational alterations reflect different sensitivity in response to azide during the opening of protein-conducting channels.

In bacteria, it has been widely recognized that SecYEG translocase is the main protein-conducting channel that mediates the insertion of the inner membrane proteins and also the membrane translocation of the exported proteins to the periplasmic space. Recently a new inner membrane protein, YidC, was discovered to be involved in membrane protein insertion. YidC works in conjunction with the Sec translocase and function in integrating proteins into the lipid bilayer, or as a chaperone to fold the proteins into their correct functional structure. YidC can also function on its own as an insertase to insert inner membrane proteins independent of the Sec translocase. In my research, I focused on the membrane insertion of proteins in E.coli using these two pathways. In chapter 2, we describe two new yidC mutants that lead to a cold-sensitive phenotype in bacterial cell growth. Both alleles impart a cold sensitive phenotype that result from point mutations localized to the third transmembrane segment of YidC, indicating this region is crucial for YidC function. We found that the yidCC423R mutant confers a weak phenotype on membrane protein insertion while a yidCP431L mutant leads to a stronger phenotype. In both cases, the affected substrates include the Pf3 coat protein and the F0C (subunit c of the F1F0 ATP synthase); CyoA (the quinol binding subunit of the cytochrome bo3 quinol oxidase complex) and wild type procoat are slightly or not affected in either cold-sensitive mutant. To determine if the different substrates require varying levels of YidC activity for membrane insertion, we performed studies where YidC was depleted using an arabinose-dependent expression system. We found that -3M-PC-Lep and Pf3 P2 required the highest amount of YidC, and CyoA-N-P2 and PC-Lep required the least while FoC required moderate YidC levels. Although the cold-sensitive mutations can preferentially affect one substrate over another, our results indicate that different substrates require different levels of YidC activity for membrane insertion. Finally, we obtained several intragenic suppressors that overcome the cold-sensitivity of the C423R mutation. One pair of mutations suggests an interaction between TM2 and TM3 of YidC. The studies reveal the critical regions of the YidC protein and provide insight into the substrate profile of the YidC insertase. In chapter 3, we proposed a new hypothesis explaining why some M13 Procoat mutants are Sec translocase dependent. Such M13 procoat mutants are proposed to bind to SecA in vivo after synthesis and are targeted to the Sec translocase for membrane insertion. In this case, membrane insertion of the M13 procoat mutant is dependent on Sec pathway. However, when SecA is not present, the M13 procoat mutant will be targeted to YidC and use the YidC only pathway for the membrane insertion. We utilized a SecA depletion strain to test our hypothesis in vivo. Different Sec dependent PCLep mutants and Sec independent PCLep mutants were studied under the condition of SecA depletion or SecA inhibition. We show that the Sec dependent proteins -5PCLep, -3MPCLep and 3NPCLep can be inserted into the inner membrane by YidC only pathway when the SecA was depleted.

SecA is an essential component for the translocation of proteins across bacterial membranes. Though SecA is known to function in the membrane the mechanism for this process remains unclear. In this study we identify two specific regions of SecA that may be important for N-terminal membrane interactions. Molecular modeling of SecA from the E. coli and B. subtilus crystal structures, previously determined, revealed that the first 30 amino acids of SecA consists of a helix of amino acids 1-11 connected by a linker (amino acids 12-16) to an amphipathic helix of amino acids 17-30. The first helix is dispensable for SecA activity; however, deletions in the second N-terminal helix, at amino acids 21-25, result in decrease of SecA activity and a deletion of 26 amino acids no longer complements E. coli ts mutant BL21.19. We show that the deletions of N-terminal amino acids that result in the decrease of SecA activity are correlated to the loss of SecA membrane binding and integration in these deletion mutants. In this study we also test the accuracy of a new membrane protein prediction software PSSM_SVM. This program predicted an embedded membrane (EM) region at SecA amino acids 110-118. The predicted sequence represents an unusual prediction for an EM region as most membrane integral regions consist of 15-30 amino acids. Molecular modeling indicated that the region 110-118 is a part of a helix composed of amino acids 107-121 in E. coli SecA, and is indicative of a membrane embedded domain. Site-directed mutagenesis was carried out with several conserved residues, which included L110, L114, and L118 to determine if substitutions at these positions would affect SecA activity. Our data shows that most SecA mutants (including some predicted to be inactive) are active in vivo; however, L110E and L114R mutations rendered the mutated SecA non-functional. All together this study shows that the N-terminal limit of SecA resides at amino acid 26 and that amino acids 21-25 may form a N-terminal membrane binding determinate. Moreover, the predicted EM region may indeed correspond to a functional embedded membrane region for SecA.

E. coli SecA is an essential component for protein translocaiton across membrane. SecA can be deleted from its N- and/or C-terminal ends without losing complementation activity. In this study, we determined the dispensity of both ends of SecA molecule. The minimal length at the SecA C-terminus is dependent on the length of the N-terminal region. SecA10-826 and SecA22-829 are the two minimal length SecAs. One more amino acid deleted at the C-terminal end completely abolished their complementation activity. A hydrophobic amino acid is required at the 826th amino acid in the minimal-length SecAs. Both SecA22-828 and SecA22-829 could form a dimer, and have decreased ATPase and protein translocation activities. The active truncated SecA mutants tended to have more soluble form than membrane-bound form, but were stably embedded in membrane. In contrast, the inactive truncated SecA mutants tended to have more membrane-bound form, but were not stable in membrane. Thus, the loss of complementation is not related to dimerization, ATPase and translocation activity but to certain extent related to their biased subcelluar localization and conformation in membrane. Isolated membranes of E coli strains were solubilized and fractionated by sucrose gradient fractionation. These membranes fractions were depleted of SecY and YidC, but contained SecD, SecF and GroEL. Proteoliposomes reconstituted from these fractionated membrane proteins were active in pOmpA translocation which required SecA and ATP. Membrane fractions from strain CK1801 in which the unc gene is deleted were reconstituted into liposomes and also showed translocation activities. Moreover, proteoliposomes reconstituted with Bacteriorodopsin alone were not active in translocation, while proteoliposomes reconstituted with Bacteriorodopsin and CK1801 membrane fractions showed elevated translocation efficiency. These data suggested that proton motive force is not obligatory for, but stimulatory to translocation of pOmA. Purified GroEL was reconstituted into lipsomes and the reconstituted proteoliposomes were active in pOmpA translocation although at lower efficiency. This translocation also required SecA and ATP. These results together suggested that translocation of pOmpA is active in the absence of SecY and YidC. In the absence of SecYEG, translocation of pOmpA requires SecA and ATP. GroEL, SecD and SecF may participate in the SecY-independent translocation.