Mapping of the SecA Signal Peptide Binding Site and DimericInterface by Using the Substituted Cysteine Accessibility Method

Meera K. Bhanu,a Ping Zhao,b Debra A. Kendallb

Department of Molecular and Cell Biologya and Department of Pharmaceutical Sciences,b University of Connecticut, Storrs, Connecticut, USA

SecA is an ATPase nanomotor critical for bacterial secretory protein translocation. Secretory proteins carry an amino-terminalsignal peptide that is recognized and bound by SecA followed by its transfer across the SecYEG translocon. While this process iscrucial for the onset of translocation, exactly where the signal peptide interacts with SecA is unclear. SecA protomers also inter-act among themselves to form dimers in solution, yet the oligomeric interface and the residues involved in dimerization are un-known. To address these issues, we utilized the substituted cysteine accessibility method (SCAM); we generated a library of 23monocysteine SecA mutants and probed for the accessibility of each mutant cysteine to maleimide-(polyethylene glycol)2-biotin(MPB), a sulfhydryl-labeling reagent, both in the presence and absence of a signal peptide. Dramatic differences in MPB labelingwere observed, with a select few mutants located at the preprotein cross-linking domain (PPXD), the helical wing domain(HWD), and the helical scaffold domain (HSD), indicating that the signal peptide binds at the groove formed between thesethree domains. The exposure of this binding site is varied under different conditions and could therefore provide an ideal mech-anism for preprotein transfer into the translocon. We also identified residues G793, A795, K797, and D798 located at the two-helix finger of the HSD to be involved in dimerization. Adenosine-5=-(�-thio)-triphosphate (ATP�S) alone and, more exten-sively, in conjunction with lipids and signal peptides strongly favored dimer dissociation, while ADP supports dimerization.This study provides key insight into the structure-function relationships of SecA preprotein binding and dimer dissociation.

About one-third of all proteins synthesized in the bacterial cy-tosol are destined to reside in the membrane or to be trans-

ported through it. A majority of these secretory proteins utilize thebacterial general secretory (Sec) pathway for their translocation.Other pathways include the YidC insertase system, used mainlyfor membrane protein insertion, and the Tat pathway for secre-tion of folded proteins into the periplasm (see reference 1 for areview). Secretory proteins are synthesized in the cytosol as pre-proteins that contain an amino-terminal signal peptide whichserves as an “address tag,” followed by the mature protein. In theSec pathway, the molecular chaperone SecB maintains newly syn-thesized preproteins in their unfolded state and then transfersthem to an ATPase nanomotor known as SecA via the signal pep-tide region. SecA is a key component of the bacterial Sec pathwaythat is critical for membrane protein transport and is crucial forcell viability. The energy required for translocation of the prepro-tein across the heterotrimeric SecYEG membrane channel is fur-nished by SecA through ATP hydrolysis (2).

SecA is a large 102-kDa cytosolic protein. Crystal structures ofSecA from different bacterial species have been resolved, and theinformation gleaned from these structures has led to the groupingof SecA regions into the preprotein cross-linking domain (PPXD),the helical wing domain (HWD), the helical scaffold domain(HSD), and the two nucleotide binding domains (NBD), the high-affinity site NBD I and the low-affinity site NBD II (3). The struc-ture of the protomer is largely the same in all these structures;however, an opening in the PPXD-HSD-HWD region is presentin some of the so-called open forms of SecA, while not in others.SecA has been crystallized both in the monomeric as well as par-allel and antiparallel dimeric forms (3–6). However, there is nogeneral consensus as to the oligomeric form and orientation of theprotomers in the homodimer. SecA cycles between its ATP- andADP-bound states to provide energy for translocation. In theADP-bound form, SecA is thought to exist in a compact form,

while ATP binding produces an elongated conformation (7). SecAis known to interact with SecB, the SecYEG channel, and anionicphospholipids in the membrane bilayer (8). In addition, SecA spe-cifically interacts with secretory preproteins by recognition of thesignal sequence (9). Most of these ligands are known to produceconformational changes within SecA, with concomitant changesin its ATPase activity (10). Experiments carried out to study theseinteractions have provided conflicting results with regard to thealteration of SecA’s monomer-dimer equilibrium (11–15). Ex-actly how SecA modulates its diverse functions and its associationwith several ligands is still under examination.

Signal peptides play a key role in the targeting and membraneinsertion of preproteins. SecA’s ATPase activity has been shown toincrease dramatically in the presence of signal peptides (16), indi-cating that they are responsible, in part, for the modulation of therate of nucleotide hydrolysis by SecA. Indeed, signal peptides withaltered amino acid hydrophobicity and length have been shown todramatically affect translocation (17). Signal peptides of prepro-teins that utilize the Sec pathway in Gram-negative bacteria aregenerally 18 to 30 residues long and contain three distinct regions:a positively charged amino terminus followed by a central hydro-phobic core region and a carboxy-terminal polar segment (18).Residues at the �1 and �3 locations of the signal peptide arecrucial for signal peptide cleavage by a membrane-embedded pro-tease, the signal peptidase enzyme (19, 20). Following cleavage,the functional mature protein is released into the membrane or

Received 3 June 2013 Accepted 7 August 2013

Published ahead of print 9 August 2013

Address correspondence to Debra A. Kendall, debra.kendall@uconn.edu.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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the periplasm, while the signal peptide is cleaved by the signalpeptide peptidase enzyme (21).

Several studies have been carried out to analyze signal peptidebinding to SecA. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiim-ide (EDAC) cross-linking studies carried out with multiple trun-cated SecA mutants and preproteins found that residues 267 to340 of the PPXD were involved in preprotein binding (22). ThePPXD region was also implicated as the signal peptide binding sitein two more subsequent studies: a nuclear magnetic resonance(NMR) study carried out with E. coli SecA and a KRR-LamB signalpeptide identified the groove formed between the PPXD and theintramolecular regulator of ATP hydrolysis-1 domain (IRA1) as asuitable candidate for a signal peptide binding site (23). Similarly,signal peptides with a photoactivatable label were found to cross-link at the PPXD region (24). However, the hydrophobic cleftformed between the PPXD and NBD II and the two � sheets con-necting the PPXD and NBD I have also been suggested (25, 26).Hence, the region on SecA that binds the signal peptide is stillmuch debated. Although there are significant differences in theapproaches utilized for analyzing SecA-signal peptide interac-tions, a frequently observed site of binding is the PPXD region;interactions with other sites remain controversial. Since this rec-ognition is a crucial prerequisite for preprotein translocation, un-derstanding the mechanism and site of action is of paramountimportance for fully comprehending bacterial protein transport.

SecA is thought to likely exist in the cytosol predominantly in adimeric state, since the dissociation constant (Kd) of SecA in so-lution is below 0.3 �M (11), while the total SecA concentration ina bacterial cell is roughly 5 �M (13). However, the functionaloligomeric state of SecA at the SecYEG translocon is unclear, withstudies identifying both monomers and dimers as the active formof SecA (27–29). SecA’s dimer interface and the alignment of itsprotomers are also unknown, including the identity of the dimericinterface residues.

We have employed the substituted cysteine accessibilitymethod (SCAM) to establish the signal peptide binding site andthe dimer interface of SecA (30). For this purpose, we generated alibrary of monocysteine E. coli SecA mutants, and the accessibilityof the single Cys residue on each mutant was probed using ma-leimide-PEG2-biotin (MPB) in the presence and absence of signalpeptide and other ligands. Using this approach, we found that Cysmutants at the PPXD-HSD-HWD region exhibited maximal MPBlabeling intensity changes in the presence of a signal peptide. Wealso found that monocysteine mutants at the two-helix finger re-gion of SecA, but not others, formed disulfide-bonded dimericspecies, and these dimers could dissociate upon interaction withdifferent ligands. These results are particularly interesting in lightof recent studies that point to the two-helix finger playing a sig-nificant role in the opening of the SecYEG channel (31).

MATERIALS AND METHODSGeneration of SecA mono-Cys mutants. Substitution mutagenesis usingthe pET-29b-T7 SecA His plasmid, encoding a C-terminal His-taggedSecA, was performed using the QuikChange site-directed mutagenesis kit(Stratagene). All four naturally occurring Cys residues were mutated toSer to generate a Cys-less SecA template, and mono-Cys mutants weregenerated using this template. All mutations were confirmed via DNAsequence analysis.

SecA purification. E. coli BL21(DE3) cells harboring the SecA vectorwere grown in LB medium with 100 �g/ml ampicillin at 37°C until anoptical density at 600 nm (OD600) of �0.6. Expression of plasmid-derived

SecA was induced with 0.5 mM IPTG (isopropyl-�-D-thiogalactopyrano-side), and cells were allowed to grow for 2 h. Cells were sedimented bycentrifugation and resuspended in lysis buffer (50 mM NaH2PO4, 300mM NaCl, pH 7.0), followed by sonication on ice for 4 min. The lysate wascentrifuged and the supernatant incubated with washed Talon metal af-finity resin beads (Clontech) for 1 to 2 h at 4°C. The beads were washedthrice with lysis buffer, followed by elution with 250 mM imidazole. Theeluate was dialyzed extensively against phosphate-buffered saline (PBS)buffer (136.89 mM NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4, 1.8 mMKH2PO4, pH 7.4). The final protein solution was stored at �80°C insingle-use aliquots, and the working protein stocks used for experimentswere stored at 4°C for less than 1 week. Protein concentrations were de-termined using OD280 with a molar extinction coefficient of 243.6 �M�1

cm�1. ATPase assays for the wild-type and 23 SecA mutants in the pres-ence of signal peptide and E. coli lipids were performed as described pre-viously (16).

SCAM experiments. SCAM experiments were carried out in a 20-�lreaction volume with 1 �M SecA protein in PBS buffer, in the presenceand absence of 10 �M KK-31 (KKKMKQSTIALALLPLLFTPVTKARTPEKKK-NH2) signal peptide. The reaction mixture was incubated at 30°Cfor 20 min. MPB (Thermo Scientific) and L-cysteine stocks were preparedimmediately before use. SecA was labeled with 1 �M MPB for 2 min, andthe labeling reaction was quenched with the addition of 5 �M L-cysteinefor 5 min. A total of 10 �l of 6� SDS-PAGE loading dye was added to thereaction mixture, followed by heating to 95°C for 5 min. A total of 15 �l ofeach reaction mixture was run on a 7.5% SDS-PAGE gel, followed byimmunotransfer onto a polyvinylidene difluoride (PVDF) membrane(Millipore). The membrane was blocked with Super Block blocking buffer(Thermo Scientific) overnight at 4°C and incubated with a 1:4,000 dilu-tion of horseradish peroxidase (HRP)-conjugated streptavidin antibody(Thermo Scientific) for 1 h, followed by 3 washes with PBS-Tween andincubation with SuperSignal West Pico chemiluminescent substrate(Thermo Scientific) for 5 min. For SecA detection, blots were incubatedwith a 1:3,000 dilution of monoclonal mouse anti-His antibody (GenScript)for 1 h, washed with PBS-Tween thrice, and incubated with a 1:6,000dilution of HRP-conjugated goat anti-mouse antibody (GenScript) for1 h. The blots were developed with an X-ray film developer or using theChemiDoc MP System (Bio-Rad). The blot intensities were analyzedusing ImageJ and GraphPad software and standardized against bovineserum albumin (BSA), which was also labeled with MPB. All proteinstructures utilized in this paper are from the RCSB Protein Data Bank(http://www.rcsb.org). Protein structures were presented using PyMol(Schrödinger, LLC) software.

Dimer interface experiments. SecA was reduced with a 1:100 dilutionof 50 mM Tris(2-carboxyethyl)phosphine (TCEP) for 30 min at roomtemperature. TCEP was removed using a Zeba desalting spin column(Thermo Scientific). A total of 0.4 �M of reduced SecA was incubated inthe presence and absence of 4 mM nucleotides, 20 �M KK-31, and 400�M E. coli polar lipid extract in liposomes (Avanti Polar Lipids) in 20 mMphosphate buffer, pH 7.4, supplemented with 50 mM KCl and 0.5 mMMgCl2 for 15 min at 30°C, followed by oxidation with 0.5 mM copperphenanthroline for 10 min at 30°C and quenching with 5 mM neocupro-ine for 5 min at room temperature (32). Samples were run on a nonre-ducing 7.5% SDS-PAGE gel and analyzed by Coomassie staining.

RESULTSGeneration of the monocysteine SecA mutant library and exper-imental strategy. To elucidate the signal peptide binding site andthe dimeric interface residues on SecA, we employed the estab-lished substituted cysteine accessibility method (SCAM). Thismethod has been used extensively to analyze membrane proteininsertion sites (30) and has been utilized to identify regions onSecA that interact with the inner membrane both in vivo and in thepresence of right-side-out inner membrane vesicles in vitro (33,34). To exploit this strategy, we generated a Cys-less SecA by mu-

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tating all four naturally occurring Cys residues to Ser producing aSecA derivative, which has been shown to have comparable activ-ity to the wild type (34). Using this template, we generated a li-brary of 23 monocysteine SecA mutants (Fig. 1A) that could becovalently modified with the sulfhydryl-reactive biotinylation re-

agent MPB (34). The residues were chosen such that all five do-mains of SecA could be investigated; however, residues selectedfrom the two nucleotide binding domains were kept at a mini-mum, since these two domains are crucial for ATP hydrolysis andare not expected to play a key role in preprotein binding. Some ofthe residues chosen were also based on previous studies (23, 25).The mutant SecA proteins displayed signal peptide-induced AT-Pase activities comparable to that of the wild type (Fig. 1D), withthe exception of the T221C, K633C, and D654C mutants, whichshowed activities roughly 70% of that of the wild type, and theT109C mutant, which was about 250% that of the wild type; thelatter is not surprising, since residue T109 is located close to the site ofATP hydrolysis. If the signal peptide interaction masks the Cysresidue, labeling with MPB would be precluded, whereas if the Cysresidue is located outside the region of signal peptide interaction,labeling should occur just as well as in the absence of signal pep-tide. Toward this end, we used the signal peptide KK-31 (Fig. 1B),which contains 21 residues of the alkaline phosphatase signal pep-tide plus four residues of the mature region and is flanked at theamino and carboxy termini by three Lys residues to enhance pep-tide solubility. The alkaline phosphatase signal peptide has beenfound to be representative of several model Sec pathway peptides(35). To ensure Cys specificity of MPB labeling of SecA, we treatedwild-type SecA, Cys-less SecA, and the M235C mutant with MPBand found that MPB could successfully label both the wild typeand the M235C mutant but was unable to label the Cys-less SecAtemplate (Fig. 1C). The MPB reagent includes a maleimide grouplinked to two polyethylene glycol (PEG) groups and a biotin; whilewe cannot rule out that its length precludes precision mapping, itis nevertheless ideal for interfering with productive signal peptide-SecA interactions that involve extensive surfaces.

Signal peptide binds at the PPXD-HSD-HWD groove. Toidentify the signal peptide binding site on SecA, we carried outMPB accessibility studies for each of the 23 mutants in the pres-ence and absence of KK-31. We categorized the mutants into threegroups, namely, strong, medium, and weak, based on labelingintensity changes before and after incubation with KK-31, of�50%, 49 to 25%, and �25%, respectively (Fig. 2A). As shown inFig. 2B, we found that different mutants exhibited different label-ing intensity changes upon incubation with KK-31, with valuesranging from 2.16% for G227C to 80.32% for E708C. We foundthat maximal labeling intensity changes were elicited in the pres-ence of KK-31 for a select few mutants at the PPXD, HWD, andHSD. These include M235C, I242C, N369C, P704C, E708C,V766C, and S773C, and one with medium labeling intensitychanges, R238C (Fig. 2C). Together, these residues comprise aregion on SecA that is ideally positioned to bind a signal peptidewithin the PPXD-HSD-HWD groove. Indeed, three additionalresidues that gave medium intensity changes are neighboring thisgroove, including F193C, T221C, and L353C. The other 12 mono-Cys mutants showed little to no difference in labeling with andwithout KK-31. SecA is known to exist in solution in both theopen and closed forms, with the accessibility of the PPXD-HSD-HWD groove being enhanced and diminished, respectively (5, 6).Thus, our results suggest that signal peptides could bind SecA inits open form at the PPXD-HSD-HWD groove.

Residues at the two-helix finger region form disulfide bonds.While performing our SCAM experiments, we observed that a fewmutants, namely, G793C, A795C, K797C, and D798C, located atthe two-helix finger region of the HSD, were incapable of labeling

FIG 1 Substituted cysteine accessibility strategy. (A) E. coli SecA monocys-teine mutants and their locations on SecA are shown; (B) sequence of theKK-31 signal peptide used in this study; (C) specificity of MPB reagent label-ing. A total of 2 �M wild-type SecA, Cys-less SecA, and M235C SecA mutantwere labeled with 1 �M MPB followed by quenching with 5 �M L-cysteine.The samples were run on an SDS-PAGE gel, followed by Western blotting withstreptavidin-HRP antibody as described in Materials and Methods. (D) Re-sults obtained from ATPase assays performed for the wild type (WT) and the23 SecA mutants are shown.

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with MPB even in the absence of signal peptide. This indicatedthat the accessibility of the Cys residue in these mutants was dra-matically reduced, suggesting that these residues could be buriedwithin the folded SecA structure or could be involved in a disulfide

bond. To determine whether lack of labeling with MPB was aresult of disulfide bond formation, we used a reducing agent,TCEP, to ensure complete reduction of disulfide-bonded species,if any. We found that mutants F193C and M235C that lie awayfrom the two-helix finger region labeled well with MPB, irrespec-tive of the presence or absence of TCEP. However, mutantsG793C, A795C, K797C, and D798C were found to label with MPBonly upon reduction with TCEP, indicating that these four mu-tants formed disulfide bonds (Fig. 3). These results indicate thatthese SecA proteins formed disulfide-bonded dimeric species,suggesting that the two-helix finger region could lie at the dimerinterface.

Dimer interface of SecA. To analyze the dimeric interface ofSecA, we examined the molecular weights of the mutants identi-fied earlier, namely, G793C, A795C, K797C, and D798C, that pro-duced disulfide-linked dimers. Using nonreducing SDS-PAGE,we found that these mutants migrated as dimeric species with amolecular mass of approximately 200 kDa (Fig. 3B), while theymigrated as monomers with TCEP treatment (data not shown).Since this experiment employs a nonreducing gel, some slightvariation in mobility is observed, likely representing small differ-ences in folding. Size exclusion chromatography and sucrose gra-dient density centrifugation experiments showed that the mono-mer-dimer equilibrium of SecA is substantially shifted towardmonomer by the deletion of residues 2 to 11 of SecA; these resi-dues are therefore thought to lie on the dimer interface (28, 29).We tested the ability of mutant L6C to form dimers and observedthat this protein existed in dimeric states in the absence of TCEP,though a small amount of monomer is also observed (Fig. 3B). Incontrast, consistent with the lack of disulfide-bonded dimers ob-served, F193C and M235C lie outside the two-helix finger region.The residues that did produce cross-linked dimers are located at

FIG 2 SecA monocysteine mutant labeling differences in the presence andabsence of KK-31. (A) Western blots illustrating representative labeling inten-sity changes. MPB labeling intensity results were classified as strong, medium,or weak based on intensity levels of �50%, 49 to 25%, and �25%, respectively.(B) The extent of reduction in labeling intensities of SecA monocysteine mu-tants in the presence of KK-31. Data represent the means � standard errors(SE) from at least three independent experiments. (C) E. coli SecA structure(PDB 2VDA) with monocysteine mutants that showed strong, medium, andweak labeling intensity differences in the presence of KK-31 are shown as red(and residues labeled), orange, and yellow spheres, respectively. The differentdomains are shown with NBD I in cyan, NBD II in blue, PPXD in green, HSDin purple, and HWD in pink.

FIG 3 SecA monocysteine mutants at the two-helix finger region form disul-fide bonds. (A) SecA monocysteine mutants L6C, G793C, A795C, K797C,D798C, F193C, and M235C were labeled with MPB before and after TCEPtreatment, followed by immunoblotting with antibiotin antibody. Total SecAconcentrations were detected using anti-His antibody as described in experi-mental procedures. (B) SecA monocysteine mutants were analyzed on a 7.5%nonreducing SDS-PAGE gel with no TCEP treatment. Small differences inrelative mobility, including apparent doublets, are likely due to slightly differ-ent folded forms that migrate as such on a nonreducing gel.

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the two-helix finger region of the HSD (or lie on the same face ofSecA, e.g., L6) that is thought to play a key role in the opening ofthe SecYEG channel (26).

Ligands alter SecA monomer-dimer equilibrium. To identifyligands that could disrupt the SecA monomer-dimer equilibrium,we reduced the mutant proteins with TCEP and incubated theproteins with signal peptides, E. coli phospholipids, ADP, orATP�S. Copper phenanthroline, a mild oxidizing agent, was thenadded to enable the reformation of exposed disulfide bonds. Ifindeed any of the ligands were able to shift the equilibrium towardthe monomeric state, the Cys residues of the two protomers wouldno longer be in close proximity, and hence disulfide bond forma-tion should not occur, and the proteins would migrate as mono-mers on a nonreducing SDS-PAGE gel. We found that, surpris-ingly, ATP�S had the greatest effect on shifting the equilibriumtoward the monomer (Fig. 4A). Phospholipids could also producemonomers, with the addition of signal peptide leading to a furtherenhancement of this shift. ADP, on the other hand, did not lead tomonomerization of SecA. Similar results were also obtained withmutant L6C, although in this case, the dimer was more easilyshifted toward the monomer in the presence of ligands. Two othermutants, namely, F193C and M235C, existed predominantly asmonomers both in the absence and presence of all ligands, consis-tent with the location of the Cys residues in these mutants, farfrom the dimer interface precluding disulfide bond formation.

Further experiments were carried out with mutant K797C toanalyze the effect of nucleotides in conjunction with other ligandson the alteration of the monomer-dimer equilibrium. Incubation

of K797C with ADP and signal peptides did not produce signifi-cant monomers, while the addition of phospholipids with andwithout signal peptide resulted in the generation of some mono-mers (Fig. 4B). On the other hand, ATP�S was able to form mono-mers in the presence of signal peptide or phospholipids, with thegreatest shift seen in the presence of all three ligands. These resultsindicate that binding of ATP leads to the formation of SecA mono-mers, while ADP favors dimerization. The addition of transloca-tion ligands, such as phospholipids and signal peptides, to ATP-bound SecA further enhances monomer production.

DISCUSSION

We employed the substituted cysteine accessibility strategy to ad-dress the location of the signal peptide interaction with SecA andits oligomeric interface. We utilized 23 monocysteine SecA mu-tants that covered much of the SecA “landscape,” with residueschosen from each of its five domains. The use of a sulfhydryllabeling reagent is advantageous in that our approach is sensitiveand specific for binding to Cys residues while also circumventingthe common pitfall of false-positive results often associated withthe use of strong cross-linking reagents such as EDAC. The resultsfrom these studies indicate that the signal peptide binds at agroove formed with residues of the PPXD, HSD, and HWD. Re-solved crystal structures of SecA show that SecA exists in the“open” or “closed” form, where the PPXD maintains tight or looseinteractions with the HWD, respectively. Such a swiveling motionof the PPXD involves a 60° rigid body rotation of the PPXD to-ward or away from NBD II (36). The PPXD has been previouslyimplicated in signal peptide binding (22–24), while other studieshave identified different regions on SecA, including the hydro-phobic cleft between the PPXD and NBD II and the � strandsconnecting the PPXD and NBD I (25, 26).

Our results identify a region on SecA that could “nestle” asignal peptide such that its hydrophobic core may interact withthe predominantly hydrophobic residues of the PPXD, while res-idues of the HWD and the HSD could interact with the amino andcarboxy termini of the signal peptide, respectively. Some of theresidues of SecA identified include M235, R238, I242, N369, P704,E708, V766, and S773; these results are in close agreement withresults obtained from an NMR study carried out in the presence ofSecA and a KRR-LamB signal peptide (23). The mature region ofthe preprotein may interact with the � strands linking PPXD andNBD I (37). Our results are consistent with proposed mechanismsfor preprotein translocation (23). SecA’s signal peptide bindinggroove may exist in the closed or open form (Fig. 5A and B);however, the signal peptide can productively bind SecA only in itsopen form. Studies carried out with cross-linked SecA-SecYEGproteins (38) have indicated that the signal peptide can be releasedfrom this site upon further opening of the PPXD-HSD-HWDgroove, along with a concomitant tight interaction of SecA withthe SecYEG channel (Fig. 5C and D). Although two of the struc-tures are from Bacillus subtilis and one is from Thermotoga mari-tima, the opening of the groove is evident from these structuresand is likely physiologically relevant. The opening of this groovemay also provide a feasible mechanism for trapping the matureregion of the preprotein at the clamp between the PPXD and NBDII (26), followed by its release into the channel.

We further established some of the residues on the dimer in-terface of SecA. Several studies have reported that SecA exists as adimer in the cytosol (39, 40); however, the ligands that alter the

FIG 4 SecA dimerizes at the two-helix finger region and various ligands mod-ulate dimer dissociation. (A) G793C, A795C, K797C, D798C, F193C, M235C,and L6C monocysteine mutants were reduced with TCEP, followed by itsremoval by desalting. Oxidation of 0.4 �M SecA was carried out in the pres-ence of 0.5 mM copper phenanthroline with and without the ligands shown,followed by quenching with 5 mM neocuproine. Final concentrations of nu-cleotides, KK-31, and E. coli phospholipids were 4 mM, 20 �M, and 400 �M,respectively. The samples were analyzed on a nonreducing SDS-PAGE gel.Dimer and monomer are indicated as D and M, respectively. (B) SecA mono-merization is induced in the presence of ATP�S, signal peptide, and lipids.Oxidation experiments with the K797C monocysteine SecA mutant were car-ried out as described for panel A. The samples were analyzed on a nonreducingSDS-PAGE gel. Dimer and monomer are indicated as D and M, respectively.

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monomer-dimer equilibrium, the functional translocation-activeoligomeric state of SecA, and the dimer interface of SecA are un-clear (11–15, 41). Monomeric SecA has been shown to be suffi-cient for maintaining translocation (13, 28), while experimentscarried out with dimeric SecA proteins indicate that monomeriza-tion is not required for maintaining translocation activity (29, 41,42). Crystal structures of SecA from different bacterial specieshave also provided differing reports on the dimeric interface andthe orientation of the protomers (3–6, 43). We found that G793C,A795C, K797C, and D798C monocysteine SecA mutants, whichlie on the two-helix finger loop region, produced disulfide-bonded dimers in the absence of reducing agents, while the F193Cand M235C mutants, which are located outside the two-helix fin-ger, did not. Our results also indicate that ATP�S and lipids re-sulted in the partial dissociation of these dimers, while completemonomerization was seen in the presence of ATP�S, lipids, andsignal peptides, strongly suggesting that monomers are producedwhen translocation is under way. While we cannot rule out thatdimeric SecA may be sufficient for translocation in the cell, basedon our experiments, we deduce that monomeric SecA plays animportant role in protein export.

Our study provides a picture of the dimer interface and de-scribes how the monomer-dimer equilibrium can be altered dur-ing translocation. We propose that residues involved in SecA

dimerization lie along the two-helix finger region. Our results arein agreement with the T. thermophilus parallel dimer crystal struc-ture (4). Earlier reports have also suggested that the C-terminalregion of SecA plays a key role in dimerization (13, 44, 45). Thetwo-helix finger is particularly important for interactions with theSecYEG channel and is thought to be directly involved in channelopening (31). As SecA engages the SecYEG channel, SecA coulddissociate into monomers, and the two-helix finger would now beavailable to open the SecYEG channel from the cis-side of themembrane (Fig. 5E) (26, 46). This conformational change mayoccur in conjunction with the opening of the PPXD-HSD-HWDgroove, release of ADP and uptake of ATP, and binding of thepreprotein, followed by its successful translocation across themembrane.

ACKNOWLEDGMENTS

This work was supported by grant GM037639 from the National Insti-tutes of Health (D.A.K.).

REFERENCES1. Pohlschroder M, Hartmann E, Hand NJ, Dilks K, Haddad A. 2005.

Diversity and evolution of protein translocation. Annu. Rev. Microbiol.59:91–111.

2. Economou A, Wickner W. 1994. SecA promotes preprotein translocation

FIG 5 Accessibility of the PPXD-HSD-HWD groove is altered under different conformational states. (A) Crystal structure of B. subtilis SecA (PDB 1M6N) in theclosed form. The asterisk indicates the location of the two-helix finger region. (B) Crystal structure of B. subtilis SecA (PDB 1TF5) in the open form; (C) crystalstructure of T. maritima SecA (PDB 3DIN) in the SecYEG-bound form; (D) overlay of the three crystal structures of SecA with the closed, open, andSecYEG-bound forms colored blue, green, and red, respectively, and illustrating the progression in opening the PPXD-HSD-HWD groove; (E) a proposed modelthat links the changes in oligomerization of SecA with the translocation of a preprotein is shown. Dimeric cytosolic SecA dissociates into monomers as it engageswith SecYEG, and the two-helix finger unlocks the channel. The signal peptide binding groove of SecA is opened, along with concomitant uptake of ATP andsignal peptide and preprotein binding.

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