Hsiuchin Yang

Ring-like pore structures of SecA: Implication for bacterial protein-conducting channels

SecA, an essential component of the general protein secretion pathway of bacteria, is present in Escherichia coli as soluble and membrane-integral forms. Here we show by electron microscopy that SecA assumes two characteristic forms in the presence of phospholipid monolayers: dumbbell-shaped elongated structures and ring-like pore structures. The ring-like pore structures with diameters of 8 nm and holes of 2 nm are found only in the presence of anionic phospholipids. These ring-like pore structures with larger 3- to 6-nm holes (without staining) were also observed by atomic force microscopic examination. They do not form in solution or in the presence of uncharged phosphatidylcholine. These ring-like phospholipid-induced pore-structures may form the core of bacterial protein-conducting channels through bacterial membranes.

The first low μM SecA inhibitors

SecA ATPase is a critical member of the Sec family, which is important in the translocation of membrane and secreted polypeptides/proteins in bacteria. Small molecule inhibitors can be very useful research tools as well as leads for future antimicrobial agent development. Based on previous virtual screening work, we optimized the structures of two hit compounds and obtained SecA ATPase inhibitors with IC(50) in the single digit micromolar range. These represent the first low micromolar synthetic inhibitors of bacterial SecA and will be very useful for mechanistic studies.

Additional In Vitro and In Vivo Evidence for SecA Functioning as Dimers in the Membrane: Dissociation into Monomers Is Not Essential for Protein Translocation in Escherichia coli

SecA is an essential component in the Sec-dependent protein translocation pathway and, together with ATP, provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. Previous studies established that SecA undergoes monomer-dimer equilibrium in solution. However, the oligomeric state of functional SecA during the protein translocation process is controversial. In this study, we provide additional evidence that SecA functions as a dimer in the membrane by (i) demonstration of the capability of the presumably monomeric SecA derivative to be cross-linked as dimers in vitro and in vivo, (ii) complementation of the growth of a secA(Ts) mutant with another nonfunctional SecA or (iii) in vivo complementation and in vitro function of a genetically tandem SecA dimer that does not dissociate into monomers, and (iv) formation of similar ring-like structures by the tandem SecA dimer and SecA in the presence of lipid bilayers. We conclude that SecA functions as a dimer in the membrane and dissociation into monomers is not necessary during protein translocation.

Packaging of chemicals in the defensive secretory glands of the sea hare Aplysia californica

SUMMARY Sea hares protect themselves from predatory attacks with several modes of chemical defenses. One of these is inking, which is an active release of a protective fluid upon predatory attack. In many sea hares including Aplysia californica and A. dactylomela, this fluid is a mixture of two secretions from two separate glands, usually co-released: ink, a purple fluid from the ink gland; and opaline, a white viscous secretion from the opaline gland. These two secretions are mixed in the mantle cavity and directed toward the attacking predator. Some of the chemicals in these secretions and their mechanism of action have been identified. In our study, we used western blots, immunocytochemistry, amino acid analysis, and bioassays to examine the distribution of these components: (1) an l-amino acid oxidase called escapin for A. californica and dactylomelin-P for A. dactylomela, which has antimicrobial activity but we believe its main function is in defending sea hares against predators that evoke its release; and (2) escapins major amino acid substrates - l-lysine and l-arginine. Escapin is exclusively produced in the ink gland and is not present in any other tissues or secretions. Furthermore, escapin is only sequestered in the amber vesicles of the ink glandand not in the red-purple vesicles, which contain algal-derived chromophores that give ink its distinctive purple color. The concentration of escapin and dactylomelin-P in ink, both in the gland and after its release, is as high as 2 mg ml-1, or 30 μmol ml-1, which is well above its antimicrobial threshold. Lysine and arginine (and other amino acids) are packaged into vesicles in the ink and opaline glands, but arginine is present in ink and opaline at <1 mmol l-1 and lysine is present in ink at <1 mmol l-1 but in opaline at 65 mmol l-1. Our previous results showed that both lysine and arginine mediate escapins bacteriostatic effects, but only lysine mediates its bactericidal effects. Given that escapins antimicrobial effects require concentrations of lysine and/or arginine >1 mmol l-1, our data lead us to conclude that lysine in opaline is the primary natural substrate for escapin in ink. Furthermore, packaging of the enzyme escapin and its substrate lysine into two separate glands and their co-release and mixing at the time of predatory attack allows for the generation of bioactive defensive compounds from innocuous precursors at the precise time they are needed. Whether lysine and/or arginine are substrates for escapins antipredatory functions remains to be determined.

SecA Alone Can Promote Protein Translocation and Ion Channel Activity SecYEG INCREASES EFFICIENCY AND SIGNAL PEPTIDE SPECIFICITY

Background: SecA has been viewed as ATPase helping precursors across SecYEG channels. Results: SecA alone could promote protein translocation and ion channel activity, but loses specificity and efficiency, which can be restored by SecYEG. Conclusion: SecA plays important structural roles and can function as low affinity protein conducting channels in membranes. Significance: Establishing SecA as channels is crucial for understanding diverse mechanisms and evolution of bacterial translocation pathways. SecA is an essential component of the Sec-dependent protein translocation pathway across cytoplasmic membranes in bacteria. Escherichia coli SecA binds to cytoplasmic membranes at SecYEG high affinity sites and at phospholipid low affinity sites. It has been widely viewed that SecYEG functions as the essential protein-conducting channel through which precursors cross the membranes in bacterial Sec-dependent pathways, and that SecA functions as a motor to hydrolyze ATP in translocating precursors through SecYEG channels. We have now found that SecA alone can promote precursor translocation into phospholiposomes. Moreover, SecA-liposomes elicit ionic currents in Xenopus oocytes. Patch-clamp recordings further show that SecA alone promotes signal peptide- or precursor-dependent single channel activity. These activities were observed with the functional SecA at about 1–2 μm. The results show that SecA alone is sufficient to promote protein translocation into liposomes and to elicit ionic channel activity at the phospholipids low affinity binding sites, thus indicating that SecA is able to form the protein-conducting channels. Even so, such SecA-liposomes are less efficient than those with a full complement of Sec proteins, and lose the signal-peptide proofreading function, resembling the effects of PrlA mutations. Addition of purified SecYEG restores the signal peptide specificity and increases protein translocation and ion channel activities. These data show that SecA can promote protein translocation and ion channel activities both when it is bound to lipids at low affinity sites and when it is bound to SecYEG with high affinity. The latter of the two interactions confers high efficiency and specificity.

Fluorescein Analogues Inhibit SecA ATPase: The First Sub-micromolar Inhibitor of Bacterial Protein Translocation

SecA is a central component of the general secretion system that is essential for bacterial growth and thus an ideal target for antimicrobial agents. A series of fluorescein analogues were first screened against the ATPase activity using the truncated unregulated SecA catalytic domain. Rose bengal (RB) and erythrosin B (EB) were found to be potent inhibitors SecA with IC50 values of 0.5 μM and 2 μM, respectively. RB and EB inhibit the catalytic SecA ATPase more effectively than the F1F0‐proton ATPase. We used three assays to test the effect of these compounds on full‐length SecA ATPase: in solution (intrinsic ATPase), in membrane preparation, and translocation ATPase. RB and EB show the following trend in terms of IC50 values: translocation ATPase

Reconstitution of functionally efficient SecA-dependent protein-conducting channels: transformation of low-affinity SecA-liposome channels to high-affinity SecA-SecYEG-SecDF·YajC channels.

Previous work showed that SecA alone can promote protein translocation and ion-channel activity in liposomes, and that SecYEG increases efficiency as well as signal peptide specificity. We now report that SecDF·YajC further increases translocation and ion-channel activity. These activities of reconstituted SecA-SecYEG-SecDF·YajC-liposome are almost the same as those of native membranes, indicating the transformation of reconstituted functional high-affinity protein-conducting channels from the low-affinity SecA-channels.

Design, synthesis and biological evaluation of rose bengal analogues as SecA inhibitors.

SecA, a key component of bacterial Sec‐dependent secretion pathway, is an attractive target for exploring novel antimicrobials. Rose bengal (RB), a polyhalogenated fluorescein derivative, was found from our previous study as a potent SecA inhibitor. Here we describe the synthesis and structure–activity relationships (SAR) of 23 RB analogues that were designed by systematical dissection of RB. Evaluation of these analogues allowed us to establish an initial SAR in SecA inhibition. The antimicrobial effects of these SecA inhibitors are confirmed in experiments using E. coli and B. subtilis.

To Be or Not to Be: Predicting Soluble SecAs as Membrane Proteins

SecA is an important component of protein translocation in bacteria, and exists in soluble and membrane-integrated forms. Most membrane prediction programs predict SecA as being a soluble protein, with the exception of TMpred and TopPred. However, the membrane associated predicted segments by TMpred and TopPred are inconsistent across bacterial species in spite of high sequence homology. In this paper we describe a new method for membrane protein prediction, PSSM_SVM, which provides consistent results for integral membrane domains of SecAs across bacterial species. This PSSM encoding scheme demonstrates the highest accuracy in terms of Q2 among the common prediction methods, and produces consistent results on blind test data. None of the previously described methods showed this kind of consistency when tested against the same blind test set. This scheme predicts traditional transmembrane segments and most of the soluble proteins accurately. The PSSM scheme applied to the membrane-associated protein SecA shows characteristic features. In the set of 223 known SecA sequences, the PSSM_SVM prediction scheme predicts eight to nine residue embedded membrane segments. This predicted region is part of a 12 residue helix from known X-ray crystal structures of SecAs. This information could be important for determining the structure of SecA proteins in the membrane which have different conformational properties from other transmembrane proteins, as well as other soluble proteins that may similarly integrate into lipid bi-layers.

Phospholipids Induce Conformational Changes of SecA to Form Membrane-Specific Domains: AFM Structures and Implication on Protein-Conducting Channels

SecA, an essential component of the Sec machinery, exists in a soluble and a membrane form in Escherichia coli. Previous studies have shown that the soluble SecA transforms into pore structures when it interacts with liposomes, and integrates into membranes containing SecYEG in two forms: SecAS and SecAM; the latter exemplified by two tryptic membrane-specific domains, an N-terminal domain (N39) and a middle M48 domain (M48). The formation of these lipid-specific domains was further investigated. The N39 and M48 domains are induced only when SecA interacts with anionic liposomes. Additionally, the N-terminus, not the C-terminus of SecA is required for inducing such conformational changes. Proteolytic treatment and sequence analyses showed that liposome-embedded SecA yields the same M48 and N39 domains as does the membrane-embedded SecA. Studies with chemical extraction and resistance to trypsin have also shown that these proteoliposome-embedded SecA fragments exhibit the same stability and characteristics as their membrane-embedded SecA equivalents. Furthermore, the cloned lipid-specific domains N39 and M48, but not N68 or C34, are able to form partial, but imperfect ring-like structures when they interact with phospholipids. These ring-like structures are characteristic of a SecA pore-structure, suggesting that these domains contribute part of the SecA-dependent protein-conducting channel. We, therefore, propose a model in which SecA alone is capable of forming a lipid-specific, asymmetric dimer that is able to function as a viable protein-conducting channel in the membrane, without any requirement for SecYEG.