Interdependent Effects of the Charge of the N-Terminal Region of the Signal Peptide,SecA, and SecB on Secretion of Alkaline Phosphatase in Escherichia coli

The cytoplasmic step of posttranslational secretion in Escherichia coli is catalyzed by export-specific chaperone SecB and translocational ATPase SecA. In addition, the efficiency of secretion depends on the charge of the signal peptide (SP). Replacement of positively charged Lys(–20) with uncharged Ala or negatively charged Glu in the N-terminal region of SP of the alkaline phosphatase precursor (prePhoA) was shown to decrease the PhoA secretion in the periplasm. The effect on secretion increased in the absence of SecB and was especially high on SecA inactivation. A change in SP charge strengthened the SecA and SecB dependences of secretion. On evidence of immunoprecipitation, the charge of the N-terminal region of SP had no effect on prePhoA interaction with the cytoplasmic secretion factors, suggesting no direct binding between this region and SecA or SecB. Yet the charge of the N-terminal region proved to affect the functions of SP as an intramolecular chaperone and a factor of prePhoA targeting to the membrane in cooperation with SecA and SecB.     

Effect of export-specific cytoplasmic chaperone, protein SecB, on secretion of Escherichia coli alkaline phosphatase

The efficiency of secretion of Escherichia coli alkaline phosphatase depends on the presence in cells of a cytoplasmic chaperone—protein SecB. Secretion increases in the presence of this chaperone at 30°C, which is the most favorable for the interaction of SecB with the export-initiation domain found previously in the N-terminal region of the mature enzyme. This interaction most likely occurs in the region of the export domain, which is located close to the signal peptide and in complex with a translocational ATPase—protein SecA.     

Effect of Membrane Phospholipid Composition and Charge of the Signal Peptide of Escherichia coli Alkaline Phosphatase on Efficiency of Its Secretion

The secretion of the Escherichia coli alkaline phosphatase with a different charge of signal peptide due to replacement of positively charged Lys(–20) has been studied depending on the phospholipid composition of the membranes and the activity of the translocational ATPase—protein SecA. Changing the signal peptide charge, along with a change in phospholipid composition, has been shown to reduce the efficiency of secretion. In the absence of phosphatidylethanolamine the membrane contains anionic phospholipids only, and the dependence of secretion on the signal peptide charge decreases. The dependence of secretion on membrane phospholipid composition and the signal peptide charge is also determined by the activity of SecA protein. If SecA is inactivated by sodium azide, then the dependence of secretion on anionic phospholipids increases; on the contrary, higher content of anionic phospholipids (in the absence of phosphatidylethanolamine) decreases the dependence of secretion on the SecA activity. The results suggest a direct interaction of positively charged signal peptide with negatively charged membrane phospholipids under initiation of secretion and also interdependent contribution of the signal peptide charge, anionic phospholipids, and translocational ATPase to secretion.     

Inactivation of the pgsA Gene Affects Biosynthesis and Secretion of Alkaline Phosphatase in Escherichia coli

Inactivation of pgsA, which is responsible for biosynthesis of anionic phospholipid phosphatidyl-glycerol (PG), was shown to affect biosynthesis and secretion of alkaline phosphatase (PhoA) in Escherichia coli. A decrease in PG, but not in total anionic phospholipids, correlated with reduction of PhoA secretion, suggesting the role of PG in this process. A dramatic decrease in PG (from 18 to 3, but not 8, percent of the total phospholipids) inhibited not only secretion, but also synthesis of PhoA. In addition, pgsA inactivation expedited repression of PhoA synthesis by exogenous orthophosphate.     

Crystal structure of SecB from Escherichia coli

The chaperone SecB from Escherichia coli is primarily involved in passing precursor proteins into the Sec system via specific interactions with SecA. The crystal structure of SecB from E. coli has been solved to 2.35 A resolution. The structure shows flexibility in the crossover loop and the helix-connecting loop, regions that have been implicated to be part of the SecB substrate-binding site. Moreover conformational variability of Trp36 is observed as well as different loop conformations for the different monomers. Based on this, we speculate that SecB can regulate the access or extent of its hydrophobic substrate-binding site, by modulating the conformation of the crossover loop and the helix-connecting loop. The structure also clearly explains why the tetrameric equilibrium is shifted towards the dimeric state in the mutant SecBCys76Tyr. The buried cysteine residue is crucial for tight packing, and mutations are likely to disrupt the tetramer formation but not the dimer formation.     

Effects of pre-protein overexpression on SecB synthesis in Escherichia coli

Protein translocation through the cytoplasmic membrane of Escherichia coli involves cytosolic chaperones. The export-dedicated chaperone SecB mediates targeting of a subset of pre-proteins. In this report, synthesis of SecB in response to plasmid-mediated overexpression of pre-proteins was studied. Overexpression of SecB-dependent pre-proteins stimulated synthesis of SecB under conditions where the cellular export capacity was saturated or uncomplexed SecB was trapped. On the contrary, overexpression of SecB-independent pre-beta-lactamase reduced the promoter activity of secB. The results suggest that uncomplexed SecB can be sequestered by synthesis of SecB-dependent pre-proteins. Furthermore, these data demonstrate the distinct action of the SecB- and signal recognition particle-dependent protein targeting pathways.     

Study of interaction of export initiation domain of Escherichia coli mature alkaline phosphatase with membrane phospholipids during secretion

The efficiency of secretion of alkaline phosphatase from Escherichia coli depending on the primary structure of its N-terminal region and the content of zwitterionic phospholipid phosphatidylethanolamine and anionic phospholipids in membranes has been studied in this work to establish the peculiarities of interaction of mature protein during its secretion with membrane phospholipids. It has been shown that the effect of phosphatidylethanolamine but not anionic phospholipids on the efficiency of alkaline phosphatase secretion is determined by the primary structure of its N-terminal region. The absence of phosphatidylethanolamine appreciably reduces the efficiency of secretion of wild type alkaline phosphatase and its mutant forms with amino acid substitutions in positions +5+6 and +13+14. In contrast, secretion of the protein with amino acid substitutions in positions +2+3, significantly decreased as a result of such mutation, in the presence of phosphatidylethanolamine, reaches the level of wild type protein secretion in the absence of phosphatidylethanolamine. The results suggest an interaction of the N-terminal region of the mature protein under its translocation across the membrane with phosphatidylethanolamine.     

Characterization of three areas of interactions stabilizing complexes between SecA and SecB, two proteins involved in protein export

The general secretory, Sec, system translocates precursor polypeptides from the cytosol across the cytoplasmic membrane in Escherichia coli. SecB, a small cytosolic chaperone, captures the precursor polypeptides before they fold and delivers them to the membrane translocon through interactions with SecA. Both SecB and SecA display twofold symmetry and yet the complex between the two is stabilized by contacts that are distributed asymmetrically. Two distinct regions of interaction have been defined previously and here we identify a third. Calorimetric studies of complexes stabilized by different subsets of these interactions were carried out to determine the binding affinities and the thermodynamic parameters that underlie them. We show here that there is no change in affinity when either one of two contact areas out of the three is lacking. This fact and the asymmetry of the binding contacts may be important to the function of the complex in protein export.     

Sites of interaction between SecA and the chaperone SecB, two proteins involved in export

SecB, a small tetrameric cytosolic chaperone in Escherichia coli, facilitates the export of precursor poly-peptides by maintaining them in a nonnative conformation and passing them to SecA, which is a peripheral member of the membrane-bound translocation apparatus. It has been proposed by several laboratories that as SecA interacts with various components along the export pathway, it undergoes conformational changes that are crucial to its function. Here we report details of molecular interactions between SecA and SecB, which may serve as conformational switches. One site of interaction involves the final C-terminal 21 amino acids of SecA, which are positively charged and contain zinc. The C terminus of each subunit of the SecA dimer makes contact with the flat beta-sheet that is formed by each dimer of the SecB tetramer. Here we demonstrate that a second interaction exists between the extreme C-terminal alpha-helix of SecB and a site on SecA, as yet undefined but different from the C terminus of SecA. We investigated the energetics of the interactions by titration calorimetry and characterized the hydrodynamic properties of complexes stabilized by both interactions or each interaction singly using sedimentation velocity centrifugation.     

Requirement of the SecB chaperone for export of a non-secretory polypeptide in Escherichia coli

Summary The SecB protein of Escherichia coli is a cytosolic component of the export machinery which can prevent some precursors from prematurely folding into export-incompatible conformations by binding to the newly synthesised polypeptide. The feature(s) of target proteins recognised by SecB, however, are unclear and have been a matter of controversy. Also, it has not been asked if binding of SecB is specific for secretory proteins. We demonstrate here that a non-secretory polypeptide, a fragment of a tail fiber protein of phage T4, fused to the signal peptide of the outer membrane protein OmpA has a very strong SecB requirement for export and that the signal peptide itself cannot, at least not alone, be responsible for this action of SecB. The data reported, together with those of the literature, suggest that SecB recognizes the polypeptide backbone of the target protein.     

Changes in the composition of anionic membrane phospholipids influence protein secretion and cell envelope biogenesis in Escherichia coli

The secretion of alkaline phosphatase (PhoA) and peculiarities of biogenesis of the cell envelope were studied in Escherichia coli strains HD30/pHD102 and HDL11 with controlled synthesis of the anionic phospholipids, phosphatidylglycerol and cardiolipin. Inactivation of the pgsA gene responsible for the synthesis of anionic phospholipids or changes in the regulation of its expression by an environmental factor caused changes in the metabolism and composition of membrane phospholipids, which resulted in a decrease in the secretion of alkaline phosphatase through the cytoplasmic membrane and an increase in PhoA secretion from the periplasm into the culture medium. An increase was observed in exopolysaccharide secretion, as well as a decrease in the contents of the outer membrane lipopolysaccharides and lipopolyproteins, which determine its barrier properties. The results obtained show that anionic phospholipids play a significant role in protein secretion and are probably involved in the interrelation between protein secretion and biogenesis of cell envelope components.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 179–184.Original Russian Text Copyright © 2005 by Anisimova, Badyakina, Vasileva, Nesmeyanova.     

Membrane phospholipid composition of Escherichia coli affects secretion of periplasmic alkaline phosphatase into the medium

Secretion of alkaline phosphatase (PhoA) encoded by a gene constituent of plasmids has been studied in Escherichia coli strains with controlled synthesis of anionic phospholipids (phosphatidylglycerol and cardiolipin, strain HDL11) and zwitterionic phospholipid (phosphatidylethanolamine, strain AD93). Changing the phospholipid composition of the membrane of these strains leads to an increase in secretion of PhoA, which is usually localized in the periplasm, into the culture medium. This correlates with a higher secretion of exopolysaccharides and lower content of lipopolysaccharide in the outer membrane. The results show the possibility of coupling protein secretion into the medium with biogenesis of cell envelope components in which phospholipids are involved.     

Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA

Protein export mediated by the general secretory Sec system in Escherichia coli proceeds by a dynamic transfer of a precursor polypeptide from the chaperone SecB to the SecA ATPase motor of the translocon and subsequently into and through the channel of the membrane‐embedded SecYEG heterotrimer. The complex between SecA and SecB is stabilized by several separate sites of contact. Here we have demonstrated directly an interaction between the N‐terminal residues 2 through 11 of SecA and the C‐terminal 13 residues of SecB by isothermal titration calorimetry and analytical sedimentation velocity centrifugation. We discuss the unusual binding properties of SecA and SecB in context of a model for transfer of the precursor along the pathway of export.     

Structure of dimeric SecA, the Escherichia coli preprotein translocase motor

SecA is the preprotein translocase ATPase subunit and a superfamily 2 (SF2) RNA helicase. Here we present the 2 A crystal structures of the Escherichia coli SecA homodimer in the apo form and in complex with ATP, ADP and adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP). Each monomer contains the SF2 ATPase core (DEAD motor) built of two domains (nucleotide binding domain, NBD and intramolecular regulator of ATPase 2, IRA2), the preprotein binding domain (PBD), which is inserted in NBD and a carboxy-terminal domain (C-domain) linked to IRA2. The structures of the nucleotide complexes of SecA identify an interfacial nucleotide-binding cleft located between the two DEAD motor domains and residues critical for ATP catalysis. The dimer comprises two virtually identical protomers associating in an antiparallel fashion. Dimerization is mediated solely through extensive contacts of the DEAD motor domains leaving the C-domain facing outwards from the dimerization core. This dimerization mode explains the effect of functionally important mutations and is completely different from the dimerization models proposed for other SecA structures. The repercussion of these findings on translocase assembly and catalysis is discussed.     

Thermostable Alkaline Phosphatase of Bacterium Meiothermus ruber: Gene Cloning,Expression in Escherichia coli,and Biochemical Characterization of the Recombinant Protein

The Meiothermus ruber alkaline phosphatase gene was cloned, expressed in Escherichia coli cells, and sequenced. The enzyme precursor, including the putative signal peptide, was shown to consist of 503 residues (deduced molecular mass 54,229 Da). The recombinant enzyme showed the maximal activity at 60–65°C, pH 11.0, K _M = 0.055 mM with p-nitrophenyl phosphate. The enzyme proved to be moderately thermostable, retaining 50% activity after 6 h incubation at 60°C and being completely inactivated in 2 h at 80°C. In substrate specificity assays, the highest activity was observed with p-nitrophenyl phosphate and dATP. Vanadate, inorganic phosphate, and SDS were inhibitory, while thiol-reducing agents had virtually no effect. The enzyme activity strongly depended on exogenous Mg²⁺ and declined in the presence of EDTA.     

Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

Genetic organization and biogenesis of adhesive fimbriae of Escherichia coli

The genetic organization of the determinants of type 1, K88ab, K99 fimbriae and P(pap)pili of Escherichia coli is presented. The functions of the various gene products are described and a model for the process of fimbriae biogenesis is presented and discussed.     

Interaction of cAMP receptor protein from Escherichia coli with cAMP and DNA studied by differential scanning calorimetry

The cyclic AMP receptor protein (CRP) regulates the expression of many genes in Escherichia coli. The protein is a homodimer, and each monomer is folded into two distinct structural domains. In this study, we have used differential scanning calorimetry (DSC) and circular dichroism (CD) to measure the enthalpy change and melting temperature of the apo-CRP and CRP complexes with cAMP or DNA sequences lac, gal, and palindromic ICAP. DSC and CD measurements showed irreversible thermal denaturation process of CRP. Enthalpy of dissociation of the protein–DNA complex, as measured by DSC, depends on the DNA sequence. The thermal transition of the protein in CRP-DNA complexes, measured by CD, indicates that the protein stability in the complex is also DNA sequence-dependent.     

大肠杆菌碱性磷酸酶的体外定向进化研究

大肠杆菌碱性磷酸酶（E.coli alkaline phosphatase, EAP, EC 3.1.3.1）是一个非特异性二聚体磷酸单酯酶. 采用易错聚合酶链反应(error prone PCR)的方法,在原有高活力突变株的基础上,对EAP远离活性中心催化三联体的区域进行定向进化,经两轮error prone PCR,获得催化活力较亲本D101S突变株提高3倍、较野生型酶提高35倍的进化酶4-186,并对该酶的催化动力学特征进行了分析. 进化酶基因的DNA测序表明4-186含两个有义氨基酸置换：K167R和S374C,二者既不位于底物结合位点,也不位于酶的金属离子结合位点.     

Why does ethanol induce cellular heat-shock response?

At the time of induction of the periplasmic protein alkaline phosphatase (AP) in Escherichia coli, the presence of ethanol (10% v/v) in the growth medium did not allow the induced AP to be translocated out to the periplasm. The nontransported AP was stored in the cytoplasm as the unfolded precursor form (AP with its amino-terminal signal sequence), which had no enzymatic activity. The presence of 10% v/v ethanol in the growth medium also induced the heat-shock response in E. coli, which was evident from the enhanced syntheses of several heat-shock proteins (HSPs) over their cellular basal levels. These results, in conjunction with our earlier findings on the occurrence of heat-shock response in an AP-signal sequence mutant of E. coli due to the export deficiency of AP precursor, suggest that the membrane protein precursors, stored in the cytoplasm due to the ethanol-mediated inhibition of translocation, behaved to the cells as abnormal proteins, which ultimately triggered the signal for the induction of heat-shock response in E. coli.