A positive residue in the hydrophobic core of the Escherichia coli lipoprotein signal peptide suppresses the secretion defect caused by an acidic amino terminus.

The signal peptide of secretory proteins requires a basic amino terminus followed by a stretch of hydrophobic residues to effect efficient translocation of precursor proteins. Replacement of the positively charged amino-terminal residues of prolipoprotein by acidic amino acids decreased the rate of precursor translocation (Inouye, S., Soberon, X., Franceschini, T., Nakamura, K., Itakura, K., and Inouye, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3438-3441; Vlasuk, G. P., Inouye, S., Ito, H., Itakura, K., and Inouye, M. (1983) J. Biol. Chem. 258, 7141-7148). We demonstrate here that an arginine residue, but not an aspartate, when localized at position 9 of the hydrophobic region of the lipoprotein signal peptide, is able to suppress intramolecularly the processing defect caused by an acidic amino terminus. Furthermore, when present at position 14 of the signal peptide, this positive residue, but not aspartate, was able to support efficient translocation of unmodified prolipoprotein. This demonstrates that a positive residue can restore the function of a severely defective signal peptide and need not be localized at the amino terminus to do so. Both aspartate and arginine substitution at position 14 of the lipoprotein signal peptide stimulated prolipoprotein synthesis. This effect was position-specific, did not require precursor translocation, and was dominant to the inhibition of synthesis caused by an acidic amino terminus.     

Effects of mutations at glycine residues in the hydrophobic region of the Escherichia coli prolipoprotein signal peptide on the secretion across the membrane

Each of the 2 glycine residues in the hydrophobic region of the prolipoprotein signal peptide of Escherichia coli was systematically deleted or substituted with a valine residue by oligonucleotide-directed site-specific mutagenesis. Functional analysis of four such mutants as well as four double mutants, resulting from combinations of any two of the single mutations, revealed that (a) glycine residues at positions 9 and 14 could be replaced individually or at the same time with a valine residue without affecting the secretion of prolipoprotein; (b) the deletion of glycine at position 9 had no effect on the secretion of prolipoprotein whereas, when glycine at position 14 was deleted, the glyceride modification and the processing of the mutant prolipoprotein occurred at a much slower rate at 42 degrees C than those of the wild type prolipoprotein; and (c) the effects of deleting glycine at position 14 could be suppressed by the deletion of glycine at position 9, which resulted in shortening the hydrophobic region of the prolipoprotein signal peptide by 2 amino acid residues. These results indicate that the hydrophobic region of the prolipoprotein signal peptide has remarkable flexibility in terms of the relationship between its primary structure and function in protein secretion.     

Exploring the conformational roles of signal sequences: synthesis and conformational analysis of lambda receptor protein wild-type and mutant signal peptides

Secretion of the Escherichia coli lambda receptor protein (LamB protein) appears from genetic evidence to be correlated with the predicted tendency of its signal sequence to adopt an alpha-helical conformation [Emr, S. D., & Silhavy, T. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4599]. We have tested this hypothesis by synthesizing major portions of signal sequences from the wild-type and mutant LamB proteins and analyzing their conformations by circular dichroism. The wild-type signal sequence contains a seven-residue hydrophobic region flanked by a proline and a glycine. Chou-Fasman rules predict that this segment will adopt an alpha-helical conformation. An export-deficient mutant is missing four residues from this region; the helix-breaking glycine and proline are thus separated by only three residues, and an alpha helix is not predicted to form. In each of the export-restored revertants, either the glycine or the proline is replaced with a residue which promotes helix formation. The helix content of the synthetic signal sequence fragments on the basis of CD measurements supports the secondary structure hypothesis described above. The relative helicity in aqueous sodium dodecyl sulfate, lysolecithin, or trifluoroethanol is as follows: wild type greater than R2 (Pro----Leu) greater than R1 (Gly----Cys) much greater than deletion mutant.     

Amino-acid residues involved in the expression of the activity of Escherichia coli TolC

The Escherichia coli TolC, composed of 471 amino-acid residues, functions as a channel tunnel in the transport of various molecules across the outer membrane. We found previously that Leu-412, the 60th amino-acid residue from the carboxy terminal end, was crucial to the transport activity of TolC. Leu-412 is located in a domain which protrudes from the main body of TolC into the periplasm. Subsequent study indicated that the hydrophobicity generated by Leu-412 played an important role in the activity of TolC (H. Yamanaka, T. Nomura, N. Morisada, S. Shinoda, and K. Okamoto, Microb. Pathog. 33: 81-89, 2002). We predicted that other hydrophobic amino-acid residues around Leu-412 were also involved in the expression of the activity of TolC. To test this possibility, we substituted several hydrophobic residues around Leu-412, (Leu-3, Val-6, Leu-212, Leu-213, Leu-223, and Leu-224), with serine and examined the activity of these mutant TolCs. The result showed that Leu-3 is involved in the activity of TolC, but the other residues are not. The involvement of Leu-3 was confirmed by the residue deletion experiment. A subsequent point-mutational analysis of the residue showed that a hydrophobic side chain is required at position 3 for TolC to express its activity. As the distance between the alpha-carbons of Leu-3 and Leu-412 is just 7.45 angstroms, hydrophobic interaction between the two leucine residues might be involved in the activity of TolC.     

The functional efficiency of a mammalian signal peptide is directly related to its hydrophobicity

We have previously shown that the signal sequence of the Saccharomyces cerevisiae vacuolar protein carboxypeptidase Y (CPY) does not function in mammalian cells unless a glycine residue in the central core is replaced by leucine. Additional mutants were constructed to investigate the features of this hydrophobic core (h) region that are important for signal sequence function in mammalian cells. We find that the degree of hydrophobicity of the h region of any particular mutant signal is directly related to the efficiency with which it directs the translocation of CPY. A minimal h region in a functional signal appears to consist of five hydrophobic residues interrupted by 1 glycine. Analysis of potential secondary structures suggests that a functional mutant signal is more likely than the nonfunctional CPY signal to adopt either a beta strand or an alpha-helical conformation.     

In vivo effect of asparagine in the hydrophobic region of the signal sequence

On the basis of the biophysical studies on the synthetic mutant (Ile-8----Asn) OmpA signal peptide in the preceding paper (Hoyt, D. C., and Gierasch, L.M. (1991) J. Biol. Chem. 266, 14406-14412), the in vivo effects of the same mutation were examined by fusing the mutant OmpA signal sequence to Staphylococcus aureus nuclease or TEM beta-lactamase. The mutation in which the isoleucine residue at position 8 of the OmpA signal sequence of Escherichia coli was replaced with a neutral polar residue, asparagine, resulted in a defective signal peptide. The mutant signal sequence was unable to be processed, and the precursor molecule accumulated in the cytoplasmic as well as in the membrane fractions, indicating that the Ile-8----Asn OmpA signal sequence is not competent for translocating nuclease A or beta-lactamase across the membrane. This result is consistent with the in vitro studies on the Ile-8----Asn OmpA signal peptide, which indicated that the mutant signal peptide was unable to penetrate into the hydrophobic core of the lipid bilayer. Other asparagine or glutamine substitution mutations in the hydrophobic region of the OmpA signal sequence were also examined. Interestingly, the OmpA signal sequence with either Ile-8----Gln, Val-10----Asn, or Leu-12----Asn mutation was completely defective as the Ile-8----Asn OmpA signal sequence, while the Ile-6----Asn and Ala-9----Asn OmpA nucleases were able to be processed to secrete nuclease, although the processing occurred at a much slower rate than the wild-type OmpA nuclease. These results indicate that the defects depend on the position of the lesion in the hydrophobic core of the OmpA signal sequence.     

E. coli cardiolipin synthase: Function of N-terminal conserved residues

The E. coli cls open reading frame (ORF) predicts a 54.8 kDa polypeptide, whereas mature cardiolipin (CL) synthase is 46 kDa. The N-terminal region extending to residue 60 contains several conserved residues but is not essential for enzyme activity. A deletion mutant that is missing residues 2-60 produces a fully active protein. These findings raise the question of why several residues in a region that is not required for enzyme activity are conserved. Recombinant DNA technology was used to introduce an EYMPE epitope (EE) tag into the interior of CL synthase. The EE tagged polypeptide retained the biological properties of wild type CL synthase, including full enzymatic activity. Site-directed mutagenesis was used to alter conserved residues in the N-terminal region. An EE tagged CL synthase in which Leu-7 and Val-8 were both replaced by Ser residues retains in vitro activity but loses most of its in vivo activity. Furthermore, the mutant protein has a higher apparent molecular mass than its parent protein. Taken together, these findings suggest that conserved residues L7 and V8 play a role in polypeptide processing, topology, or both.     

An alternate pathway for the processing of the prolipoprotein signal peptide in Escherichia coli

Previous studies showed that when the signal sequence plus 9 amino acid residues from the amino terminus of the major lipoprotein of Escherichia coli was fused to beta-lactamase, the resulting hybrid protein was modified, proteolytically processed, and assembled into the outer membrane as was the wild-type lipoprotein (Ghrayeb, J., and Inouye, M. (1983) J. Biol. Chem. 259, 463-467). We have constructed several hybrid proteins with mutations at the cleavage site of the prolipoprotein signal peptide. These mutations are known to block the lipid modification of the lipoprotein at the cysteine residue, resulting in the accumulation of unprocessed, unmodified prolipoprotein in the outer membrane. The mutations blocked the lipid modification of the hybrid protein. However, in contrast to the mutant lipoproteins, the cleavage of the signal peptides for the mutant hybrid proteins did occur, although less efficiently than the unaltered prolipo-beta-lactamase. The mutant prolipo-beta-lactamase proteins were cleaved at a site 5 amino acid residues downstream of the prolipoprotein signal peptide cleavage site. This new cleavage between alanine and lysine residues was resistant to globomycin, a specific inhibitor for signal peptidase II. This indicates that signal peptidase II, the signal peptidase which cleaves the unaltered prolipo-beta-lactamase, is not responsible for the new cleavage. The results demonstrate that the cleavage of the signal peptide is a flexible process that can occur by an alternative pathway when the normal processing pathway is blocked.     

Genetic analyses of processing involving C-terminal cleavage in penicillin-binding protein 3 of Escherichia coli.

The processing of Escherichia coli penicillin-binding protein 3 (PBP 3) was investigated by gene manipulation for producing hybrid and truncated PBP 3 molecules. The hybrid PBP 3 was processed when the N-terminal 40 residues of PBP 3 were replaced by the murein lipoprotein signal peptide which lacked the cysteine residue for processing and followed by seven extra linker residues. In contrast, the PBP 3 molecules truncated at Thr-560 (28-residue deletion) or at Thr-497 (91-residue deletion) were not processed, and those truncated at Phe-576 (12-residue deletion) were processed at a greatly reduced rate. The results indicate that the C-terminal part, rather than the N-terminal part, is involved in the processing. This was supported by the result that the purified mature PBP 3 retained the complete N-terminal sequence with Met for translation initiation. The cleavage at the C-terminal region was shown by the loss of [35S]cysteine label when the cysteine-free hybrid PBP 3 joined to a cysteine-rich extra peptide tail was processed into the mature form. Confirmative assays for processing of PBP 3 were aided by a newly found prc mutant, defective in the processing involving the C-terminal region. A plasmid that directs PBP 3 truncated at Thr-560 complemented a thermosensitive PBP 3 mutation, but the truncated product was unstable in vivo. This suggests the importance of C-terminal hydrophobic regions that terminate at Leu-558 to PBP 3 functioning and the requirement of further-distal peptides for the stability of PBP 3.     

The differential effect on two hybrid proteins of deletion mutations within the hydrophobic region of the Escherichia coli OmpA signal peptide

Oligonucleotide-directed site-specific mutagenesis was used to systematically shorten the hydrophobic region within the signal peptide of the Escherichia coli outer membrane protein OmpA. DNA encoding the wild type and mutant OmpA signal peptides were then fused in frame to DNA encoding the mature regions of Staphylococcus aureus nuclease A and TEM beta-lactamase. The ability of these signal peptides to direct processing of the resulting hybrid proteins was dependent on both their length and the protein to which they were fused. Deletion of two or more residues progressively slowed processing of pro-OmpA-nuclease. By contrast, pro-OmpA-beta-lactamase was less sensitive to the length of the hydrophobic region than to the nature of the deleted residue(s). Deletion of an Ala residue tended to reduce processing efficiency of pro-OmpA-beta-lactamase, while deletion of an Ile residue, together with the Ala residue, resulted in improvement. The loss of either 3 or 4 residues abolished processing of both hybrids. These data indicate that both the length as well as the identity of residues in the hydrophobic region are important. The relative importance of these two factors depends on the mature region of the protein being secreted.     

Evidence for a signal sequence at the N terminus of the common precursor to adrenocorticothrophin and beta-lipotropin in mouse pituitary cells

The precursor to corticotropin and beta-endorphin was synthesized in a reticulocyte cell-free system under the direction of mRNA from mouse AtT-20 pituitary tumor cells in the presence of [3H]proline, [3H]phenylalanine, [3H]leucine, [3H]valine, [3H]isoleucine or [35S]methionine. Automatic Edman degradation of the radioactive cell-free product showed the following N-terminal sequence: Pro-1, Met-2, Leu-11, Leu-12, Leu-13, Leu-15, Leu-16, Leu-17, Ile-21 and Val-23. The corticotropin-endorphin precursor was also labeled in AtT-20 cells with [3H]valine, [3H]leucine, [3H]tryptophan, [3H]serine, [35S]methionine or [35S]cysteine. Automatic Edman degradation of the radioactive intact cell form gave the following N-terminal sequence: Trp-1, Cys-2, Leu-3, Ser-5, Ser-6, Val-7, Cys-8, Leu-11, Leu-17, Leu-18 and tentatively Met-27. The sequence of the intact cell form from AtT-20 cells matches the sequence of the cell-free form of bovine pituitary precursor beginning at Trp-27, as determined by recombinant DNA technology [Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A. C. Y., Cohen, S. N., and Numa, S. (1979) Nature (Lond.) 278, 423-427]. The sequence of the mouse pituitary mRNA-directed cell-free translation product also matches the bovine precursor beginning at Pro-2. The results suggest that both the mouse and bovine precursors possess a signal sequence of 26 amino acids which is cleaved in intact cells. CNBr cleavage of [35S]cysteine-labelled intact cell precursor gave rise to an N-terminal fragment of a size compatible with the presence of a methionyl residue at or near position 27.     

The glycine-rich sequence of the beta subunit of Escherichia coli H(+)-ATPase is important for activity

A short sequence motif rich in glycine residues, Gly-X-X-X-X-Gly-Lys-Thr/Ser, has been found in many nucleotide-binding proteins including the beta subunit of Escherichia coli H(+)-ATPase (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residues 149-156). The following mutations were introduced in this region of the cloned E. coli unc operon carried by a plasmid pBWU1: Ala-151----Pro or Val; insertion of a Gly residue between Lys-155 and Thr-156; and replacement of the region by the corresponding sequence of adenylate kinase (Gly-Gly-Pro-Gly-Ser-Gly-Lys-Gly-Thr) or p21 ras protein (ras) (Gly-Ala-Gly-Gly-Val-Gly-Lys-Ser). All F0F1 subunits were synthesized in the deletion strain of the unc operon-dependent on pBWU1 with mutations, and essentially the same amounts of H(+)-ATPase with these mutant beta subunits were found in membranes. The adenylate kinase and Gly insertion mutants showed no oxidative phosphorylation or ATPase activity, whereas the Pro-151 mutants had higher ATPase activity than the wild-type, and the Val-151 and ras mutants had significant activity. It is striking that the enzyme with the ras mutation (differing in three amino acids from the beta sequence) had about half the membrane ATPase activity of the wild-type. These results together with the simulated three-dimensional structures of the wild-type and mutant sequences suggest that in mutant beta subunits with no ATPase activity projection of Thr-156 residues was opposite to that in the wild-type, and that the size and direction of projection of residue 151 are important for the enzyme activity.     

Effects of prolipoprotein signal peptide mutations on secretion of hybrid prolipo-beta-lactamase in Escherichia coli

Hybrid proteins were constructed by coupling beta-lactamase to the signal sequence (plus nine amino acids) of selected mutant prolipoproteins of Escherichia coli. The mutant prolipoprotein signal peptides contained lesions in two structural domains of the signal peptide, the basic amino-terminal domain and the hydrophobic core domain. We then compared the processing and localization of the mutant prolipo-beta-lactamases to the processing and localization of the comparable mutant prolipoproteins. We show that a mutant signal sequence with an anionic amino terminus exhibits similar limitations in the processing of prolipo-beta-lactamase as previously observed in prolipoprotein. Deletion of four hydrophobic residues from hydrophobic core results in a signal peptide which slowly translocates a fraction of the total mutant hybrid protein synthesized. This signal peptide was previously shown to translocate lipoprotein efficiently. Alteration of this hydrophobic core, which stimulated synthesis of mutant prolipoproteins, does not stimulate synthesis of prolipo-beta-lactamase. Finally mutations that slowed processing of prolipoprotein by affecting the proposed helical structure of the signal peptide had no significant effect on the processing of prolipo-beta-lactamase. These results suggest that the positively charged amino-terminal domain of the signal peptide has a common role in protein secretion regardless of the secretory protein. On the other hand, other domains of the signal peptide exhibit different phenotypes when the secretory protein is changed.     

Functioning of a hybrid BRP-β-lactamase protein in the release of cloacin DF13 and lysis of Escherichia coli cells

The gene encoding a hybrid BRP-Bla protein consisting of the pCloDF13 encoded BRP signal sequence, 25 of the 28 amino acid residues of the mature bacteriocin release protein (BRP) and the mature portion of beta-lactamase (Bla) was subcloned in the expression vector pEB112. A similar construct was made using a mutant gene encoding a BRP-Bla protein in which the cysteine residue at the +1 position was changed into a glycine residue. The expression, processing, functioning and subcellular localization of the 'wild-type' and mutant hybrid protein at high-level expression conditions were studied. The 'wild-type' BRP-Bla protein was mainly found in the outer membranes and possessed all the activities of the BRP itself; the protein was able to bring about the release of cloacin DF13 and caused apparent cell-lysis after high-level synthesis. The mutant hybrid protein was predominantly located in the inner membranes, was inactive in the release of cloacin DF13, but caused apparent cell-lysis only after strong induction.     

The protein sequence responsible for lipoprotein membrane localization in Escherichia coli exhibits remarkable specificity

Structural information defining an N-terminal sequence required for the membrane sorting of bacterial lipoproteins has been previously garnered through the study of a hybrid outer membrane (OM) lipo-beta-lactamase (LL) (Ghrayeb and Inouye (1984) J. Biol. Chem. 259, 463-467). Introduction of an aspartate as the second residue of mature LL (D2 mutant) causes an inner membrane (IM) localization of this protein (Yamaguchi, K., Yu, F., and Inouye, M. (1988) Cell 53, 423-432). Introduction of as aspartate at the third residue of mature LL (D3) causes a weaker IM sorting signal and when present as the fourth residue (D4), normal OM sorting occurs. A positively charged residue at the second position (K2) has no effect on OM localization. Remarkably, glutamate substitution at either the second (E2) or third (E3) position does not interfere with OM sorting. Sorting of the mutant D2 LL can be partially suppressed by introduction of a positively charged histidine (D2H3) or lysine (D2K3) at residue 3 of the mature protein. These results indicate that both the negative charge of the aspartate residue and some structural feature not present in a glutamate residue are required for sorting to the IM. The suppression of IM localization of the D2H3 LL double mutant can be eliminated by growing Escherichia coli at pH 8.4 to reduce the histidine partial positive charge. This result supports the essentiality of a negative charge in IM localization and indicates that the committed step in lipoprotein sorting is made in a cellular compartment, the periplasm, at equilibrium with the external pH.     

The interactions of phenytoin and its binding site in DI-S6 segment of Na+ channel voltage-gated peptide by NMR spectroscopy and molecular modeling study.

Nuclear magnetic resonance (NMR) spectra of a model peptide (BL-DIS6), in the presence of anticonvulsant diphenyl drug, phenytoin (DPH), were measured to obtain the interactions between the selected drug and the model peptide. BL-DIS6's sequence corresponds to the S6 segment in domain I of rat brain type IIA Na+-channel. NMR studies have demonstrated that the magnitude of the chemical shifts of amide- and alpha-protons can be used as a measurement of the complex stability and binding site of the peptide. Our NMR results propose a 3(10)-helical structure for BL-DIS6, and suggest a binding cavity for DPH that involves the hydrophobic particles of residues Ans-7, Leu-8, Val-11, and Val-12. Furthermore, molecular modeling was performed to provide a possible complex conformation that the phenyl portion of DPH is accommodated in the proximity of the C-terminal residues Ala-11 and Val-12, and simultaneously the heterocyclic amine ring of DPH is perching at the residue Asn-7 periphery and stabilizing the phenyl portion deep insertion into the peptide.     

Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus

The Staphylococcus aureus transpeptidase sortase A (SrtA) is responsible for anchoring a range of virulence- and colonization-associated proteins to the cell wall. SrtA recognizes substrates that contain a C-terminal LPXTG motif. This sequence is cleaved following the threonine, and an amide bond is formed between the threonine and the pentaglycine cross-bridge of branched lipid II. Previous studies have implicated the beta6/beta7 loop region of SrtA in LPXTG recognition but have not systematically characterized this domain. To better understand the individual roles of the residues within this loop, we performed alanine-scanning mutagenesis. Val-168 and Leu-169 were found to be important for substrate recognition, and Glu-171 was also found to be important, consistent with its hypothesized role as a Ca(2+)-binding residue. Gly-167 and Asp-170 were dispensable for catalysis, as was Gln-172. The role of Arg-197 in SrtA has been the subject of much debate. To explore its role in catalysis, we used native chemical ligation to generate semi-synthetic SrtA in which we replaced Arg-197 with citrulline, a non-ionizable analog. This change resulted in a decrease of <3-fold in k(cat)/K(m), indicating that Arg-197 utilizes a hydrogen bond, rather than an electrostatic interaction. Our results are consistent with a model for LPXTG recognition wherein the Leu-Pro sequence is recognized primarily by hydrophobic contacts with SrtA Val-168 and Leu-169, as well as a hydrogen bond from Arg-197. This model contradicts the previously proposed mechanism of binding predicted by the x-ray crystal structure of SrtA.     

Hydrophobic content and lipid interactions of wild-type and mutant OmpA signal peptides correlate with their in vivo function

Peptides corresponding to the wild-type signal sequence of the Escherichia coli outer membrane protein OmpA and several mutants have been synthesized and characterized biophysically. The mutations were designed collaboratively with Inouye and co-workers to test the understanding of the critical characteristics of signal sequences required for their functions. The in vivo results for these mutants have been reported [Lehnhardt, S., Pollitt, S., & Inouye, M. (1987) J. Biol. Chem. 262, 1716-1719; Goldstein, J., Lehnhardt, S., & Inouye, M. (1990) J. Bacteriol. 172, 1225-1231; Goldstein, J., Lehnhardt, S., & Inouye, M. (1991) J. Biol. Chem. 266, 14413-14417], and the present paper compares the conformational and membrane-interactive properties of six of the OmpA signal peptides. Peptides corresponding to functional OmpA signal sequences in vivo are predominantly alpha-helical in membrane-mimetic environments and insert readily into phospholipid bilayers. Nonfunctional OmpA signal peptides may have high helical content but do not penetrate deeply into the acyl chain region of bilayers. The ability of the signal peptides to insert into membranes and their in vivo function correlate with the residue-average hydrophobicity of their hydrophobic cores. The results obtained on OmpA signal peptides parallel closely our previous observations on peptides corresponding to the LamB signal sequence and mutants, arguing that the critical biophysical properties of signal sequences are general despite their lack of primary sequence identity.