Fluorescence spectroscopy of soluble E. coli SPase I Δ2‐75 reveals conformational changes in response to ligand binding

The bacterial Sec pathway is responsible for the translocation of secretory preproteins. During the later stages of transport, the membrane‐embedded signal peptidase I (SPase I) cleaves the signal peptide from a preprotein. We used tryptophan fluorescence spectroscopy of a soluble, catalytically active E. coli SPase I Δ2‐75 enzyme to study its dynamic conformational changes while in solution and when interacting with lipids and signal peptides. We generated four single Trp SPase I Δ2‐75 mutants, W261, W284, W300, and W310. Based on fluorescence quenching experiments, W300 and W310 were found to be more solvent accessible than W261 and W284 in the absence of ligands. W300 and W310 inserted into lipids, consistent with their location at the enzyme's proposed membrane‐interface region, while the solvent accessibilities of W261, W284, and W300 were modified in the presence of signal peptide, suggesting propagation of structural changes beyond the active site in response to peptide binding. The signal peptide binding affinity for the enzyme was measured via FRET experiments and the K_d determined to be 4.4 μM. The location of the peptide with respect to the enzyme was also established; this positioning is crucial for the peptide to gain access to the enzyme active site as it emerges from the translocon into the membrane bilayer. These studies reveal enzymatic structural changes required for preprotein proteolysis as it interacts with its two key partners, the signal peptide and membrane phospholipids. Proteins 2014; 82:596–606. © 2013 Wiley Periodicals, Inc.     

The role of the membrane-spanning domain of type I signal peptidases in substrate cleavage site selection

Type I signal peptidase (SPase I) catalyzes the cleavage of the amino-terminal signal sequences from preproteins destined for cell export. Preproteins contain a signal sequence with a positively charged n-region, a hydrophobic h-region, and a neutral but polar c-region. Despite having no distinct consensus sequence other than a commonly found c-region "Ala-X-Ala" motif preceding the cleavage site, signal sequences are recognized by SPase I with high fidelity. Remarkably, other potential Ala-X-Ala sites are not cleaved within the preprotein. One hypothesis is that the source of this fidelity is due to the anchoring of both the SPase I enzyme (by way of its transmembrane segment) and the preprotein substrate (by the h-region in the signal sequence) in the membrane. This limits the enzyme-substrate interactions such that cleavage occurs at only one site. In this work we have, for the first time, successfully isolated Bacillus subtilis type I signal peptidase (SipS) and a truncated version lacking the transmembrane domain (SipS-P2). With purified full-length as well as truncated constructs of both B. subtilis and Escherichia coli (Lep) SPase I, in vitro specificity studies indicate that the transmembrane domains of either enzyme are not important determinants of in vitro cleavage fidelity, since enzyme constructs lacking them reveal no alternate site processing of pro-OmpA nuclease A substrate. In addition, experiments with mutant pro-OmpA nuclease A substrate constructs indicate that the h-region of the signal peptide is also not critical for substrate specificity. In contrast, certain mutants in the c-region of the signal peptide result in alternate site cleavage by both Lep and SipS enzymes.     

Leishmania major: Disruption of signal peptidase type I and its consequences on survival, growth and infectivity

Leishmania major (L. major) signal peptidase type I (SPase I) is an endopeptidase encoded by a single-copy gene. In all organisms, SPase I is responsible for removing the signal peptide from secretory pre-proteins and releasing mature proteins to cellular or extra-cellular space. In this study, the role of SPase I in L. major is investigated by gene deletion using homologous recombination (HR). The null mutant of SPase I was not possible to create, suggesting that SPase I is an essential gene for parasite survival.The obtained heterozygote mutant by disrupting one allele of SPase I in L. major showed significantly reduced level of infectivity in bone marrow-derived macrophages. In addition, the heterozygote mutants are unable to cause cutaneous lesion in susceptible BALB/c mice. This is the first report showing that SPase I may have an important role in Leishmania infectivity, e.g. in differentiation and survival of amastigotes. Apparently, the SPase I expression is not essential for in vitro growth of the parasite.     

Structural studies of a signal peptide in complex with signal peptidase I cytoplasmic domain: the stabilizing effect of membrane-mimetics on the acquired fold

A protein destined for export from the cell cytoplasm is synthesized as a preprotein with an amino-terminal signal peptide. In Escherichia coli, typically signal peptides that guide preproteins into the SecYEG protein conduction channel are subsequently removed by signal peptidase I. To understand the mechanism of this critical step, we have assessed the conformation of the signal peptide when bound to signal peptidase by solution nuclear magnetic resonance. We employed a soluble form of signal peptidase, which laks the two transmembrane domains (SPase I Δ2-75), and the E. coli alkaline phosphatase signal peptide. Using a transferred NOE approach, we found clear evidence of a weak peptide-enzyme complex formation. The peptide adopts a U-turn shape originating from the proline residues within the primary sequence that is stabilized by its interaction with the peptidase and leaves key residues of the cleavage region exposed for proteolysis. In dodecylphosphocholine (DPC) micelles the signal peptide also adopts a U-turn shape comparable with that observed in association with the enzyme. In both environments this conformation is stabilized by the signal peptide phenylalanine side chain-interaction with enzyme or lipid mimetic. Moreover, in the presence of DPC, the N-terminal core region residues of the peptide adopt a helical motif and based on PRE (paramagnetic relaxation enhancement) experiments are shown to be buried within the membrane. Taken together, this is consistent with proteolysis of the preprotein occurring while the signal peptide remains in the bilayer and the enzyme active site functioning at the membrane surface.     

Solution NMR of signal peptidase, a membrane protein

Useful solution nuclear magnetic resonance (NMR) data can be obtained from full-length, enzymatically active type I signal peptidase (SPase I), an integral membrane protein, in detergent micelles. Signal peptidase has two transmembrane segments, a short cytoplasmic loop, and a 27-kD C-terminal catalytic domain. It is a critical component of protein transport systems, recognizing and cleaving amino-terminal signal peptides from preproteins during the final stage of their export. Its structure and interactions with the substrate are of considerable interest, but no three-dimensional structure of the whole protein has been reported. The structural analysis of intact membrane proteins has been challenging and only recently has significant progress been achieved using NMR to determine membrane protein structure. Here we employ NMR spectroscopy to study the structure of the full-length SPase I in dodecylphosphocholine detergent micelles. HSQC-TROSY spectra showed resonances corresponding to approximately 3/4 of the 324 residues in the protein. Some sequential assignments were obtained from the 3D HNCACB, 3D HNCA, and 3D HN(CO) TROSY spectra of uniformly ²H, ¹³C, ¹⁵N-labeled full-length SPase I. The assigned residues suggest that the observed spectrum is dominated by resonances arising from extramembraneous portions of the protein and that the transmembrane domain is largely absent from the spectra. Our work elucidates some of the challenges of solution NMR of large membrane proteins in detergent micelles as well as the future promise of these kinds of studies.     

Signal peptidase I overproduction results in increased efficiencies of export and maturation of hybrid secretory proteins in Escherichia coli

Summary The effects of 25-fold overproduction ofEscherichia coli signal peptidase I (SPase I) on the processing kinetics of various (hybrid) secretory proteins, comprising fusions between signal sequence functions selected from theBacillus subtilis chromosome and the mature part of TEM-β-lactamase, were studied inE. coli. One precursor (pre[A2d]-β-lactamase) showed an enhanced processing rate, and consequently, a highly improved release of the mature enzyme into the periplasm. A minor fraction of a second hybrid precursor (pre[Al3i]-β-lactamase), which was not processed under standard conditions of SPase I synthesis, was shown to be processed under conditions of SPase I overproduction. However, this did not result in efficient release of the mature β-lactamase into the periplasm. In contrast, the processing rates of wild-type pre-β-lactamase and pre(A2)-β-lactamase, already high under standard conditions, were not detectably altered by SPase I overproduction. These results demonstrate that the availability of SPase I can be a limiting factor in protein export inE. coli, in particular with respect to (hybrid) precursor proteins showing low (SPase I) processing efficiencies.     

The effects of mutations in the carboxyl-terminal region on the catalytic activity of Escherichia coli signal peptidase I

Escherichia coli signal peptidase I (SPase I) is a membrane-bound serine endopeptidase that catalyses the cleavage of signal peptides from the pre-forms of membrane or secretory proteins. Our previous studies using chemical modification and site-directed mutagenesis suggested that Trp(300) and Arg(77), Arg(222), Arg(315) and Arg(318) are important for the proper and stable conformation of the active site of SPase I. Interestingly, many of these residues reside in the C-terminal region of the enzyme. As a continuation of these studies, we investigated in the present study the effects of mutations in the C-terminal region including amino acid residues at positions from 319 to 323 by deletions and site-directed mutagenesis. As a result, the deletion of the C-terminal His(323) was shown to scarcely affect the enzyme activity of SPase I, whereas the deletion of Gly(321)-His(323) or Ile(319)-His(323) as well as the point mutation of Ile(322) to alanine was shown to decrease significantly both the activity in vitro and in vivo without a big gross conformational change in the enzyme. These results suggest a significant contribution of Ile(322) to the construction and maintenance of the proper and critical local conformation backing up the active site of SPase I.     

Complete maturation of the plastid protein translocation channel requires a type I signal peptidase

The protein translocation channel at the plastid outer envelope membrane, Toc75, is essential for the viability of plants from the embryonic stage. It is encoded in the nucleus and is synthesized with a bipartite transit peptide that is cleaved during maturation. Despite its important function, the molecular mechanism and the biological significance of the full maturation of Toc75 remain unclear. In this study, we show that a type I signal peptidase (SPase I) is responsible for this process. First, we demonstrate that a bacterial SPase I converted Toc75 precursor to its mature form in vitro. Next, we show that disruption of a gene encoding plastidic SPase I (Plsp1) resulted in the accumulation of immature forms of Toc75, severe reduction of plastid internal membrane development, and a seedling lethal phenotype. These phenotypes were rescued by the overexpression of Plsp1 complementary DNA. Plsp1 appeared to be targeted both to the envelope and to the thylakoidal membranes; thus, it may have multiple functions.     

Altered -3 substrate specificity of Escherichia coli signal peptidase 1 mutants as revealed by screening a combinatorial peptide library

Signal peptidase functions to cleave signal peptides from preproteins at the cell membrane. It has a substrate specificity for small uncharged residues at -1 (P1) and aliphatic residues at the -3 (P3) position. Previously, we have reported that certain alterations of the Ile-144 and Ile-86 residues in Escherichia coli signal peptidase I (SPase) can change the specificity such that signal peptidase is able to cleave pro-OmpA nuclease A in vitro after phenylalanine or asparagine residues at the -1 position (Karla, A., Lively, M. O., Paetzel, M. and Dalbey, R. (2005) J. Biol. Chem. 280, 6731-6741). In this study, screening of a fluorescence resonance energy transfer-based peptide library revealed that the I144A, I144C, and I144C/I86T SPase mutants have a more relaxed substrate specificity at the -3 position, in comparison to the wild-type SPase. The double mutant tolerated arginine, glutamine, and tyrosine residues at the -3 position of the substrate. The altered specificity of the I144C/I86T mutant was confirmed by in vivo processing of pre-beta-lactamase containing non-canonical arginine and glutamine residues at the -3 position. This work establishes Ile-144 and Ile-86 as key P3 substrate specificity determinants for signal peptidase I and demonstrates the power of the fluorescence resonance energy transfer-based peptide library approach in defining the substrate specificity of proteases.     

Signal peptidase I: cleaving the way to mature proteins

Signal peptidase I (SPase I) is critical for the release of translocated preproteins from the membrane as they are transported from a cytoplasmic site of synthesis to extracytoplasmic locations. These proteins are synthesized with an amino-terminal extension, the signal sequence, which directs the preprotein to the Sec- or Tat-translocation pathway. Recent evidence indicates that the SPase I cleaves preproteins as they emerge from either pathway, though the steps involved are unclear. Now that the structure of many translocation pathway components has been elucidated, it is critical to determine how these components work in concert to support protein translocation and cleavage. Molecular modeling and NMR studies have provided insight on how the preprotein docks on SPase I in preparation for cleavage. This is a key area for future work since SPase I enzymes in a variety of species have now been identified and the inhibition of these enzymes by antibiotics is being pursued. The eubacterial SPase I is essential for cell viability and belongs to a unique group of serine endoproteases which utilize a Ser-Lys catalytic dyad instead of the prototypical Ser-His-Asp triad used by eukaryotes. As such, SPase I is a desirable antimicrobial target. Advances in our understanding of how the preprotein interfaces with SPase I during the final stages of translocation will facilitate future development of inhibitors that display a high efficacy against SPase I function.     

Chaperone-assisted Post-translational Transport of Plastidic Type I Signal Peptidase 1

Type I signal peptidase (SPase I) is an integral membrane Ser/Lys protease with one or two transmembrane domains (TMDs), cleaving transport signals off translocated precursor proteins. The catalytic domain of SPase I folds to form a hydrophobic surface and inserts into the lipid bilayers at the trans-side of the membrane. In bacteria, SPase I is targeted co-translationally, and the catalytic domain remains unfolded until it reaches the periplasm. By contrast, SPases I in eukaryotes are targeted post-translationally, requiring an alternative strategy to prevent premature folding. Here we demonstrate that two distinct stromal components are involved in post-translational transport of plastidic SPase I 1 (Plsp1) from Arabidopsis thaliana, which contains a single TMD. During import into isolated chloroplasts, Plsp1 was targeted to the membrane via a soluble intermediate in an ATP hydrolysis-dependent manner. Insertion of Plsp1 into isolated chloroplast membranes, by contrast, was found to occur by two distinct mechanisms. The first mechanism requires ATP hydrolysis and the protein conducting channel cpSecY1 and was strongly enhanced by exogenously added cpSecA1. The second mechanism was independent of nucleoside triphosphates and proteinaceous components but with a high frequency of mis-orientation. This unassisted insertion was inhibited by urea and stroma extract. During import-chase assays using intact chloroplasts, Plsp1 was incorporated into a soluble 700-kDa complex that co-migrated with the Cpn60 complex before inserting into the membrane. The TMD within Plsp1 was required for the cpSecA1-dependent insertion but was dispensable for association with the 700-kDa complex and also for unassisted membrane insertion. These results indicate cooperation of Cpn60 and cpSecA1 for proper membrane insertion of Plsp1 by cpSecY1.     

Competitive Inhibition of the Endoplasmic Reticulum Signal Peptidase by Non-cleavable Mutant Preprotein Cargos

Upon translocation across the endoplasmic reticulum (ER) membrane, secretory proteins are proteolytically processed to remove their signal peptide by signal peptidase (SPase). This process is critical for subsequent folding, intracellular trafficking, and maturation of secretory proteins. Prokaryotic SPase has been shown to be a promising antibiotic target. In contrast, to date, no eukaryotic SPase inhibitors have been reported. Here we report that introducing a proline immediately following the natural signal peptide cleavage site not only blocks preprotein cleavage but also, in trans, impairs the processing and maturation of co-expressed preproteins in the ER. Specifically, we find that a variant preproinsulin, pPI-F25P, is translocated across the ER membrane, where it binds to the catalytic SPase subunit SEC11A, inhibiting SPase activity in a dose-dependent manner. Similar findings were obtained with an analogous variant of preproparathyroid hormone, demonstrating that inhibition of the SPase does not depend strictly on the sequence or structure of the downstream mature protein. We further show that inhibiting SPase in the ER impairs intracellular processing of viral polypeptides and their subsequent maturation. These observations suggest that eukaryotic SPases (including the human ortholog) are, in principle, suitable therapeutic targets for antiviral drug design.     

Identification of arginine residues important for the activity of Escherichia coli signal peptidase I

Escherichia coil signal peptidase I (leader peptidase, SPase I) is an integral membrane serine protease that catalyzes the cleavage of signal (leader) peptides from pre-forms of membrane or secretory proteins. We previously demonstrated that E. coil SPase I was significantly inactivated by reaction with phenylglyoxal with concomitant modification of three to four of the total 17 arginine residues in the enzyme. This result indicated that several arginine residues are important for the optimal activity of the enzyme. In the present study, we have constructed 17 mutants of the enzyme by site-directed mutagenesis to investigate the role of individual arginine residues in the enzyme. Mutation of Arg127, Arg146, Arg198, Arg199, Arg226, Arg236, Arg275, Arg282, and Arg295 scarcely affected the enzyme activity in vivo and in vitro. However, the enzymatic activity toward a synthetic substrate was significantly decreased by replacements of Arg77, Arg222, Arg315, or Arg318 with alanine/lysine. The kcat values of the R77A, R77K, R222A, R222K, R315A, R318A, and R318K mutant enzymes were about 5.5-fold smaller than that of the wild-type enzyme, whereas the Km values of these mutant enzymes were almost identical with that of the wild-type. Moreover, the complementing abilities in E. Arg222, Arg315, coil IT41 were lost completely when Arg77, or Arg318 was replaced with alanine/lysine. The circular dichroism spectra and other enzymatic properties of these mutants were comparable to those of the wild-type enzyme, indicating no global conformational changes. However, the thermostability of R222A, R222K, R315A, and R318K was significantly lower compared to the wild type. Therefore, Arg77, Arg222, Arg315, and Arg318 are thought to be important for maintaining the proper and stable conformation of SPase I.     

In vitro and in vivo self-cleavage of Streptococcus pneumoniae signal peptidase I.

We have previously demonstrated that Streptococcus pneumoniae signal peptidase (SPase) I catalyzes a self-cleavage to result in a truncated product, SPase37-204 [Peng, S.B., Wang, L., Moomaw, J., Peery, R.B., Sun, P.M., Johnson, R.B., Lu, J., Treadway, P., Skatrud, P.L. & Wang, Q.M. (2001) J. Bacteriol.183, 621-627]. In this study, we investigated the effect of phospholipid on invitro self-cleavage of S. pneumoniae SPase I. In the presence of phospholipid, the self-cleavage predominantly occurred at one cleavage site between Gly36-His37, whereas the self-cleavage occurred at multiple sites in the absence of phospholipid, and two additional self-cleavage sites, Ala65-His66 and Ala143-Phe144, were identified. All three self-cleavage sites strongly resemble the signal peptide cleavage site and follow the (-1, -3) rule for SPase I recognition. Kinetic analysis demonstrated that self-cleavage is a concentration dependent and intermolecular event, and the activity in the presence of phospholipid is 25-fold higher than that in the absence of phospholipid. Biochemical analysis demonstrated that SPase37-204, the major product of the self-cleavage totally lost activity to cleave its substrates, indicating that the self-cleavage resulted in the inactivation of the enzyme. More importantly, the self-cleavage was demonstrated to be happening in vivo in all the growth phases of S. pneumoniae cells. The bacterial cells keep the active SPase I at the highest level in exponential growth phase, suggesting that the self-cleavage may play an important role in regulating the activity of the enzyme under different conditions.     

Not just an antibiotic target: Exploring the role of type I signal peptidase in bacterial virulence

The looming antibiotic crisis has prompted the development of new strategies towards fighting infection. Traditional antibiotics target bacterial processes essential for viability, whereas proposed antivirulence approaches rely on the inhibition of factors that are required only for the initiation and propagation of infection within a host. Although antivirulence compounds have yet to prove their efficacy in the clinic, bacterial signal peptidase I (SPase) represents an attractive target in that SPase inhibitors exhibit broad-spectrum antibiotic activity, but even at sub-MIC doses also impair the secretion of essential virulence factors. The potential consequences of SPase inhibition on bacterial virulence have not been thoroughly examined, and are explored within this review. In addition, we review growing evidence that SPase has relevant biological functions outside of mediating secretion, and discuss how the inhibition of these functions may be clinically significant.     

Solution NMR of signal peptidase, a membrane protein

Useful solution nuclear magnetic resonance (NMR) data can be obtained from full-length, enzymatically active type I signal peptidase (SPase I), an integral membrane protein, in detergent micelles. Signal peptidase has two transmembrane segments, a short cytoplasmic loop, and a 27-kD C-terminal catalytic domain. It is a critical component of protein transport systems, recognizing and cleaving amino-terminal signal peptides from preproteins during the final stage of their export. Its structure and interactions with the substrate are of considerable interest, but no three-dimensional structure of the whole protein has been reported. The structural analysis of intact membrane proteins has been challenging and only recently has significant progress been achieved using NMR to determine membrane protein structure. Here we employ NMR spectroscopy to study the structure of the full-length SPase I in dodecylphosphocholine detergent micelles. HSQC-TROSY spectra showed resonances corresponding to approximately 3/4 of the 324 residues in the protein. Some sequential assignments were obtained from the 3D HNCACB, 3D HNCA, and 3D HN(CO) TROSY spectra of uniformly 2H, 13C, 15N-labeled full-length SPase I. The assigned residues suggest that the observed spectrum is dominated by resonances arising from extramembraneous portions of the protein and that the transmembrane domain is largely absent from the spectra. Our work elucidates some of the challenges of solution NMR of large membrane proteins in detergent micelles as well as the future promise of these kinds of studies.     

Membrane topology of Escherichia coli prolipoprotein signal peptidase (signal peptidase II)

The lsp gene of Escherichia coli encodes the inner membrane enzyme, signal peptidase II (SPase II). SPase II is comprised of 164 amino acid residues and contains four hydrophobic domains. A series of lsp-phoA and lsp-lacZ gene fusions have been constructed in vitro to determine the topology of SPase II. The fusion junction for each of these gene fusions was determined by DNA sequencing. The lengths of the SPase II fragment in the fusions varied from 12 to 159 amino acid residues. Strains containing SPase II-PhoA fusions to the two predicted periplasmic loops exhibited higher levels of alkaline phosphatase activity than fusions to the predicted cytoplasmic domains. In contrast, SPase II-LacZ fusions at the cytoplasmic and the periplasmic domains of SPase II showed high and low levels of beta-galactosidase activity, respectively, a result opposite to those shown by SPase II-PhoA fusions located at precisely the same amino acid of SPase II. Taken together, these results strongly support the predicted model for SPase II topology, i.e. this enzyme spans the cytoplasmic membrane four times with both the amino and the carboxyl termini facing the cytoplasm.     

Analysis of type I signal peptidase affinity and specificity for preprotein substrates

Type I signal peptidases (SPases) are membrane-bound endopeptidases responsible for the catalytic cleavage of signal peptides from secretory proteins. Here, we analysed the interaction between a bacterial type I SPase and preprotein substrates using surface plasmon resonance. The use of a home-made biosensor surface based on a mixed self-assembled monolayer of thiols on gold allowed qualitative and kinetic analysis. In vitro binding of purified preproteins to a covalently immobilised bacterial SPase was found to be rather efficient (apparent K(D)=10(-7)-10(-8)M). The signal peptide was shown to be a prerequisite for SPase binding and the nature of the mature part of the preprotein significantly affected SPase binding affinity. The developed biosensor containing immobilised SPase is of great importance for analysis of specificity at substrate binding level and for drug screening. In fact, this is the first report of a membrane protein that was covalently attached to a biosensor surface and that retained binding capacity.