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.     

Structure-based site-directed mutagenesis of the UDP-MurNAc-pentapeptide-binding cavity of the FemX alanyl transferase from Weissella viridescens

Weissella viridescens FemX (FemX(Wv)) belongs to the Fem family of nonribosomal peptidyl transferases that use aminoacyl-tRNA as the amino acid donor to synthesize the peptide cross-bridge found in the peptidoglycan of many species of pathogenic gram-positive bacteria. We have recently solved the crystal structure of FemX(Wv) in complex with the peptidoglycan precursor UDP-MurNAc-pentapeptide and report here the site-directed mutagenesis of nine residues located in the binding cavity for this substrate. Two substitutions, Lys36Met and Arg211Met, depressed FemX(Wv) transferase activity below detectable levels without affecting protein folding. Analogues of UDP-MurNAc-pentapeptide lacking the phosphate groups or the C-terminal D-alanyl residues were not substrates of the enzyme. These results indicate that Lys36 and Arg211 participate in a complex hydrogen bond network that connects the C-terminal D-Ala residues to the phosphate groups of UDP-MurNAc-pentapeptide and constrains the substrate in a conformation that is essential for transferase activity.     

The Cys319 loop modulates the transition between dehydrogenase and hydrolase conformations in inosine 5'-monophosphate dehydrogenase

X-ray crystal structures of enzyme-ligand complexes are widely believed to mimic states in the catalytic cycle, but this presumption has seldom been carefully scrutinized. In the case of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase (IMPDH), 10 structures of various enzyme-substrate-inhibitor complexes have been determined. The Cys319 loop is found in at least three different conformations, suggesting that its conformation changes as the catalytic cycle progresses from the dehydrogenase step to the hydrolase reaction. Alternatively, only one conformation of the Cys319 loop may be catalytically relevant while the others are off-pathway. Here we differentiate between these two hypotheses by analyzing the effects of Ala substitutions at three residues of the Cys319 loop, Arg322, Glu323, and Gln324. These mutations have minimal effects on the value of k(cat) (≤5-fold) that obscure large effects (>10-fold) on the microscopic rate constants for individual steps. These substitutions increase the equilibrium constant for the dehydrogenase step but decrease the equilibrium between open and closed conformations of a mobile flap. More dramatic effects are observed when Arg322 is substituted with Glu, which decreases the rates of hydride transfer and hydrolysis by factors of 2000 and 130, respectively. These experiments suggest that the Cys319 loop does indeed have different conformations during the dehydrogenase and hydrolase reactions as suggested by the crystal structures. Importantly, these experiments reveal that the structure of the Cys319 loop modulates the closure of the mobile flap. This conformational change converts the enzyme from a dehydrogenase into hydrolase, suggesting that the conformation of the Cys319 loop may gate the catalytic cycle.     

Effect of distance and orientation between arginine-302, histidine-322, and glutamate-325 on the activity of lac permease from Escherichia coli

lac permease of Escherichia coli was modified by site-directed mutagenesis in order to investigate the effects of polarity, distance, and orientation between the components of a putative H+ relay system (Arg302/His322/Glu325) postulated to be involved in lactose-coupled H+ translocation. The importance of polarity between His322 and Glu325 was studied by interchanging the residues, and the modified permease--H322E/E325H--is inactive in all modes of translocation. The effect of distance and/or orientation between His322 and Glu325 was investigated by interchanging Glu325 with Val326, thereby moving the carboxylate one residue around putative helix X. The resulting permease molecule--E325V/V326E--is also completely inactive; control mutations, E325V [Carrasco, N., Püttner, I. B., Antes, L. M., Lee, J. A., Larigan, J. D., Lolkema, J. S., Roepe, P. D., & Kaback, H. R. (1989) Biochemistry (second paper of three in this issue)], and E325A/V326E, indicate that a Glu residue at position 326 inactivates the permease. The wild-type orientation between His and Glu was then restored by further mutation of E325V/V326E to introduce a His residue into position 323 or by interchanging Met323 with His322. The resulting permease molecules--M323H/E325V/V326E and H322M/M323H/E325V/V326E--contain the wild-type His/Glu orientation, but the His/Glu ion pair is rotated about the helical axis by 100 degrees relative to Arg302 in putative helix IX. Both mutants are inactive with respect to all modes of translocation. The results provide strong support for the contention that the polarity between His322 and Glu325 and the geometric relationship between Arg302, His322, and Glu325 are critical for permease activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The carboxyl terminus of coffee bean alpha-galactosidase is critical for enzyme activity

The role of the carboxyl (C)-terminal region of coffee bean alpha-galactosidase (alpha-GAL) has been studied by expressing C-terminal deletion mutants in the methylotrophic yeast strain Pichia pastoris. A previous study of human alpha-galactosidase determined that enzyme activity increased when up to 10 amino acid residues were deleted. Deleting 11 residues reduced activity, and deleting 12 residues abolished activity. In our studies, alpha-GAL activity is reduced when one or two amino acids are deleted, as is enzyme secretion directed by P. pastoris signal sequences. The pH profile is similar to that of the wild-type enzyme. Deleting 3 or more residues from the C-terminal end results in a complete loss of both enzyme secretion and activity. The C-terminus of alpha-GAL seems to play an important role in overall enzyme conformation and may directly affect the proper conformation of the active site.     

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.     

Differential effects of mutations in the chromophore pocket of recombinant phytochrome on chromoprotein assembly and Pr-to-Pfr photoconversion.

Site-directed mutagenesis was performed with the chromophore-bearing N-terminal domain of oat phytochrome A apoprotein (amino acid residues 1-595). Except for Trp366, which was replaced by Phe (W366F), all the residues exchanged are in close proximity to the chromophore-binding Cys321 (i.e. P318A, P318K, H319L, S320K, H322L and the double mutant L323R/Q324D). The mutants were characterized by their absorption maxima, and the kinetics of chromophore-binding and the Pr-->Pfr conversion. The strongest effect of mutation on the chromoprotein assembly, leading to an almost complete loss of the chromophore binding capability, was found for the exchanges of His322 by Leu (H322L) and Pro318 by Lys (P318K), whereas a corresponding alanine mutant (P318A) showed wild-type behavior. The second histidine (H319) is also involved in chromophore fixation, as indicated by a slower assembly rate upon mutation (H319L). For the other mutants, an assembly process very similar to that of the wild-type protein was found. The light-induced Pr-->Pfr conversion kinetics is altered in the mutations H319L and S320K and in the double mutant L323R/Q324D, all of which exhibited a significantly faster I700 decay and accelerated Pfr formation. P318 is also involved in the Pr-->Pfr conversion, the millisecond steps (formation of Pfr) being significantly slower for P318A. Lacking sufficient amounts of W366F, assembly kinetics could not be determined in this case, while the fully assembled mutant underwent the Pr-->Pfr conversion with kinetics similar to wild-type protein.     

The specificity of carboxypeptidase Y may be altered by changing the hydrophobicity of the S'1 binding pocket.

The S'1 binding pocket of carboxypeptidase Y is hydrophobic, spacious, and open to solvent, and the enzyme exhibits a preference for hydrophobic P'1 amino acid residues. Leu272 and Ser297, situated at the rim of the pocket, and Leu267, slightly further away, have been substituted by site-directed mutagenesis. The mutant enzymes have been characterized kinetically with respect to their P'1 substrate preferences using the substrate series FA-Ala-Xaa-OH (Xaa = Leu, Glu, Lys, or Arg) and FA-Phe-Xaa-OH (Xaa = Ala, Val, or Leu). The results reveal that hydrophobic P'1 residues bind in the vicinity of residue 272 while positively charged P'1 residues interact with Ser297. Introduction of Asp or Glu at position 267 greatly reduced the activity toward hydrophobic P'1 residues (Leu) and increased the activity two- to three-fold for the hydrolysis of substrates with Lys or Arg in P'1. Negatively charged substituents at position 272 reduced the activity toward hydrophobic P'1 residues even more, but without increasing the activity toward positively charged P'1 residues. The mutant enzyme L267D + L272D was found to have a preference for substrates with C-terminal basic amino acid residues. The opposite situation, where the positively charged Lys or Arg were introduced at one of the positions 267, 272, or 297, did not increase the rather low activity toward substrates with Glu in the P'1 position but greatly reduced the activity toward substrates with C-terminal Lys or Arg due to electrostatic repulsion. The characterized mutant enzymes exhibit various specificities, which may be useful in C-terminal amino acid sequence determinations.     

Cumulative effect of amino acid replacements results in enhanced thermostability of potato type L alpha-glucan phosphorylase

The thermostability of potato type L alpha-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations-Phe39-->Leu (F39L), Asn135-->Ser (N135S), and Thr706-->Ile (T706I)-by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60 degrees C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65 degrees C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.     

Involvement of the carboxy-terminal residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site that involves His-15, which is phosphorylated to form a N delta 1-P-histidine, Arg-17, and the carboxy-terminal residue Glu-85. Mutant HPrs with alterations to the three C-terminal residues, Glu-85, Leu-84, and Glu-83, were produced by site-directed mutagenesis. The properties of these mutants were assessed by kinetic analysis of enzyme I, enzyme IImannose, enzyme IIN-acetylglucosamine, and enzyme IImannitol, and the phosphohydrolysis properties of the HPr mutants. The results show that it is the C-terminal alpha-carboxyl of Glu-85 that is involved in the active site, and this involvement may be restricted to the phosphoryl donor action of HPr. The contribution of this alpha-carboxyl group is modest as the deletion of Glu-85 resulted in the reduction of the enzyme II activity (kcat/Km) to about 33%. Removal of both Glu-85 and Leu-84 yields an HPr that is an impaired substrate of both the enzyme I and enzyme II reactions. Glu-83 appears to have no role in the active site.     

Cloning, expression, and carbon catabolite repression of the bamM gene encoding beta-amylase of Bacillus megaterium DSM319

The bamM gene from Bacillus megaterium DSM319 encoding an extracellular beta-amylase was isolated and completely sequenced. Chromosomal inactivation by deletion mutagenesis resulted in total loss of amylolytic activity, indicative of a single starch-degrading enzyme. Functional characterization of the expressed protein revealed a maltogenic enzyme exhibiting optimal activities at pH 7.5 and 50 degrees C. Amylase expression is subject to catabolite repression by glucose. A putative cis-acting catabolite-responsive element (CRE) was identified; it is located within the bamM coding region, matching the position of the predicted signal peptide processing site. Base substitutions introduced by site-directed mutagenesis within the bamM-CRE--retaining unchanged the amino acid sequence--provoked a remarkable relief from carbon catabolite repression (CCR), thereby proving functionality of the CRE for CCR.     

Mitochondrial targeting sequence of the influenza A virus PB1-F2 protein and its function in mitochondria

The influenza A virus PB1-F2 protein predominantly localizes in the mitochondria of virus-infected cells. A series of cDNAs encoding N- and C-terminal deletion mutants and site-directed mutagenesis of the basic residues of PB1-F2 appended to 3xFLAG revealed the domain from residues 46 to 75 to be both necessary and sufficient for mitochondrial targeting. In addition, the subdomain residues 63-75 and both Lys73 and Arg75 are minimally required for mitochondrial localization. Transfection of untagged- and 3xFLAG tagged-PB1-F2 into Vero, HeLa and MDCK cells changed the mitochondrial morphology from a filamentous to a dotted structure and suppressed the inner-membrane potential.     

An essential residue in the flexible peptide linking the two idiosynchratic domains of bacterial tyrosyl-tRNA synthetases

Tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus comprises three sequential domains: an N-terminal catalytic domain, an alpha-helical domain with unknown function, and a C-terminal tRNA binding domain (residues 320-419). The properties of the polypeptide segment that links the alpha-helical and C-terminal domains, were analyzed by measuring the effects of sequence changes on the aminoacylation of tRNA(Tyr) with tyrosine. Mutations F323A (Phe323 into Ala), S324A, and G325A showed that the side chain of Phe323 was essential but not those of Ser324 and Gly325. Insertions of Gly residues between Leu322 and Phe323 and the point mutation L322P showed that the position and precise orientation of Phe323 relative to the alpha-helical domain were important. Insertions of Gly residues between Gly325 and Asp326 and deletion of residues 330-339 showed that the length and flexibility of the sequence downstream from Gly325 were unimportant but that this sequence could not be deleted. Mutations F323A, -L, -Y, and -W showed that the essential property of Phe323 was its aromaticity. The Phe323 side chain contributed to the stability of the initial complex between TyrRS and tRNA(Tyr) for 2.0 +/- 0.2 kcal x mol(-1) and to the stability of their transition state complex for 4.2 +/- 0.1 kcal x mol(-1), even though it is located far from the catalytic site. The results indicate that the disorder of the C-terminal domain in the crystals of TyrRS is due to the flexibility of the peptide that links it to the helical domain. They identified Phe323 as an essential residue for the recognition of tRNA(Tyr).     

Activation of the AT1 angiotensin receptor is dependent on adjacent apolar residues in the carboxyl terminus of the third cytoplasmic loop

The C-terminal region of the third intracellular loop of the AT(1) angiotensin receptor (AT(1)-R) is an important determinant of G protein coupling. The roles of individual residues in agonist-induced activation of G(q/11)-dependent phosphoinositide hydrolysis were determined by mutational analysis of the amino acids in this region. Functional studies on mutant receptors transiently expressed in COS-7 cells showed that alanine substitutions of the amino acids in positions 232-240 of the third loop had no major effect on signal generation. However, deletion mutations that removed Ile(238) or affected its position relative to transmembrane helix VI significantly impaired angiotensin II-induced inositol phosphate responses. Substitution of Ile(238) with an acidic residue abolished the ability of the receptor to mediate inositol phosphate production, whereas its replacement with basic or polar residues reduced the amplitude of inositol phosphate responses. Substitutions of Phe(239) with polar residues had relatively minor effects on inositol phosphate signal generation, but its replacement by aspartic acid reduced, and by positively charged residues (Lys, Arg) significantly increased, angiotensin II-induced inositol phosphate responses. The internalization kinetics of the Ile(238) and Phe(239) mutant receptors were impaired in parallel with the reduction in their signaling responses. These findings have identified Ile(238) and Phe(239) as the critical residues in the C-terminal region of the third intracellular loop of the AT(1)-R for receptor activation. They also suggest that an apolar amino acid corresponding to Ile(238) of the AT(1)-R is a general requirement for activation of other G protein-coupled receptors by their agonist ligands.     

The C1-C2 interface residue lysine 50 of pig kidney fructose-1, 6-bisphosphatase has a crucial role in the cooperative signal transmission of the AMP inhibition.

To understand the mechanism of signal propagation involved in the cooperative AMP inhibition of the homotetrameric enzyme pig-kidney fructose-1,6-bisphosphatase, Arg49 and Lys50 residues located at the C1-C2 interface of this enzyme were replaced using site-directed mutagenesis. The mutant enzymes Lys50Ala, Lys50Gln, Arg49Ala and Arg49Gln were expressed in Escherichia coli, purified to homogeneity and the initial rate kinetics were compared with the wild-type recombinant enzyme. The mutants exhibited kcat, Km and I50 values for fructose-2,6-bisphosphate that were similar to those of the wild-type enzyme. The kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive (wild-type) to noncompetitive in the mutant enzymes. The Lys50Ala and Lys50Gln mutants showed a biphasic behavior towards AMP, with total loss of cooperativity. In addition, in these mutants the mechanism of AMP inhibition with respect to fructose-1,6-bisphosphate changed from noncompetitive (wild-type) to uncompetitive. In contrast, AMP inhibition was strongly altered in Arg49Ala and Arg49Gln enzymes; the mutants had > 1000-fold lower AMP affinity relative to the wild-type enzyme and exhibited no AMP cooperativity. These studies strongly indicate that the C1-C2 interface is critical for propagation of the cooperative signal between the AMP sites on the different subunits and also in the mechanism of allosteric inhibition of the enzyme by AMP.     

A Suppressor Analysis of Residues Involved in Cation Transport in the Lactose Permease: Identification of a Coupling Sensor

Four amino acids critical for lactose permease function were altered using site-directed mutagenesis. The resulting Quad mutant (E269Q/R302L/H322Q/E325Q) was expressed at 60% of wild-type levels but found to have negligible transport activity. The Quad mutant was used as a parental strain to isolate suppressors that regained the ability to ferment the α-galactoside melibiose. Six different suppressors were identified involving five discrete amino acid changes and one amino acid deletion (Q60L, V229G, Y236D, S306L, K319N and ΔI298). All of the suppressors transported α-galactosides at substantial rates. In addition, the Q60L, ΔI298 and K319N suppressors regained a small but detectable amount of lactose transport. Assays of sugar-driven cation transport showed that both the Q60L and K319N suppressors couple the influx of melibiose with cations (H⁺ or H₃O⁺). Taken together, the data show that the cation-binding domain in the lactose permease is not a fixed structure as proposed in previous models. Rather, the data are consistent with a model in which several ionizable residues form a dynamic coupling sensor that also may interact directly with the cation and lactose.     

Proteolytic sensitivity of a recombinant phospholipase D from cabbage: identification of loop regions and conformational changes

A recombinant phospholipase D from white cabbage (PLD2) composed of 812 amino acid residues was studied by site-directed mutagenesis and limited proteolysis to obtain first information on its tertiary structure. Limited proteolysis by thermolysin resulted in the formation of some large fragments of PLD2. From mass spectrometry and N-terminal sequencing of the peptides, the cleavage sites could be identified (1. Thr41-Ile42, 2. Asn323-Leu324 or Gly287-Leu288 and Ser319-Ile320 in case of the mutant L324S-PLD2). This suggested an exposed loop in the C2 domain of PLD2 and a large flexible region close to the N-terminal side of the first catalytic (HKD) motif. Calcium ions, the substrate 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and the competitive inhibitor 1,3-dipalmitoylglycero-2-phosphocholine influenced the proteolytic cleavage. Calcium ions exerted a destabilizing effect on the conformation of PLD2.     

Conserved amino acid residues in ribosome-inactivating proteins from plants.

The amino acid sequences of eleven RIPs sequenced to date have been compared in the expectation that this would be useful in the location of functionally and/or structurally important sites of these molecules. In addition to several highly conserved hydrophobic amino acids, thirteen absolutely conserved residues have been found in ricin A-chain: Tyr21, Phe24, Arg29, Tyr80, Tyr123, Gly140, Ala165, Glu177, Ala178, Arg180, Glu208, Asn209 and Trp211. The role of these residues as well as of the C-terminal region have been discussed based on the results of chemical and enzymatic modifications, site-directed mutagenesis, and deletion studies.     

The C-terminal tail of the dual-specificity Cdc25B phosphatase mediates modular substrate recognition.

Cdc25 is a dual-specificity phosphatase that catalyzes the activation of the cyclin-dependent kinases (Cdk/cyclins), thus triggering initiation and progression of successive phases of the cell cycle. In our efforts to elucidate the interaction between Cdc25B and the natural substrate, bis-phosphorylated Cdk2/CycA (Cdk2-pTpY/CycA), we have previously found that the 17 residues of the C-terminal tail mediate a factor of 10 in substrate recognition. In the studies reported here, we localize the majority of this interaction using site-directed mutagenesis to two arginine residues (Arg556 and Arg562) located within this C-terminal region. We also show that the catalytic domain of Cdc25C, which differs most significantly from Cdc25B in this tail region, has a 100-fold lower activity toward Cdk2-pTpY/CycA. We further demonstrate that the proper presentation of the C-terminal tail of Cdc25B can be achieved in a "gain-of-function" chimeric protein consisting of the C-terminal tail of Cdc25B fused onto the catalytic core of Cdc25C. The >10-fold increase in activity seen only in the chimeric protein containing the two critical arginine residues demonstrates that the modular C-terminal tail of Cdc25B is the basis for most of the catalytic advantage of Cdc25B versus Cdc25C toward the Cdk2-pTpY/CycA substrate.     

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.