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Processing of Escherichia coli Alkaline Phosphatase

2002; Elsevier BV; Volume: 277; Issue: 52 Linguagem: Inglês

10.1074/jbc.m205781200

1083-351X

Andrey V. Kajava, Sergey N. Zolov, Konstantin Pyatkov, Andrey E. Kalinin, M. A. Nesmeyanova,

Endoplasmic Reticulum Stress and Disease

Analysis of the precursors of bacterial exported proteins revealed that those having bulky hydrophobic residues at position −5 have a high incidence of Pro residues at positions −6 and −4, Val at position −3, and Ser at positions −4 and −2. This led to a hypothesis that the previously observed inhibition of processing by bulky residues at position −5 can be suppressed by introduction of Pro, Ser, or Val in the corresponding nearby positions. Subsequent mutational analysis of Escherichia coli alkaline phosphatase showed that, as it was predicted, Pro on either side of bulky hydrophobic −5 Leu, Ile, or Tyr completely restores efficiency of the maturation. Introduction of Val at position −3 also partially suppresses the inhibition imposed by −5 Leu, while a Ser residue at position −4 or −2 does not restore processing. In addition, effective maturation of a mutant with Pro residues at positions from −6 throughout −4 proved that polyproline conformation of this region is permissive for processing. To understand the effects of the mutations, we modeled a peptide substrate into the active site of the signal peptidase using the known position of the β-lactam inhibitor. The inhibitory effect of the −5 residue and its suppression by either Pro −6 or Pro −4 can be explained if we assume that Pro-containing −6 to −4 regions adopt a polyproline conformation whereas the region without Pro residues has a β-conformation. These results permit us to specify sequence requirements at −6, −5, and −4 positions for efficient processing and to improve the prediction of yet unknown cleavage sites. Analysis of the precursors of bacterial exported proteins revealed that those having bulky hydrophobic residues at position −5 have a high incidence of Pro residues at positions −6 and −4, Val at position −3, and Ser at positions −4 and −2. This led to a hypothesis that the previously observed inhibition of processing by bulky residues at position −5 can be suppressed by introduction of Pro, Ser, or Val in the corresponding nearby positions. Subsequent mutational analysis of Escherichia coli alkaline phosphatase showed that, as it was predicted, Pro on either side of bulky hydrophobic −5 Leu, Ile, or Tyr completely restores efficiency of the maturation. Introduction of Val at position −3 also partially suppresses the inhibition imposed by −5 Leu, while a Ser residue at position −4 or −2 does not restore processing. In addition, effective maturation of a mutant with Pro residues at positions from −6 throughout −4 proved that polyproline conformation of this region is permissive for processing. To understand the effects of the mutations, we modeled a peptide substrate into the active site of the signal peptidase using the known position of the β-lactam inhibitor. The inhibitory effect of the −5 residue and its suppression by either Pro −6 or Pro −4 can be explained if we assume that Pro-containing −6 to −4 regions adopt a polyproline conformation whereas the region without Pro residues has a β-conformation. These results permit us to specify sequence requirements at −6, −5, and −4 positions for efficient processing and to improve the prediction of yet unknown cleavage sites. cleavage region alkaline phosphatase alkaline phosphatase precursor bacterial type I signal peptidase root-mean-square In prokaryotes and eukaryotes, most exported proteins are synthesized as precursors with an amino-terminal extension called the leader or signal peptide. The signal peptide directs protein translocation across membranes and is removed by a membrane-bound peptidase after transition through the membrane (1Dalbey R.E. Von Heijne G. Trends Biochem. Sci. 1992; 17: 474-478Abstract Full Text PDF PubMed Scopus (177) Google Scholar). Despite their common purpose, signal peptides have very little amino acid sequence similarity, although they do share general features. Typically 15–30 amino acids long, signal peptides of prokaryotic proteins consist of three distinct regions: a 1–5-residue amino-terminal positively charged segment, a 10–15-residue central hydrophobic core, and a more polar 5–7-residue carboxyl-terminal cleavage region (c-region)1 (2Perlman D. Halvorson H.O. J. Mol. Biol. 1983; 167: 391-409Crossref PubMed Scopus (736) Google Scholar, 3von Heijne G. J. Mol. Biol. 1985; 184: 99-105Crossref PubMed Scopus (1535) Google Scholar). In addition, most bacterial proteins have a 14–18-residue region in the mature part immediately downstream of the signal sequence, which has a negative or neutral net charge (4Summers R.G. Knowles J.R. J. Biol. Chem. 1989; 264: 20074-20081Abstract Full Text PDF PubMed Google Scholar, 5Kuhn A. Kiefer D. Kohne C. Zhu H.Y. Tschantz W.R. Dalbey R.E. Eur. J. Biochem. 1994; 226: 891-897Crossref PubMed Scopus (17) Google Scholar, 6Kajava A.V. Zolov S.N. Kalinin A.E. Nesmeyanova M.A. J. Bacteriol. 2000; 182: 2163-2169Crossref PubMed Scopus (56) Google Scholar). As a result of extensive research over the last two decades, the role of each region of the exported proteins has been mainly elucidated (for review see Ref. 7Fekkes P. Driessen A.J. Microbiol. Mol. Biol. Rev. 1999; 63: 161-173Crossref PubMed Google Scholar). The export of proteins is initiated by interactions of the positively charged amino terminus with negatively charged phospholipid headgroups of the cytoplasmic membrane (8Nesmeyanova M.A. Bogdanov M.V. FEBS Lett. 1989; 257: 203-207Crossref PubMed Scopus (30) Google Scholar, 9de Vrije T. de Swart R.L. Dowhan W. Tommassen J. de Kruijff B. Nature. 1988; 334: 173-175Crossref PubMed Scopus (206) Google Scholar, 10Killian J.A. de Jong A.M. Bijvelt J. Verkleij A.J. de Kruijff B. EMBO J. 1990; 9: 815-819Crossref PubMed Scopus (89) Google Scholar, 11Nesmeyanova M.A. Karamyshev A.L. Karamysheva Z.N. Kalinin A.E. Ksenzenko V.N. Kajava A.V. FEBS Lett. 1997; 403: 203-207Crossref PubMed Scopus (57) Google Scholar, 12Van Voorst F. De Kruijff B. Biochem. J. 2000; 347: 601-612Crossref PubMed Scopus (63) Google Scholar) and by insertion of the hydrophobic core of the signal peptide into the apolar environment of the membrane (3von Heijne G. J. Mol. Biol. 1985; 184: 99-105Crossref PubMed Scopus (1535) Google Scholar, 13Chou M.M. Kendall D.A. J. Biol. Chem. 1990; 265: 2873-2880Abstract Full Text PDF PubMed Google Scholar). The insertion of the signal peptide into the lipid bilayer proceeds in association with proteins of the Sec translocation machinery (7Fekkes P. Driessen A.J. Microbiol. Mol. Biol. Rev. 1999; 63: 161-173Crossref PubMed Google Scholar, 14Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (465) Google Scholar, 15Pugsley A.P. Microbiol. Rev. 1993; 57: 50-108Crossref PubMed Google Scholar). The positive charge of the amino terminus can also govern the Nin–Cout orientation of the signal peptide within the membrane (16Von Heijne G. J. Mol. Biol. 1986; 192: 287-290Crossref PubMed Scopus (123) Google Scholar). In this orientation, the c-region of the signal peptide is exposed on the periplasmic side where it can be recognized and cleaved by the signal peptidase (SPase) between positions −1 and +1 (1Dalbey R.E. Von Heijne G. Trends Biochem. Sci. 1992; 17: 474-478Abstract Full Text PDF PubMed Scopus (177) Google Scholar, 17Zimmermann R. Watts C. Wickner W. J. Biol. Chem. 1982; 257: 6529-6536Abstract Full Text PDF PubMed Google Scholar, 18Dalbey R.E. Lively M.O. Bron S. van Dijl J.M. Protein Sci. 1997; 6: 1129-1138Crossref PubMed Scopus (212) Google Scholar). A sequence motif with small residues at positions −3 and −1 defines the cleavage site (2Perlman D. Halvorson H.O. J. Mol. Biol. 1983; 167: 391-409Crossref PubMed Scopus (736) Google Scholar, 3von Heijne G. J. Mol. Biol. 1985; 184: 99-105Crossref PubMed Scopus (1535) Google Scholar, 19Paetzel M. Dalbey R.E. Strynadka N.C. Nature. 1998; 396: 186-190Crossref PubMed Scopus (2) Google Scholar). The conformational characteristics of the signal peptide are also mainly established. There is a consensus view based on several in vitro experimental studies (20Izard J.W. Kendall D.A. Mol. Microbiol. 1994; 13: 765-773Crossref PubMed Scopus (195) Google Scholar, 21Plath K. Mothes W. Wilkinson B.M. Stirling C.J. Rapoport T.A. Cell. 1998; 94: 795-807Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar) that the region of the signal peptide inserted into the membrane adopts a α-helical conformation. It is now known that the −3 to −1 region has an extended β-structural conformation, which is recognized by SPase (19Paetzel M. Dalbey R.E. Strynadka N.C. Nature. 1998; 396: 186-190Crossref PubMed Scopus (2) Google Scholar, 22Paetzel M. Dalbey R.E. Strynadka N.C. J. Biol. Chem. 2002; 277: 9512-9519Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Despite this progress, the critical physical and structural characteristics of residues −6, −5, and −4 that delineate the hydrophobic core and peptidase recognition site of the signal peptide are still poorly understood. Bacterial signal peptides frequently have α-helix-breaking residues such as proline and glycine at −6 to −4 positions (16Von Heijne G. J. Mol. Biol. 1986; 192: 287-290Crossref PubMed Scopus (123) Google Scholar), and this suggested that the disruption of the helical conformation in this region is an important requirement for efficient processing. A number of experimental data supported this conclusion (23Koshland D. Sauer R.T. Botstein D. Cell. 1982; 30: 903-914Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 24Barkocy-Gallagher G.A. Cannon J.G. Bassford P.J., Jr. J. Biol. Chem. 1994; 269: 13609-13613Abstract Full Text PDF PubMed Google Scholar). Based on the analysis of natural sequences (16Von Heijne G. J. Mol. Biol. 1986; 192: 287-290Crossref PubMed Scopus (123) Google Scholar) and experimental evidence, it was also proposed that the hydrophilicity of this region rather than its conformation may be important for the maturation (25Laforet G.A. Kendall D.A. J. Biol. Chem. 1991; 266: 1326-1334Abstract Full Text PDF PubMed Google Scholar). However, none of these rules has absolute support from the recent collection of natural sequences: there are exported proteins with c-regions consisting of only apolar or helix-fostering residues. The conformation of the −4 to −6 region is also unknown. The Pro, Gly, and Ser residues that frequently occupy −4, −5, and −6 positions (2Perlman D. Halvorson H.O. J. Mol. Biol. 1983; 167: 391-409Crossref PubMed Scopus (736) Google Scholar, 16Von Heijne G. J. Mol. Biol. 1986; 192: 287-290Crossref PubMed Scopus (123) Google Scholar) are typical for β-turns of globular proteins (26Chou P.Y. Fasman G.D. Biophys. J. 1979; 26: 367-373Abstract Full Text PDF PubMed Scopus (259) Google Scholar). This observation resulted in a widely accepted opinion that this region has a β-turn conformation (2Perlman D. Halvorson H.O. J. Mol. Biol. 1983; 167: 391-409Crossref PubMed Scopus (736) Google Scholar, 27Rosenblatt M. Beaudette N.V. Fasman G.D. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3983-3987Crossref PubMed Scopus (77) Google Scholar). Furthermore, some mutagenesis studies of exported proteins showed that a decrease of the processing efficiency in mutant proteins correlates with a low probability of β-turn formation (24Barkocy-Gallagher G.A. Cannon J.G. Bassford P.J., Jr. J. Biol. Chem. 1994; 269: 13609-13613Abstract Full Text PDF PubMed Google Scholar, 28Shen L.M. Lee J.I. Cheng S.Y. Jutte H. Kuhn A. Dalbey R.E. Biochemistry. 1991; 30: 11775-11781Crossref PubMed Scopus (75) Google Scholar). However, it was shown that when Pro residues are simultaneously present at both the −5 and −4 positions of alkaline phosphatase from E. coli, this protein is processed properly (29Karamyshev A.L. Karamysheva Z.N. Kajava A.V. Ksenzenko V.N. Nesmeyanova M.A. J. Mol. Biol. 1998; 277: 859-870Crossref PubMed Scopus (76) Google Scholar). The steric constraints of this Pro-Pro tandem allow only a β-conformation of the −5 residue, and, as a consequence, this result cast doubt on the presence of the β-turn in the −6 to −4 region. Rather, it was suggested that the c-region has an extended β-conformation (29Karamyshev A.L. Karamysheva Z.N. Kajava A.V. Ksenzenko V.N. Nesmeyanova M.A. J. Mol. Biol. 1998; 277: 859-870Crossref PubMed Scopus (76) Google Scholar). In this conformation, the −5 residue may have contact with SPase, and this can explain why the processing is sensitive to the size of the −5 residue (29Karamyshev A.L. Karamysheva Z.N. Kajava A.V. Ksenzenko V.N. Nesmeyanova M.A. J. Mol. Biol. 1998; 277: 859-870Crossref PubMed Scopus (76) Google Scholar). The determination of the three-dimensional structure of the bacterial type I SPase co-crystallized with its inhibitor (19Paetzel M. Dalbey R.E. Strynadka N.C. Nature. 1998; 396: 186-190Crossref PubMed Scopus (2) Google Scholar) allows a final rejection of the β-turn hypothesis and favors the extended conformation of the c-region. However, despite the knowledge of the active site of the SPase and docking of the peptide substrate into its binding pocket, the exact conformation of the −6 to −4 region remains unknown. This could be considered a minor academic problem if it was not known that amino acid substitutions within this region can significantly diminish or even block the maturation of exported proteins (23Koshland D. Sauer R.T. Botstein D. Cell. 1982; 30: 903-914Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 29Karamyshev A.L. Karamysheva Z.N. Kajava A.V. Ksenzenko V.N. Nesmeyanova M.A. J. Mol. Biol. 1998; 277: 859-870Crossref PubMed Scopus (76) Google Scholar, 30Palzkill T., Le, Q.Q. Wong A. Botstein D. J. Bacteriol. 1994; 176: 563-568Crossref PubMed Google Scholar, 31Kadonaga J.T. Pluckthun A. Knowles J.R. J. Biol. Chem. 1985; 260: 16192-16199Abstract Full Text PDF PubMed Google Scholar). The goal of this work is to define the sequence requirements and conformation of the −6 to −4 region and its interactions with SPase during the processing. We approached this problem by using sequence analysis of exported proteins, mutational analysis, and molecular modeling. Sequences from Gram-negative bacteria were taken from SwissProt using Sequence Retrieval System software (www.ebi.ac.uk/srs/) and then checked manually. They were 110 proteins from E. coli (68 with known and 42 with well-predicted cleavage sites) and 81 proteins from other Gram-negative bacteria with known cleavage sites. The collection did not include highly homologous sequences with more than 80% identity. Anomalous signal sequences (those whose lengths of the hydrophobic core did not fall into the range between 7 and 17 residues), and proteins, secreted by other or modified secretion machineries (hydrogenases having a RR**F*K pattern within the signal sequence, where * denotes any residue Ref. 32Niviere V. Wong S.L. Voordouw G. J. Gen. Microbiol. 1992; 138: 2173-2183Crossref PubMed Scopus (71) Google Scholar; pili, Ref. 15Pugsley A.P. Microbiol. Rev. 1993; 57: 50-108Crossref PubMed Google Scholar; and lipoproteins) were also excluded. The collection of the 191 sequences is available over the World Wide Web (cmm.cit.nih.gov/kajava/gram-negat.dat). The data sets of 114 exported proteins from Gram-positive bacteria and 1011 human exported proteins have been taken from the SIGNALP data base www.cbs.dtu.dk/services/SignalP/sp_matrices.html (33Nielsen H. Engelbrecht J. von Heijne G. Brunak S. Proteins. 1996; 24: 165-177Crossref PubMed Scopus (71) Google Scholar). E. coli strain E15 (Hfr ΔphoA8 fadL701tonA22 garB10 ompF627 relA1pit-10spoT1T2) (34Bachmann B.J. Neidhardt F.C. Escherichia coli and Salmonella typhimurium: Cellular and molecular biology. American Society for Microbiology, Washington, D. C.1987Google Scholar) was used as a host strain for the expression of wild-type and mutant phoA genes cloned in plasmids. E. coli strain Z85 (thi Δ (lac-proAB) Δ (srl-recA)hsdR::Tn10 (F′ traD proAB lacIqΔZM15)) (35Zaitsev E.N. Zaitseva E.M. Bakhlanova I.V. Gorelov V.N. Kuz'min N.P. Genetika. 1986; 22: 2721-2727PubMed Google Scholar) was used to construct mutantphoA genes. Wild-type alkaline phosphatase gene (phoA) was cloned intoHindIII/BamHI sites of vector p15SK(−) containing multiple cloning sites identical to pBluescript SK (Stratagene), p15A ori of replication and chloramphenicol-acetyltransferase gene. 2R. Fischer and W. Hengstenberg, unpublished observations. The resulting phagemid was used to construct and express mutant phoA genes. Helper phage R408 was used to isolate single-strand recombinant phagemids. The plasmid harboring the gene of amber suppressor tRNA2Ala from E. coli in the vector pGFIB (36Kleina L.G. Masson J.M. Normanly J. Abelson J. Miller J.H. J. Mol. Biol. 1990; 213: 705-717Crossref PubMed Scopus (117) Google Scholar) was provided by Dr. J. Miller. Bacteria for cloning and oligonucleotide-directed mutagenesis were grown on LB or 2YT medium at 37 °C. All media were supplemented with 25 μg/ml chloramphenicol to either select for or maintain phoA-containing plasmids. To screen for colonies expressing active alkaline phosphatase, E. coli cells were grown on agar plates made of LB medium free of inorganic phosphate and containing 40 μg/ml 5-bromo-4-chloro-3-indolyl-phosphate (37Inouye H. Michaelis S. Wright A. Beckwith J. J. Bacteriol. 1981; 146: 668-675Crossref PubMed Google Scholar). For alkaline phosphatase expression, cells were grown on minimal medium (38Torriani A. Cantoni G.L. Davis R. Procedures in nucleic acid research. Harper and Row, New York1966: 224-234Google Scholar) with 1 mm K2HPO4 and 0.1% peptone to the mid-log phase and transferred to medium without orthophosphate and peptone. To generate mutant forms of phoA, we used a new two step method, which allowed us to omit hybridization with labeled nucleotides during selection of clones containing mutant genes (6Kajava A.V. Zolov S.N. Kalinin A.E. Nesmeyanova M.A. J. Bacteriol. 2000; 182: 2163-2169Crossref PubMed Scopus (56) Google Scholar, 39Kalinin A.E. Mikhaleva N.I. Karamyshev A.L. Karamysheva Z.N. Nesmeyanova M.A. Biochemistry-Russia. 1999; 64: 1021-1029PubMed Google Scholar). Isolation of single-strand phagemid DNA and plasmid DNA, electrophoresis of DNA fragments in agar gels, phosphorylation of oligonucleotides, and transformation ofE. coli cells were performed by standard procedures (40Sambrook J. Fritsch E.F. Maniatis T. Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor Laboratory Press, New York1989Google Scholar). Mutations (Table I) were confirmed by DNA sequencing (41Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52668) Google Scholar).Table IMutant E. coli alkaline phosphatasesProtein−6−5−4−3−2−1Mutagenic oligonucleotidesWild-typeTPVTKAL(−5)TLVTKA5′-GGCTTTTGTCACCAGGGTAAACAGTAACG-3′P(−6)L(−5)PLVTKA5′-GCTTTTGTCACCAGCGGAAACAGTAACGGTAAG-3′L(−5)P(−4)TLPTKA5′-GGCTTTTGTCGGCAGGGTAAACAGTAAC-3′L(−5)S(−4)TLSTKA5′-CGGGCTTTTGTGGACAGGGTAAACAGTAACG-3′L(−5)V(−3)TLVVKA5′-CCGGGCTTTAACCACCAGGGTAAACAGTAACG-3′L(−5)S(−2)TLVTSA5′-GTGTCCGGGCAGATGTCACCAGGGTAAACAGTAAC-3′I(−5)TIVTKA5′-GGCTTTTGTCACAATGGTAAACAGTAACG-3′P(−6)I(−5)PIVTKA5′-GGCTTTTGTCACAATCGGAAACAGTAACGGTAAG-3′I(−5)P(−4)TIPTKA5′-CCGGGCTTTTGTCGGAATGGTAAACAGTAACGGTA-3′Y(−5)TYVTKA5′-GGCTTTTGTCACGTAGGTAAACAGTAACG-3′P(−6)Y(−5)PYVTKA5′-GGCTTTTGTCACGTACGGAAACAGTAACGGTAAG-3′Y(−5)P(−4)TYPTKA5′-CCGGGCTTTTGTCGGGTAGGTAAACAGTAACGGTA-3′P(−6, −5, −4)PPPTKA5′-CGGGCTTTTGTCGGCGGCGGAAACAGTAACGG-3′ Open table in a new tab Pulse-chase experiments were used to analyze the alkaline phosphatase maturation. E. coli cells grown to the mid-log phase in the minimal medium with 1 mm K2HPO4 were harvested, washed, and incubated for 10 min in the same medium without orthophosphate to induce alkaline phosphatase synthesis. The cells were labeled with 50 μCi/ml [35S]methionine for 60 s and chased for 0.1, 1.0, 5.0, or 60.0 min by addition of unlabeled methionine to a final concentration of 0.05%. Proteins were precipitated with 10% trichloroacetic acid. Alkaline phosphatase and its precursor were immunoprecipitated with rabbit antibodies and separated by 10% SDS-PAGE followed by autoradiography. Proteins were quantified using a LKB UltroScan laser densitometer. The relative quantity of mature alkaline phosphatase and its precursor was calculated with adjustment for the difference in number of methionine residues between the precursor and mature form. Cells expressing alkaline phosphatase were harvested and converted to spheroplasts in 20 mm Tris-HCl, pH 7.5, 10 mm EDTA, 50 mm sucrose, and 1 mg/ml lysozyme for 15 min on ice. Periplasmic fraction was separated from the cell debris by centrifugation at 12,000 × g for 5 min. The samples were analyzed by non-denaturing electrophoresis in 7.5% PAGE (42Davis B.J. Ann. N. Y. Acad. Sci. 1964; 121: 404-427Crossref PubMed Scopus (15957) Google Scholar). Staining of the alkaline phosphatase isoforms was performed by incubation of the gel with α-naphthyl phosphate (Sigma, N-7255) and Fast Red Dye TR (Chemapol, Czech Republic) (43Lojda Z. Grossrau R. Schibler T.H. Enzyme histochemistry: A laboratory manual. Springer-Verlag, Berlin, Germany1979Crossref Google Scholar). The alkaline phosphatase activity was determined by measuring the rate ofp-nitrophenylphosphate hydrolysis, taking the activity of hydrolysis of 1 μmol of substrate per 1 min at 37 °C as a unit of enzymatic activity (unit). Total cell protein was assayed by the Lowry method (44Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Initial docking of a peptide corresponding to the −3 to +1 region of the alkaline phosphatase into the active site of SPase was made manually based on the known position of the β-lactam inhibitor and using Insight II program (45Dayring H.E. Tramonato A. Sprang S.R. Fletterick R.J. J. Mol. Graphics. 1986; 4: 82-87Crossref Scopus (162) Google Scholar). Possible conformations of the region −6 to −4 were selected based on two constraints: first, the absence of steric clashes within the peptide chain and between the peptide and SPase; second, direction of signal peptide α-helix (residues −21 to −7) into the cytoplasmic membrane. Then the complexes between SPase and alkaline phosphatase precursor (−21 to +2) were subjected to energy minimization using DISCOVER module of Insight II (300 steps of minimization based on the steepest descent algorithm and the next 500 steps using conjugate gradients algorithm). The CHARMM force field (46Brooks B.R. Bruccoleri R.E. Olafson B.D. States D.J. Swaminathan S. Karplus M. J. Computat. Chem. 1983; 4: 187-217Crossref Scopus (13964) Google Scholar) and the distance-dependent dielectric constant were used for the energy calculations. During the minimization (i) the backbone atoms of SPase were tethered to their positions in the crystal structure, (ii) a carbonyl carbon atom in the −1 residue was covalently linked to the oxygen atom of the Ser-90 side chain forming a tetrahedral intermediate, and (iii) several hydrogen bonds (between the oxygen of the peptide group of −1 residue and hydroxyl group of Ser-88, between the backbone oxygen of −2 residue and NH group of Ile-144, between the backbone nitrogen of −2 residue and backbone oxygen of Asp-142) were enforced by setting the distance constraints with moderate force (K = 50), in order to improve their geometry. In addition, when the region −6 to −4 in the β-conformation was energy minimized the distance constraints were imposed on hydrogen bonds between the backbone CO group of Gln-85 and NH group of the −3 residue; the backbone NH group of Gln-85 and CO group of −4 residue; the CO group of Pro-83 and NH group of −5 residue. To allay the concern that these constraints generated significant tension in the minimized structure, the last calculation was performed without any restrictions to an RMS derivative of 0.4 kcal/(mol·Å). A module “Struct_Check” of Insight II program (45Dayring H.E. Tramonato A. Sprang S.R. Fletterick R.J. J. Mol. Graphics. 1986; 4: 82-87Crossref Scopus (162) Google Scholar) was used to check the quality of the modeled complexes. The figures were generated with Molscript (47Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar). Our previous study showed that the introduction of bulky residues at position −5 of E. coli alkaline phosphatase causes a decrease in the efficiency of its maturation (29Karamyshev A.L. Karamysheva Z.N. Kajava A.V. Ksenzenko V.N. Nesmeyanova M.A. J. Mol. Biol. 1998; 277: 859-870Crossref PubMed Scopus (76) Google Scholar). In agreement with this result, the occurrence of large hydrophobic residues Trp, Ile, Phe, Leu, Met, and Tyr (written in the order of decreasing hydrophobicity, Ref. 48Fauchere J. Pliska V. Eur. J. Med. Chem. 1983; 18: 369-375Google Scholar) at position −5 for the c-regions of eukaryotic and Gram-positive bacterial proteins is lower than at positions −6 and −4 (17% versus 45 and 27%, and 7%versus 20 and 18% correspondingly). Surprisingly, the exported proteins of Gram-negative bacteria have an opposite distribution: 21% of large residues at position −5 against 14 and 17% at positions −6 and −4. We analyzed a subset of bacterial proteins with bulky hydrophobic residues at position −5 and found that they have a higher incidence of Pro residue at positions −6 and −4, Val residue at position −3, and a Ser residue at positions −4 and −2 compared with the complete collection of these proteins (Fig. 1). This observation led to the hypothesis that the inhibitory effect of bulky hydrophobic residue in position −5 can be suppressed by introducing Pro, Ser, or Val in the corresponding nearby positions. In accordance with this, a series of mutant E. coli alkaline phosphatases were obtained (TableI) to test the hypothesis. In addition, a mutant having Pro residues in all −6, −5, and −4 positions was also obtained. The −6 to −4 region of such a protein is sterically constrained in the polyproline conformation, and it was of interest to determine whether it was processed. The fact that this tandem of three Pro was not found in natural sequences provided an additional motivation to study this mutant. All mutant proteins were enzymatically active in cells (data not shown). This result implies that the mutants were translocated across the cytoplasmic membrane, because it is known that alkaline phosphatase becomes active only after translocation into the periplasm, where disulfide bond formation and enzyme dimerization take place (49Michaelis S. Inouye H. Oliver D. Beckwith J. J. Bacteriol. 1983; 154: 366-374Crossref PubMed Google Scholar). The effect of the amino acid substitutions on alkaline phosphatase maturation was assessed by the rate of conversion of a pulse-labeled mutant protein precursor into the mature form in vivo using the standard pulse-chase method. As shown in Fig. 2, the presence of bulky Leu, Ile, or Tyr residue at position −5 (proteins L(−5), I(−5) and Y(−5)) notably impaired the maturation of the precursor in comparison with wild-type protein. Even after 60 min of chase almost half of the mutant protein precursor remained unprocessed. In agreement with our hypothesis, introduction of a Pro residue on either side of −5 Leu, Ile, or Tyr restored efficiency of maturation (proteins P(−6)L(−5), L(−5)P(−4), P(−6)I(−5), I(−5)P(−4), P(−6)Y(−5), and Y(−5)P(−4)). Introduction of a Ser residue at position −4 or −2 or Val residue at position −3 also partially suppressed the effect of the −5 Leu mutation (proteins L(−5)S(−4), L(−5)S(−2) and L(−5)V(−3), correspondingly). Pre-PhoA with a stretch of Pro residues in positions from −6 to −4 (protein P(−6,−5, −4)), was converted into the mature form with almost the same efficiency as the wild-type precursor. An unprocessed protein can reside in the cytoplasm or be translocated to the periplasmic side. It is known that the unprocessed but translocated precursor of E. coli alkaline phosphatase has an enzymatic activity, while precursor, which remains in the cytoplasm, does not (50Boyd D. Guan C.-D. Willard S. Wright W. Strauch K. Beckwith J. Torriani-Gorini A. Rothman F.G. Silver S. Wright A. Yagil E. Phosphate Metabolism and Cellular Regulation in Microorganisms. American Society for Microbiology, Washington, D. C.1987: 89-93Google Scholar). This property of alkaline phosphatase was used to distinguish which of these two situations is true for the unprocessed portions of the analyzed mutants. We visualized alkaline phosphatase isoforms (I, II, and III) in gel after electrophoresis under non-denaturing conditions. Active alkaline phosphatase can be stained in the gel by treatment with the enzyme substrate α-naphthyl phosphate and an appropriate dye. Furthermore, active mature protein and the translocated precursor can be distinguished on non-denaturing gel, since they have different electrophoretic mobilities (29Karamyshev A.L. Karamysheva Z.N. Kajava A.V. Ksenzenko V.N. Nesmeyanova M.A. J. Mol. Biol. 1998; 277: 859-870Crossref PubMed Scopus (76) Google Scholar, 51Karamyshev A.L. Kalinin A.E. Tsfasman I.M. Ksenzenko V.N. Nesmeianova M.A. Mol. Biol. (Mosk). 1994; 28: 362-373PubMed Google Scholar). The precursor translocated across the membrane can be found at the top of the gel, probably due to aggregation caused by the presence of hydrophobic signal peptide. Such active precursor was detected (Fig. 3, a series of mutants containing Leu in −5 position are shown) in all cases when significant amount of unprocessed pre-PhoA was present after 60 min of chase (Fig. 2, proteins L(−5), I(−5), Y(−5), L(−5)S(−4), and L(−5)S(−2)). This implies that these mutant precursors are located at the periplasmic side of the cytoplasmic membrane. Thus, we showed that inefficient processing of L(−5), I(−5)