J. Mol. Biol.

(1992) 226, 931.-933

Functional Analysis of the Intramolecular Chaperone Mutational Hot Spots in the Subtilisin Pro-peptide and a Second-site Suppressor Mutation within the Subtilisin Molecule Tatsuhiko Kobayashi and Masayori Inouye’f Department University

of Biochemistry, Robert Wood Johnson Medical School of Medicine and Dentistry of New Jersey at Rutgers Piscataway, NJ 08854, U.S.A.

(Received

20 February

1992; accepted 15 April

1992)

The N-terminal pro-peptide of 77 amino acid residues is essential for the folding of subtilisin, an alkaline serine protease from Bacillus subtilis. The synthetic pro-peptide has been shown to be capable of guiding the proper folding of denatured subtilisin to enzymatically active enzyme. Thus the pro-peptide serves as an intramolecular chaperone, which is removed by an autoprocessing reaction after the completion of the folding. With use of localized polymerase chain reaction random mutagenesis a total of 25 amino acid substitution mutations that affected subtilisin activities were isolated. These mutations occurred in a high frequency at the hydrophobic regions of the pro-peptide. For one of the mutations, M( -6O)T, a second-site suppressor mutation, S( 188)L, was isolated within the mature region. These results suggest that the pro-peptide consists of a few functional regions which interact with specific regions of the mature region of subtilisin during the folding process. Keywords:

protein folding; pro-peptide;

Subtilisin E is an alkaline serine protease, produced by Bacillus subtilis, of 275 amino acid residues. It is produced from pre-pro-subtilisin E (Boyer & Carton, 1968). The pre-sequence consisting of 29 residues functions as the signal peptide for secretion of subtilisin across the membrane (Wong & Doi, 1986). The pro-peptide, consisting of 77 residues, exists between the signal peptide and the mature protease (Wong & Doi, 1986; Stahl & Ferrari, 1984). The pro-peptide has been shown to be essential for the production of active subtilisin (Ikemura et al., 1987; Ikemura & Inouye, 1988; Zhu et al., 1989; Ohta & Inouye, 1990). Denatured prosubtilisin E can be renatured and once it is refolded, the pro-peptide is removed by autoprocessing (Ikemura & Inouye, 1988). In contrast, denatured subtilisin E cannot be renatured unless the synthetic pro-peptide is exogenously added during the renaturation procedure (Ohta et al., 1991). Thus, the pro-peptide is proposed to function as an intramolecular chaperone for production of active subtilisin (Ohta et al., 1991). In a preliminary experiment, we have isolated four amino acid substitution mutations within the pro-peptide, which were incapable of producing t Author addressed.

to whom all correspondence

(M,22-283e/92/160931~3

should

chaperone; subtilisin

active subtilisin (Lerner et al., 1990). Here, we extensively employed localized random polymerase chain reaction (PCRS) mutagenesis using Taq DNA polymerase (Lerner et al., 1990) within the propeptide coding region. For this purpose, the Hind111 site within the mature subtilisin coding region was eliminated by changing the codon for Ala(46), GCA to GCT. Subsequently, the sequence CAATCTGTT for Gln-Ser-Val was altered by oligonucleotide mutagenesis to CAAAGCTTT to create a new Hind111 site (underlined). This alteration resulted in an amino acid substitution at’ position +4 from Val to Phe which caused no effect on the subtilisin activity as assessed by the size of halo produced around a colony grown on a caseincontaining plate (Takagi et al., 1989). The plasmid containing the new Hind111 site was designated pHI215T. Using pHI215T, PCR random mutagenesis was carried out between the XbaI site (immediately upstream from the initiation codon) and the new Hind111 site (Lerner et al., 1990). Out of 1300 transformants, 54 halo-less colonies were obtained (4.2%). Of 54, 31 were in-frame base-substitution mutations within the pro-peptide of which 33 were independent, as shown in Figure 1. Some mutations

be $ Abbreviation

$08.00/0

931

used: PCR, polymerase 0

1992

Academic

chain reaction. Press

Limited

932

T. Kobayashi and M. Inouye -60 -40 -HI . Hz. AGKSSTEK~~~QTMSAMSSAKKKDVISEKGGKVQKQF~VN~T~EKAVKELKKDP~EEDHIAHEY +I( is\ t t if + 3 r,t+\vx v lx* C T+A T* TE*D A E RS TT+TT V C S I S V

-20

H, . ++ L A*

I t N

Figure 1. Mutations in the pro-peptide of subtilisin E. Localized PCR random mutagenesis (Lerner et al., 1990) was carried out within the region coding the pro-peptide, using the XbaI-Hind111 fragment from pHI215-T (see the text). Halo-less colonies were obtained as subtilisin-negative mutants and the mutations were determined by DNA sequencing of the entire XbaI-Hind111 fragment. The entire amino acid sequence of the subtilisin E pro-peptide is shown. The large arrowhead indicates the cleavage site of the pro-peptide. The sequence is numbered from - 1 (the C-terminal end of the pro-peptide), every tenth residue from the - I residue is indicated by a dot. Arrows indicate the amino acid substitutions and the numbers with amino acids indicates numbers of independent mutations isolated. Amino acid residues marked with an asterisk indicate those mutations isolated previously (Lerner et al., 1990). Mutations I( - 67)T and A( - 3 I)T were isolated together, and are marked with a plus. The hydrophobic sequence H,, H, and H, are boxed.

occurred more than once, as indicated by the numbers, and both I( -67)T mutations were accompanied with the A( -3l)T mutation. Four mutations previously isolated are also shown in Figure 1, of which V( - 13)A was isolated again in the present study. All mutations resulted from a single base substitution except that R at position - 34 (CGT) was derived from the codon GTT (for V) by two base substitutions. Altogether, mutations occurred at 20 different positions and at four positions more than one mutation was isolated; G at -76 to R, C or S; I at -67toVorT;Iat -48toT,SorV;andVat -34 to R or I. The subtilisin E pro-peptide has a primary sequence distinctly different from the subtilisin sequence. The notable features of the propeptide are summarized as follows (see Fig. 1): (1) it contains 15 Lys (no Arg), two His, four Asp and seven Glu, making 36% of the pro-peptide residues charged in contrast to 12% of residues in the mature region; (2) the distribution of these charged residues of the pro-peptide is extremely uneven; the N-terminal 27-residue sequence (-77 to -51) has seven positive charges, while the C-terminal 16-residue sequence ( - 16 to - 1) has five negative charges at neutral pH; (3) Ser and Thr are also unevenly distributed; seven out of ten residues are within the N-terminal 24-residue sequence; (4) there is one Pro residue at - 15; (5) it contains three short hydrophobic sequences, H,, H, and H,, as indicated in Figure 1. Out of 26 mutations, 12 (46%) were found in the hydrophobic sequences, H,, H2 and H,, indicating that these sequences are hot spots for mutations. In particular, substitutions of every Ala residue in the H, region with Thr resulted in a defective propeptide (in the case of A( -3l)T, accompanied with I( -67)T). It is tempting to speculate that these hydrophobic sequences play a key role in guiding the subtilisin folding. Outside the H regions, there are three mutations at hydrophobic residues; M( -6O)T, I( -48)T, S or V, and V( -4l)A. In addition, there are five charge mutations involving charged residues; G( - 76)R, K( - 45)E, G( - 44)D, K( - 36)E, and D( -7)N. It is also interesting to note that the mutation P(-15)L at the only Pro

residue in the pro-peptide occurred most frequently. Mutations at positions -77 and -76 may have some effects on the cleavage of the signal peptide, thus, secondarily resulting in defective pro-peptides. Next, using the M( -6O)T mutation, we attempted to isolate second-site suppressor mutations within the mature subtilisin region. This was achieved by PCR random mutagenesis in the mature subtilisin coding region. The 093 kb HindIII-BamHI fragment of pH1215T [M( -6O)T]. which encompasses the coding region of the mature subtilisin, was replaced with the PCR-amplified HindIII-BamHI fragment. Out of 1500 transformants, one halo-forming colony in a casein plate was isolated. DNA sequencing of this plasmid DNA from this colony revealed that Ser at position 188 was replaced by a Leu residue (TCA to TTA). The suppressor S( 188)L mutation appears to be compensatory to M( - 6O)T, and it is possible that these two residues at - 60 and 188 may indeed be located very close to one another in the pro-subtilisin structure. It is interesting to point out that Ser188 locates on the surface of the subtilisin molecule near the active center cleft (Wright et al., 1969; Drenth ef al., 1972). Since the synthetic pro-peptide functions as a competitive inhibitor with a Ki of 54 x lo-’ M (Ohta et al., 1991), the pro-peptide is likely to bind to the side where the active center is exposed. It should be noted that charge distribution on the surface of the subtilisin molecule is uneven; the surface region where the substrate-binding site locates is hvdrophobic with few charged residues. Thus, it is tempting to speculate that the highly charged propeptide covers the hydrophobic surface of subtilisin in such a way that charged residues are more evenly distributed on the pro-subtilisin surface. If so, the interaction between the pro-peptide and subtilisin is dominated by hydrophobic interactions, and this is consistent with the present results that the hydrophobic sequences in the pro-peptide play an important role in the pro-subtilisin folding. Besides subtilisin, a few proteins are now being demonstrated to absolutely require the prosequences for the production of active enzymes such as a-lytic protease (Silen & Agard, 1989), and carboxypeptidase Y (Winther & Sorensen, 1991; for

Communication5 a review, see Inouye, 1992). This supports the general notion that the pro-peptide functions as an intramolecular chaperone (Ohta et al., 1991), that plays an essential role in the protein folding. However, after the folding is completed, the intramolecular chaperone is cleaved off and thus not a part of the final mature enzyme. It should be noted that the intramolecular chaperones are distinctly different from the intermolecular chaperones (Rothman, 1989) such as heat shock proteins; the former work only’ for a single molecule of a specific protein, while the latter work catalytically to facilitate or assist protein folding. The intramolecular chaperones are essential, whereas the intermolecular chaperones are not essential for protein folding. The functional analysis of the subtilisin propeptide by the genetic approach described here, and biochemical characterization of mutant prosubtilisins, are expected to identify intermediates of pro-subtilisin folding and to provide clues to determine the pathway of pro-subtilisin folding and the exact role of the pro-peptide. The authors wish to thank Drs A. Stock and U. Shinde for the critical reading of the manuscript. The present work was partially supported by a grant from Ajinomoto Co., Ltd. References Boyer, H. W. & Carton, B. C. (1968). Production of two proteolytic enzymes by a transformable strain of Bacillus subtilis. Arch. Biochem. Biophys. 128, 442-455. Drenth, J., HOI, W. G. J., Jansoniu, J. & Koekuek, R. (1972). Subtilisin novo: the three-dimensional structure and its comparison with subtilisin BPN’. Eur. J. B&hem. 26, 177-181. Ikemura, H. & Inouye, M. (1988). In vitro processing of pro-subtilisin produced in Escherichia coli. J. Biol. Chem. 263, 12959-12963. Ikemura, H., Takagi? H. & Inouye, M. (1987). Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli. J. Biol. Chem. 262,

933

Inouye, M. (1992). Intramolecular chaperone: the role of the pro-peptide in protein folding. Enzyme, in the press. Lerner, C. G., Kobayashi, T. & Inouye, M. (1990). Isolation of subtilisin pro-sequence mutations that affect formation of active protease by localized random polymerase chain reaction mutagenesis. J. Biol.

Chem.

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20085-20086.

Ohta, Y. & Inouye, M. (1990). Pro-subtilisin E: purification and characterization of its autoprocessing to active subtilisin E in vitro. Mol. Microbial. 4, 295-304.

Ohta, Y., Hojo, H., Aimoto, S., Kobayashi, T., Zhu, X., Jordan, F. & Inouye, M. (1991). Pro-peptide as an intramolecular chaperone: renaturation of denatured subtilisin E with a synthetic pro-peptide. Mol. Microbial. 5, 1507-1510. Rothman, J. E. (1989). Polypeptide chain binding proteins: catalysis of protein folding and related process in cells. Cell, 59, 591-601. Silen, J. L. & Agard, D. A. (1989). The a: lytic protease pro-region does not require physical linkage to activate the protease domain in vivo. Nature (London), 341, 462-464. Stahl, M. L. & Ferrari, E. (1984). Replacement of the Bacillus subtilis subtilisin structural gene with an in vitro-derived mutation. J. Boxteriol. 158, 411-418. Takagi, H., Morinaga, Y., Ikemura, H. & Inouye, M. (1989). The role of Pro-239 in the catalysis of heat stability of subtilisin E. J. Biochem. (Tolcyo), 105, 953-956. Winther, J. R. & Sorensen, P. (1991). Pro-peptide of carboxypeptidase Y provide a chaperone like function as well as inhibition of the enzymatic activity. Proc. Nat. Acad. Sci., U.S.A. 88, 9330-9334. Wong, S. L. t Doi, R. H. (1986). Determination of the signal peptidase cleavage site in the preprosubtilisin of Bacillus subtilis. J. Biol. Chem. 261, 10176-10180. Wright, C. S., Alden, R. A. & Kraut, J. (1969). Structure of subtilisin BPN’ at 2.5 .& resolution. Nature (London), 221, 235-242. Zhu, X., Ohta, Y., Jordan, F. & Inouye, M. (1989). Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process. Nature (London), 339, 483-484.

7859-7864.

Edited by A. R. Fersht

Functional analysis of the intramolecular chaperone. Mutational hot spots in the subtilisin pro-peptide and a second-site suppressor mutation within the subtilisin molecule.

The N-terminal pro-peptide of 77 amino acid residues is essential for the folding of subtilisin, an alkaline serine protease from Bacillus subtilis. T...
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