Determination of the Cleavage Site of the Presequence by Mitochondrial Processing Peptidase on the Substrate Binding Scaffold and the Multiple Subsites inside a Molecular Cavity*

Sakae KitadaDagger, Eiki Yamasaki, Katsuhiko Kojima§, and Akio Ito

From the Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan

Received for publication, September 10, 2002, and in revised form, November 11, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mitochondrial processing peptidase (MPP) recognizes a large variety of basic presequences of mitochondrial preproteins and cleaves the single site, often including arginine, at the -2 position (P2). To elucidate the recognition and specific processing of the preproteins by MPP, we mutated to alanines at acidic residues conserved in a large internal cavity formed by the MPP subunits, alpha -MPP and beta -MPP, and analyzed the processing efficiencies for various preproteins. We report here that alanine mutations at a subsite in rat beta -MPP interacting with the P2 arginine cause a shift in the processing site to the C-terminal side of the preprotein. Because of reduced interactions with the P2 arginine, the mutated enzymes recognize not only the N-terminal authentic cleavage site with P2 arginine but also the potential C-terminal cleavage site without a P2 arginine. In fact, it competitively cleaves the two sites of the preprotein. Moreover, the acidified site of alpha -MPP, which binds to the distal basic site in the long presequence, recognized the authentic P2 arginine as the distal site in compensation for ionic interaction at the proximal site in the mutant MPP. Thus, MPP seems to scan the presequence from beta - to alpha -MPP on the substrate binding scaffold inside the MPP cavity and finds the distal and P2 arginines on the multiple subsites on both MPP subunits. A possible mechanism for substrate recognition and cleavage is discussed here based on the notable character of a subsite-deficient mutant of MPP in which the substrate specificity is altered.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nuclear-encoded mitochondrial proteins generally carry N-terminal transport signal sequences (presequences) to target into mitochondria (1, 2), and inside the organelle, the presequences are removed by the mitochondrial processing peptidase (MPP)1 (3-5). The presequences vary in length and sequence, but each is specifically recognized during protein transport and the maturation event by transport receptors and MPP, respectively. Because protease processing is irreversible, the maturation step should be highly regulated and specifically catalyzed by MPP for the biosynthesis of mitochondria.

MPP consists of two structurally related subunits, alpha - and beta -MPP (6, 7). The beta -MPPs have a zinc binding motif, HXXEHX76E, in which histidine and the final glutamate residues participate in metal binding, and the first glutamate residue is involved in water activation for the hydrolysis of the peptide bond (8). Although beta -MPP has an active site containing a zinc ion, the recombinant subunit alone has no apparent peptidase activity (9-12). alpha -MPPs are mainly responsible for the binding of the basic site in the long presequence; a preprotein was cross-linked to alpha -MPP (13), and acidic amino acids in the C-terminal domain are mainly required for binding (14). The cooperative function of the subunits is involved not only in cleavage but also in the high affinity interaction to the presequence (15). Thus, alpha -MPP regulates the catalytic activity of beta -MPP and also participates in substrate binding.

Mammal and fungi MPP subunits are highly homologous to core I and II proteins in the mitochondrial cytochrome bc1 complex and, in some species of higher plants, the MPP and core complexes are identical (16, 17). A recent report demonstrated that recombinant bovine core proteins also have processing activity themselves (18). Previous reports of simulation models of rat and potato MPP structures built using bovine and avian core proteins showed that the MPP complex forms a crack leading to a large internal cavity in the dimer (19, 20). A metal binding site of beta -MPP and a substrate binding site of alpha -MPP are located inside the cavity. Moreover, the wall of the cavity is lined with negatively charged amino acids. More recently, the crystal structures of recombinant yeast MPP and a cleavage-deficient mutant of MPP-complexed synthetic presequence peptides have been determined (21). MPP binds the short peptide in the extended conformation within a large polar cavity. The substrate peptide mainly interacts to the beta -MPP side, and the N-terminal site of the peptide almost reaches toward alpha -MPP. Recognition sites for residues in the peptide are identified around an active site in the N-terminal domain of beta -MPP.

The decrease in the ionic strength dependence of MPP activity suggests that the enzyme associates with the basic presequence through electrostatic interaction (22). Indeed, distal and proximal basic amino acids from cleavage points are required for effective processing of the presequence (23, 24). The distal basic sites are required as positively charged amino acids, lysine and arginine, and the positions of the basic residues in the presequence are not restricted. On the other hand, the proximal basic site should be an arginine residue located at the -2 position2 (P2). Hydrophobic and hydrophilic residues at P1 and P2-P3, respectively, are also important for effective catalysis (25-27). Thus, a general sequence efficiently cleaved by MPP is represented as the sequence motif (R/K)XnRXdown-arrow Phi Psi Psi (Phi  and Psi  indicate hydrophobic and hydrophilic residues, respectively). However, a defect of an element in the motif is allowed without a complete loss of cleavage by MPP. Besides the motif sequence, glycine-induced flexible conformation of the presequence is preferred for processing (28, 29). The requirement of flexibility for processing was demonstrated as the common structure of the presequence bound on MPP using fluorescence energy transfer in the analysis (30), and the extended structure of the presequence was observed in complex crystals (21). Thus, MPP recognizes the flexible presequence on the multi-binding site arranged inside a molecular cavity.

So far, mutation analyses for MPP subunits have been reported (8, 14, 31), and the target residues are usually acidic amino acids because MPP recognizes basic sites on the presequences. MPP has a large polar cavity where a variety of presequences can bind; thus, acidic binding sites should be located inside the cavity. However, the specific acidic residues involved in binding are largely unknown. In this report, we focused on acidified sites3 in the cavity expected by the modeling structure of rat MPP and mutated the residues that are conserved in MPP and the core complex among the different species. In alanine-scanning mutations, we observed that a mutant of MPP alternatively cleaves two sites of a preprotein. We conclude that acidic amino acids in beta -MPP are involved in a determinant for the specific cleavage of a single site through the interaction with P2 arginine and discuss the substrate recognition mechanism.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Selection of Acidic Amino Acid Residues Conserved in the MPP Cavity-- We built a model of rat MPP complex based on structures of bovine and avian core proteins of cytochrome bc1 complex as reference proteins using the Insight II/Homology Software Package (Molecular Simulations Inc.) as previously described (19). We displayed structures of the modeling MPP and bovine core complex using structure-viewing software, WebLab viewer (Molecular Simulations Inc.), and picked up acidic amino acids existing in the internal cavities. The amino acid sequences of the MPP subunits and core proteins were aligned using ClustalW and selected acidic amino acid residues conserved among MPPs and core proteins from various species. We excluded residues that had already mutated to neutral ones (8, 14, 31) and finally selected eight acidic residues lined on the wall of the internal cavity in the MPP structure.

Mutation, Expression, and Purification of MPP-- To obtain recombinant MPP, we wanted to purify recombinant rat MPP from Escherichia coli cells. However, rat alpha -MPP was mostly produced as inclusion bodies or insoluble aggregates in the cells, in contrast to the beta -MPP subunit that was mostly found in soluble form, and the amount of active dimer was insufficient for purification. We then used yeast alpha -MPP, most of which was expressed in a soluble monomeric form, as the partner to the rat beta -MPP. The yeast alpha -MPP and rat beta -MPP genes were mutated using the QuikChange site-directed mutagenesis system (Promega). Hexahistidine-tagged MPP subunits in the C termini were individually expressed in bacteria and purified using a nickel-chelating column, described previously (8). Briefly, each subunit gene was cloned into the expression vector, pTrc99A (Amersham Biosciences), and transformed into an E. coli strain BL21 (DE3). The subunits were individually expressed in LB medium for 24 h at 25 °C, and the harvested cells were lysed by sonication. After centrifugation, the recombinant subunits were purified from the resultant supernatant using a nickel-chelating Sepharose column (Amersham Biosciences; His trap) until the purity exceeded 95%. We then mixed an equal molar concentration of subunits to reconstitute the active enzyme complex in vitro. The recombinant MPP exhibited processing activity to various precursor proteins translated in vitro, and the kinetic parameters for a synthetic oligopeptide substrate were essentially the same as those for MPP purified from bovine liver or yeast mitochondria, as previously reported (8, 14).

Protein Synthesis and Processing of Preproteins-- The genes of preproteins were cloned into the pGEM-4Z vector (Promega) and mutated using the QuikChange site-directed mutagenesis system (Promega). Radiolabeled preproteins were synthesized in rabbit reticulocyte lysate containing [35S]methionine (TNT coupled reticulocyte lysate system; Promega). The preprotein and recombinant MPP were incubated for an appropriate time at 30 °C in a processing buffer (10 mM HEPES-KOH, pH 7.4, 0.1% Tween 20, and 0.5 mM MnCl2), and the reaction was stopped by adding SDS-PAGE sample buffer (7). The processing product was separated on SDS-PAGE and visualized using an Imaging Analyzer (Fuji film; Bas1000). The processing efficiency of MPP was determined by the quantification of the radioactivity of the cleaved protein in the total protein using Image Gauge 3.0 (Fuji Photo Film Co., Ltd. and Koshin Graphic Systems).

Processing of Recombinant palpha and the N-terminal Protein Sequences-- To overproduce the preprotein of yeast alpha -MPP, palpha , of which mature residues from 144 to 425 were deleted, a restriction NcoI site and a hexahistidine were introduced at the N and C termini of the preprotein gene, respectively, using the PCR method. The gene was subcloned into a protein expression vector, pET-3d, and the protein was produced in bacteria at 25 °C overnight. After lysing the cells by sonication, the protein solution was adjusted to 6 M guanidine and loaded on a nickel-chelating Sepharose column equilibrated with binding buffer (20 mM HEPES-KOH, pH 7.5, containing M guanidine, 0.5 M NaCl, 5 mM imidazole, and 1 mM 2-mercaptethanol). The column was washed with binding buffer containing 6 M urea instead of guanidine, and then palpha protein was eluted with 20 mM HEPES-KOH, pH 7.5, containing 6 M urea, 500 mM imidazole, and 1 mM 2-mercaptethanol. The processing reaction was started by a 50× dilution of the purified palpha (1 µg) into the processing buffer-premixed recombinant MPP (3 µg). After incubation for 1 h at 30 °C, the proteins in the reaction solution were precipitated by trichloroacetic acid and separated by SDS-PAGE. For the N-terminal sequence analysis, about 30 pmol of protein on immobilized membrane (Immobilon-PSQ Transfer Membrane; Millipore) was applied to the Protein Sequencing System 491A (PerkinElmer Life Sciences).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Substitutions of Negatively Charged Residues Inside the MPP Cavity on the Processing Activity-- To elucidate characteristic sites in which MPP interacts with the basic presequence, we focused our attention on acidic residues that are located inside the central cavity of MPP. We selected eight acidic residues conserved between MPPs and core proteins and did alanine-scanning mutations on the MPP subunits. After each subunit was purified homogeneously with a nickel-chelating column, we then analyzed the processing activity of the in vitro reconstituted enzyme using various mitochondrial preproteins with different presequences containing from 9 (in the case of palpha ) to 50 (in the case of pAd) amino acid residues (Fig. 1A). The relative processing efficiency of the enzymes varied for the preproteins (Fig. 1B). Generally, mutations in alpha -MPP caused essentially no or small reductions in the processing efficiency, except for decreases in the processing of pAd by MPP mutated at the Glu383. In contrast, mutations in beta -MPP resulted in a decrease in processing activity for palpha , pMDH, and pAd. In particular, beta E191A, beta D324A, beta D413A, and beta E420/D421A MPPs showed little processing activity toward pAd; that is, less than 10% of the wild-type MPP. The reduced activity for the preproteins does not result from large defects of MPP conformation by the mutations because the mutant subunits can associate with their non-tagged counterpart subunit by pull-down assay using a nickel-chelating column (data not shown).


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Fig. 1.   Comparison of various mitochondrial presequences and the processing activity of alanine-mutated MPPs toward the preproteins. A, comparison of the N-terminal presequences of four mitochondrial preproteins. The N-terminal amino acid sequences of the preproteins are shown with the cleavage sites. The radiolabeled preproteins translated in vitro in reticulocyte lysate were used in the processing assay as substrate for MPP. B, effect of mutations on processing activity. Processing efficiencies toward the preproteins were examined as described under "Materials and Methods." The amounts of MPP used for the assay were 12.5, 0.5, 100, and 20 ng for palpha , pMDH, pSCC, and pAd, respectively. Around 40-50% of the preproteins were processed by the amount of wild-type (WT) enzyme. Two-site processing of palpha by beta E191A MPP is inserted in the left panel. p, the precursor form; m, the mature form; x, the cleavage product newly generated by the mutated MPP.

Mutations at the Glu191 and Asp195 in beta -MPP Produced an Abnormal Processing Product of Preprotein-- When beta E191A MPP was reacted with palpha , we noticed an extra processing product (xalpha ) that migrates to a smaller molecular weight than the mature one (malpha ) on SDS-PAGE (indicated by an arrow with x in Fig. 1B). This product was never observed during incubation with the wild-type MPP (Fig. 1B and Fig. 2A). The beta E191A MPP cleaved another three preproteins at the mature sites alone (data not shown). The abnormal processing product did not result from proteolysis by endogenous bacterial protease(s), which may be contaminated in the beta E191A MPP preparation, because no processing occurs by the subunit only. Thus, the mutated enzyme complex should cleave palpha at the two sites and generate an additional smaller molecular weight polypeptide with a mature one, indicating that the substrate specificity seems to be altered by the mutation. It is notable that a single-site mutation within the protease changes the cleavage site of substrate. We then analyzed the detailed character of the mutated MPP because it must give us a new insight into the mechanism of recognition and cleavage for presequences by MPP.


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Fig. 2.   Shift of the cleavage site in the preprotein by the mutated MPPs. A, two-site cleavage of the palpha by MPPs mutated at the Glu191 and Asp195. Wild type (WT) and each mutated MPP (100 ng of protein) were incubated with radiolabeled palpha at 30 °C for 10 min, and the processing products were subjected to SDS-PAGE and visualized by an imaging analyzer. The processing efficiency was determined. p, precursor form (palpha ); m, mature form (malpha ); x, cleavage product newly generated by the mutant MPP (xalpha ). B, cleavage sites of palpha by the mutant MPP. Recombinant palpha protein denatured in 6 M urea (lanes 1, 2, and 4) or no substrate (lanes 3 and 5) was rapidly diluted in the reaction buffer and incubated with MPP (lanes 2 and 4) or no enzyme (lane 1) at 30 °C for 1 h. The proteins were separated on SDS-PAGE gel and stained with Coomassie Brilliant Blue R-250. The N-terminal amino acid sequence of palpha is shown with the processing sites (arrows with m and x) that were determined by direct protein sequencing. The P2 and P1'-P2' positions for each cleavage site are underlined. The underlined sequences denote two types of processing motifs, RXdown-arrow Phi Psi Psi and XXdown-arrow Phi Psi Psi , by MPP.

At first, we made sure of the location of the rat beta Glu191 corresponding to the site in the yeast MPP structure using the recently reported crystal structure of MPP bound with presequence peptides. The structure denoted that beta Glu160 and beta Asp164 in the S2 pocket cooperatively bound to the guanidino group of P2 arginine. These acidic residues are highly conserved among beta -MPPs, corresponding to Glu191 and Asp195 in rat beta -MPP. If the abnormal processing by beta E191A MPP resulted from the partial defect of the P2-S2 interaction, mutation at the beta Asp195 also causes the shift of cleavage. Therefore, we mutated the Asp195 to an alanine residue (D195A) and to double alanines with Glu191 (E191A/D195A), and the mutants of MPP reacted with palpha . We observed an extra fragment processed by D195A MPP in addition to the authentic processed product (Fig. 2A). The two cleaved polypeptides showed similar sizes to malpha and xalpha fragments processed by E191A MPP, as judged by SDS-PAGE, suggesting that the cleavage sites are the same, but efficiencies in the processing at the two sites are different between E191A and D195A MPPs. Moreover, the double alanine mutant of MPP, E191A/D195A, converted palpha to xalpha with little malpha (Fig. 2A). The wild-type MPP specifically recognizes and cleaves at the motif in the presequence, but in the mutants of MPP, the substrate specificity seems to be lost. To ensure site specificity of the mutated MPP, we determined the N-terminal amino acid sequences of xalpha and malpha using recombinant palpha purified from bacterial extract. The recombinant preprotein was incubated with the wild-type or E191A MPP, and the processing products were separated on SDS-PAGE. The wild-type MPP produced a single processing product, and the mutated enzyme gave two products, similar to the cleavage products of the radiolabeled preprotein substrate (Fig. 2B). Edman sequence analyses for the two polypeptides revealed that the N-terminal five residues were YSNIX and LS(S/N)LA for malpha and xalpha , respectively. This indicates that the cleavage sites were between residues 9 and 10 (RLdown-arrow YSN) and 20 and 21 (FKdown-arrow LSS) (Fig. 2B). Thus, the mutated MPP cleaves at a new site (xalpha ) in addition to the P2 arginine site (malpha ). The new processing site is included in an extended processing signal of no arginine at P2 with hydrophobic and hydrophilic residues at P1' and P2'-P3', respectively (the XXdown-arrow Phi Psi Psi motif), and this sequence is less efficiently cleaved than the P2 arginine motif by MPP. Potentially, palpha includes a weak cleavage signal at the C-terminal site from an authentic RXdown-arrow Phi Psi Psi cleavage site. The wild type and any other mutants of MPP described in the previous section never process the C-terminal site. Why do the S2-defective mutants of MPP cleave at the P2 arginine-deficient site more dominantly than at the P2 arginine site?

Competitive Recognition for Two Sites in the Preprotein by the Mutated MPP-- Because mutations to alanines at Glu191 and Asp195 reduce the interaction of MPP to P2 arginine, the S2-defective mutants of MPP may pass through P2 arginine, thereby missing an important interaction necessary for correct processing. As a result, the MPPs cause a shift of the cleavage site to the C-terminal XXdown-arrow Phi Psi Psi site. Alternatively, the mutated MPPs may simply cleave malpha to xalpha by stepwise processing. To ensure whether the S2-defective mutants of MPP cleave two sites of the preprotein by competitive processing or stepwise processing, we first analyzed the kinetics of the processing reaction. The xalpha polypeptides were produced early in the processing reaction by E191A or D195A MPPs, and no accumulation of malpha was observed during the initial period of the reaction, even with the E191A/D195A enzyme (Fig. 3A), suggesting a decreasing probability of stepwise processing. We next determined if the mutated MPPs could cleave the mature protein, malpha . Then the mutated enzymes were added to the reaction solution after the accumulation of malpha by processing of the wild-type enzyme alone. Practically no production of xalpha was observed (Fig. 3B, lanes 5-7). More directly, none of the mutants cleaved malpha that was constructed with the deletion of the nine N-terminal residues of palpha (Fig. 3B, lanes 10-12). The malpha polypeptide was no longer a substrate for the mutated enzymes. Thus, the E191A and D195A MPPs seem to cleave the two N-terminal sites of palpha competitively rather than stepwise.


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Fig. 3.   Competitive processing of palpha by MPPs mutated at the Glu191 and Asp195. A, kinetic analysis of processing by the mutated MPPs. The mutated MPPs (25 ng of protein), E191A, D195A, and E191A/D195A, were incubated with radiolabeled palpha at 30 °C for the indicated times and the processing products on SDS-PAGE. The processing ratios were quantified using an imaging analyzer. B, cleavage of malpha by the MPPs. Radioactive palpha was incubated with no MPP (lane 1) or 100 ng of wild-type (WT) MPP (lane 2 and 4-7) at 30 °C for 30 min to accumulate malpha product. Then the reactions were stopped (lane 1-3) or continued for 10 min (lane 4-7) after the addition of 100 ng of each MPP into the reaction solutions. The preprotein was reacted with E191A MPP at 30 °C for 30 min, and the incubation was stopped (lane 3). In vitro translated malpha was reacted with no MPP (lane 8) or 100 ng of MPP proteins (lane 9-10) at 30 °C for 10 min. Each reaction was analyzed on SDS-PAGE and the imaging analyzer. An asterisk indicates the non-processed product, because it was translated during protein synthesis in a rabbit reticulocyte lysate system using the control vector, pGEM-4Z.

Because the mutated MPP could cleave palpha to xalpha but not attack malpha , the nine N-terminal residues of palpha are involved in the proteolytic production of xalpha . We have previously reported that the N-terminal arginines distal to the cleavage site in the long presequence could interact mainly with the twin acidic site (Asp390/Glu391) in yeast alpha -MPP (14). Then we speculated that arginines in the nine N-terminal residues were recognized by alpha -MPP as the distal basic sites. To examine this hypothesis, the MPP complex was reconstituted with wild-type or mutated (E390Q/D391N) alpha -MPP and wild-type or the mutated (E191A) beta -MPP, and then we tested in vitro processing of palpha using various combinations of the wild-type and the mutated subunits (Fig. 4A). The MPP reconstituted from alpha E390Q/D391N and the wild-type beta -MPP had essentially no effect on the palpha processing activity, the same observation as in our previous report stating that the mutated MPP complex is insensitive to processing of such a short presequence. When we used a combination of alpha E390Q/D391N and beta E191A, the reconstituted enzyme cleaved from palpha to malpha but hardly produced any xalpha fragments. Individual alanine substitutions at the two N-terminal arginines (Arg3 and Arg8) in the palpha presequence revealed that Arg8 is required not only for processing to malpha by wild-type MPP but also for cleavage to xalpha by beta E191A MPP (data not shown). The data suggest that the mutated MPP recognizes the Arg8 as the basic distal site on the twin acidic site of alpha -MPP (Fig. 4B). Thus, the shift of the processing site is induced by the competitive recognition of the mutated MPP for two sites in the preprotein. It must be due to both effects, the defective interaction to P2 arginine on beta -MPP and, instead of P2-S2 interaction, ionic recognition for the basic distal site on the acidic subsite of alpha -MPP.


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Fig. 4.   Shift of recognition for the proximal (P2) arginine of the presequence to an acidified subsite on the alpha -MPP. A, processing of palpha by MPPs reconstituted from various combinations of the subunits. Radiolabeled palpha was incubated with MPPs (25-200 ng of proteins) reconstituted from wild-type (WT) or mutated subunits at 30 °C for 30 min. The processing products were separated on SDS-PAGE and detected using an imaging analyzer. B, a putative model for interactions between the acidic sites in MPP and the basic sites in the presequence. The palpha protein is illustrated by a thick gray line. The dashed lines indicate potential interactions between MPP subunits and arginines in the N-terminal region of palpha . In the middle panel, the arrows show the competitive processing reaction on the MPP at the two cleavage sites, m or x.

Comparison of the Processing Rates between the Cleavages at Normal and New Sites by the S2-defective Mutants of MPP-- We have so far analyzed abnormal processing by the S2-defective mutant of MPP using a preprotein, palpha , as a substrate. The preprotein has a short N-terminal presequence separated by a cleavage site with P2 arginine. There is a potential processing site without P2 arginine in the N-terminal of the mature protein. The two processing motifs belong to RXdown-arrow Phi Psi Psi and XXdown-arrow Phi Psi Psi , respectively, but the sequences of P1 to P3' are not identical to each other (Fig. 2B). In the motifs, P3' of the former site is asparagine, and the latter site is serine; the latter is the more strict processing signal by MPP. To compare the rates of the competitive cleavage at the former and latter sites by the S2-defective mutants of MPP, we constructed cDNA encoding two artificial preproteins, which have identical sequence among the former and latter sites, except for with or without P2 arginine. One is the pMDH derivative, pMDH2cs, in which a segment of AASdown-arrow FSTS was introduced just after the nine residues of the authentic cleavage site with P2 arginine (Fig. 5A). The other one is pAd2cs, in which a cleavage signal with P2 arginine is inserted into the presequence, and the authentic P2 site is mutated to alanine. As a result, the preprotein has tandem cleavage sites of RTdown-arrow LSVS and ATdown-arrow LSVS (Fig. 5A). Then we analyzed the processing of the preproteins by the wild-type and S2-deficient mutant of MPPs. The mutated MPPs, E191A, D195A, and E191A/D195A, cleaved the preproteins both at the former site with P2 arginine and at the latter site with no arginine (Fig. 5B). The E191A and D195A MPPs cleaved at the former and latter sites in pMDH2cs with about 25 and 10% efficiency, respectively. The E191A/D195A enzyme converted pMDH2cs to an almost equal amount of the two polypeptides. Wild-type MPP cleaved pMDH2cs at almost the former site with slight processing at the latter site. Very similar processing patterns were observed using pAd2cs, resulting in cleavage almost at the former site by the wild type, dominant cleavage at the former site with partial cleavage at the latter site by E191A and D195A, and even processing at the two sites by E191A/D195A (Fig. 5B). These results suggest that the S2-deficient mutant of MPPs frequently passes though former cleavage signals containing P2 arginine. Thus, Glu191 and Asp195 of beta -MPP function as determinants for the single site cleavage through the interaction with P2 arginine, possibly with a scanning of the presequence from the N-terminal side.


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Fig. 5.   Processing of preproteins that have tandem cleavage signals, RXdown-arrow Phi Psi Psi and XXdown-arrow Phi Psi Psi . A, N-terminal amino acid sequences of pMDH2cs and pAd2cs. Segments introduced into the presequence instead of the original sequences are shown in italic letters. The potential cleavage sites are hyphenated and indicated by arrows with each cleavage motif, P2 arginine (RXdown-arrow Phi Psi Psi ) and P2 no arginine (XXdown-arrow Phi Psi Psi ), underlined. B, two-site cleavage of pMDH2cs and pAd2cs by MPPs mutated at Glu191 and Asp195. The radiolabeled pMDH or pMDH2cs were incubated with wild-type (WT) or mutant MPPs (0.5 ng) at 30 °C for 10 min. In the processing of pAd and pAd2cs, 500 ng of MPP was used and reacted at 30 °C for 30 min. p, precursor form; m, mature form; x, cleavage product at C-terminal P2 no-arginine site.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Here we described a notable character of the S2-deficient mutant of MPP that causes not only reduced recognition at the RXdown-arrow Phi Psi Psi (Phi  and Psi  indicate hydrophobic and hydrophilic residues, respectively) site but also a shift of cleavage to the XXdown-arrow Phi Psi Psi site. In a previous report, alanine mutations at both rat beta Glu191 and beta Asp195 resulted in no loss of MPP activity toward the substrate without P2 arginine. Moreover, the processing activity could be partially compensated for by the beta Arg191 enzyme toward P2 acidic preproteins, suggesting an ionic interaction between the residue Glu191 and P2 (32). The crystal structure of a cleavage-deficient mutant of yeast MPP interacting with the synthetic peptide of the presequence has recently denoted that the two acidic amino acid residues corresponding to Glu191 and Asp195 in rat beta -MPP interact with P2 arginine (21). From these findings, Glu191 and Asp195 at the S2 of rat MPP are the essential residues required for the specific determination of the cleavage site.

We initially analyzed the effect of alanine mutations for acidic amino acid residue-conserved inside cavities of MPP and core proteins on the processing activity toward various preproteins. The relative processing efficiency of the mutants varied for the preproteins, and therefore, it is difficult to achieve consensus on the function for each acidic amino acid in the processing reaction. However, it is notable that the processing of pAd is sensitive to mutations at multiple acidic sites in the MPP cavity. Because the pAd has nine basic sites in the long presequence region containing 50 amino acid residues, such a highly basic long presequence may require many acidic sites in the cavity to stabilize the enzyme-substrate complex through ionic interactions. Indeed, our preliminary experiment shows that long synthetic peptides based on the presequence of pAd have a much lower value of Kd than those of the shorter peptide for binding to MPP. Detailed analysis for each mutated enzyme will be required to elucidate the functions of the acidic amino acids in the processing mechanism.

The processing of preproteins by S2-deficent MPP indicates alternate substrate specificity and drastic change of the substrate recognition system of the MPP. It follows that the characterization of the shift of cleavage described here may give a new insight into the recognition and cleavage of the presequence. Thereby, we propose a possible mechanism for the search and determination of processing site using the substrate binding scaffold and the multiple subsite inside a MPP cavity. Observations from the crystal structure show two synthetic prepeptides bound to MPP in an extended conformation. Both peptides form main-chain hydrogen bonds with the edges of the two beta  sheets (21). One of the sheets is located near the active site, and the other is in the C domain of beta -MPP (strands 1 and 2 of red ribbons in Fig. 6A). Because the N-terminal sides of the peptides of the cytochrome c oxidase subunit IV presequence almost reach the alpha -MPP, the edges of the two other beta  sheets of alpha -MPP (strands 3 and 4 of yellow ribbons in Fig. 6A) seem to be available to bind to longer presequences within the cavity. The proposed "substrate binding scaffold" formed from the four beta  sheets inside the MPP cavity denotes a highly ordered substrate binding state, and it could lead to the directional insertion of the peptide into the MPP cavity through a narrow crack between both subunits. The mechanism of entry of the presequence into the cavity is unknown, but electrostatic interactions would contribute to capturing the positively charged presequence into the cavity, because its inside is an electronegative environment due to the arrangement of many aspartate and glutamate residues. During the insertion, MPP may scan the extended substrate from the N to C termini on the substrate binding scaffold and determine the cleavage site. If a cleavage signal of RXdown-arrow Phi Psi Psi is included in a short stretch of the N-terminal side, as palpha and pMDH, the MPP uses the edges of the beta  strands 1 and 2 to bind the presequence backbone with clipping P2 arginine to acidic S2 on beta -MPP and immediately cleaving off the peptide (Fig. 6B, upper panel). Unlike with the shorter peptide, the MPP can scan a longer presequence to find the cleavage signal with interactions to the edges of another one or two beta  strands in alpha -MPP (Fig. 6B, lower panel). Moreover, distal basic amino acids bind to symmetric acidic sites for S2 to clip the N-terminal side of the longer presequence to strands 3 and 4 on alpha -MPP, offering stabilization of the substrate-enzyme complex. Thus, the long presequence is pasted along the scaffold, clipping the proximal and distal basic residues to acidic S2 on beta -MPP and the acidic side on alpha -MPP, respectively. Besides, S1'-P1', S2'-P2', and S3'-P3' interactions would also be required for an overall high affinity Michaelis complex and for the strict recognition of the processing site. The multiple subsite based on the scaffold restricts the protein processing and allows a defect of subsites in the enzyme without a complete loss of processing activity, although the mutation could cause a shift in the processing site.


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Fig. 6.   Putative model for determination of the cleavage site using substrate binding scaffold and multiple subsite inside cavity of MPP. A, structure of yeast MPP complexed with yeast cytochrome c oxidase subunit IV presequence. The structure of the MPP subunits (ribbon models) and cytochrome c oxidase subunit IV (Cox IV) presequence peptide (stick and space-filling models) are visualized from x-ray crystallographic data in a protein data bank (ID code 1HR9) using the WebLab ViewerPro 3.7 software package. The alpha - and beta -MPPs are shown in blue and green ribbons, respectively. Four strands of yellow ribbons in alpha -MPP and red ribbons in beta -MPP indicate a substrate binding scaffold inside the cavity. The space-filling models colored in red indicate acidic subsite residues required for binding to proximal and distal basic residues in the presequence. B, differential modes for substrate recognition of MPP toward shorter (upper panel) and longer (lower panel) presequences. See "Discussion" for details. The alpha - and beta -MPPs are painted in blue and green, respectively. The substrate binding scaffold is indicated by four orange arrows, and the internal acidic cavity is gradually colored in red. Scissors indicate the active site of beta -MPP. Bold and narrow lines of preprotein indicate the presequence and mature portion, respectively. The plus (+) symbols represent basic amino acid residues in the presequence, and the minus (-) symbols indicate binding sites of beta - and alpha -MPPs for proximal (S2) and distal basic sites in the presequence.

It is unknown what regulates the directional insertion of the presequence into the cavity through a narrow crack and the putative scanning of the sequence. Boteva et al. (33) report ATP and ADT binding on Neurospora MPP subunits. In the report they indicate putative ATP binding sequences that resemble to some extent the consensus pattern for "segment A and B" of typical ATP-binding proteins. One of the sequences is part of a Gly-rich loop of about a 20-amino-acid stretch that is highly conserved in alpha -MPPs. A deletion of the Gly-rich structure in yeast enzyme resulted in a great decrease in the processing activity with a reduction of substrate binding, indicating that the loop is essential for presequence cleavage and binding (19). Most interesting is the crystallographic data showing weak electron densities for the loop near the cleavage site of the substrate peptide, suggesting that it is flexible and/or flapping, interacting with the presequence (21). Additionally, we have observed an increase in prepeptide cleavage activity of the enzyme in the presence of ATP and a decrease in the activity after apyrase treatment of MPP.4 Although there is no experimental evidence of the linkage between nucleotide binding and the loop-flapping, nucleotide-induced structure changes in the loop may be required for the directional insertion of the presequence and for the movable presequence inside the cavity. Further analyses on protein structure and the dynamics will contribute to an understanding of a putative model for the determination of cleavage sites through the scanning of the presequence on the substrate binding scaffold and multiple subsite with or without nucleotide binding.

    FOOTNOTES

* This work was supported in part by Grant-in-aid for Scientific Research 12780456 from the Ministry of Education, Science, Sports, and Culture of Japan (to S. K.) and by a grant-in-aid from Takeda Science Foundation in Japan (to S. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 81-92-642-4182; Fax: 81-92-642-2607; E-mail: s.kitscc@mbox.nc.kyushu-u.ac.jp.

§ Present address: Dept. of Microbiology and Immunology, Shinshu University School of Medicine, Matsumoto 390-8621, Japan.

Published, JBC Papers in Press, November 13, 2002, DOI 10.1074/jbc.M209263200

2 Following Schechter and Berger (34), the enzyme binding sites are denoted S1, S2,  ... , Si and S1', S2',  ... , Si' away from the scissile peptide bond toward the N and C termini, respectively. Amino acid residues in the substrates are referred to as P1, P2,  ... , Pi and P1', P2',  ... , Pii', in accordance with the binding site.

3 The residues were numbered according to the full-length MPP precursors.

4 S. Kitada, E. Yamasaki, K. Kojima, and A. Ito, unpublished results.

    ABBREVIATIONS

The abbreviations used are: MPP, mitochondrial processing peptidase; alpha -MPP, a alpha  subunit of MPP; beta -MPP, a beta  subunit of MPP; pAd, a precursor form of Ad; palpha , a precursor form of alpha -MPP; pMDH, a precursor form of MDH; pSCC, a precursor form of bovine cytochrome P450scc.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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