A Proposed Common Structure of Substrates Bound to Mitochondrial
Processing Peptidase*
Katsuhiko
Kojima,
Sakae
Kitada,
Tadashi
Ogishima, and
Akio
Ito
From the Department of Chemistry, Faculty of Science, Kyushu
University, Fukuoka 812-8581, Japan
Received for publication, April 12, 2000, and in revised form, August 2, 2000
 |
ABSTRACT |
Mitochondrial processing peptidase (MPP), a
metalloendopeptidase consisting of
- and
-subunits, specifically
cleaves off the N-terminal presequence of the mitochondrial protein
precursor. Structural information of the substrate bound to MPP was
obtained using fluorescence resonance energy transfer (FRET)
measurement. A series of the peptide substrates, which have distal
arginine residues required for effective cleavage at positions
7,
10,
14, and
17 from the cleavage site, were synthesized and
covalently labeled with 7-diethyl aminocoumarin-3-carboxylic
acid at the N termini and
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) at position +4, as fluorescent donor and acceptor,
respectively. When the peptides were bound to MPP, substantially the
same distances were obtained between the two probes, irrespective of
the length of the intervening sequence between the two probes. When
7-diethylamino-3-(4'-maleimidyl phenyl)-4-methyl coumarin was
introduced into a single cysteine residue in
-MPP as a donor and
IANBD was coupled either at the N terminus or the +4 position of the
peptide substrate as an acceptor, intermolecular FRET measurements also
demonstrated that distances of the donor-acceptor pair were essentially
the same among the peptides with different lengths of intervening
sequences. The results indicate that the N-terminal portion and the
portion around the cleavage site of the presequence interact with
specific sites in the MPP molecule, irrespective of the length of the
intervening sequence between the two portions, suggesting the structure
of the intervening sequence is flexible when bound to the
MPP.
 |
INTRODUCTION |
Numerous mitochondrial proteins are translated on cytoplasmic
ribosomes as larger precursors. An N-terminal presequence of the
mitochondrial protein precursor functions as a targeting signal for
their transport to mitochondria (1-3). During import of precursors into mitochondria, the presequences are recognized by multiple proteins
(4, 5), such as molecular chaperones, translocases of the mitochondrial
outer and inner membranes, and peptidases from inside mitochondria.
Despite the identification of various proteins that interact with the
mitochondrial precursors, the mechanism of recognition of the
presequence by these components has not yet been understood. The lack
of sequence homology of the presequences, even though they are
characterized by the positively charged residues and the formation
property of amphiphilic
-helices, has inhibited clarification of the
recognition mechanism (6).
Mitochondrial processing peptidase
(MPP),1 located in the matrix
of the mitochondria, cleaves off most presequences of the imported
precursors. MPP consists of two structurally related subunits,
-MPP
and
-MPP. Complex formation with the two subunits is essential for
both enzymatic activity (7, 8) and substrate binding (9).
Earlier studies indicated that some structural elements of the
presequence are required for recognition by MPP. An arginine residue at
position
2, the so-called "proximal arginine," from the cleavage
site, which is usually found among most precursor proteins, plays a
critical role in cleavage reaction (10-12). Distal basic amino acid
residue(s) around position
10 are also important for effective
cleavage (10-12). The length between the proximal arginine and the
distal basic residues is not so fixed, and 4-10 amino acids are
allowed (13). Our more recent studies have shown a requirement for
effective cleavage of flexible linker sequences containing proline and
glycine between the two basic residues (13, 14), a hydrophobic residue
at position +1 (12, 13), and serine or threonine residues at position
+2 and/or +3 (12, 15).
Some functional amino acid residues in MPP were determined using
mutational analysis; His-101, Glu-104, and His-105 in rat
-MPP,
which form a metal binding site, HxxEH, conserved among a
pitrilysin metallopeptidase superfamily (16), are the catalytic center
of MPP (17, 18). Glu-181 is the third metal-binding residue (19).
Glu-174 may participate in the catalytic reaction (18). Glu-124, which
is in a characteristic acidic amino acid cluster conserved in
-MPP,
may interact with the N-terminal portion of the presequence in the
cleavage reaction (18). On the other hand, Glu-390 and Asp-391 in yeast
-MPP interact with the distal arginine residues, which are
required especially for cleavage of precursors with a longer
presequence (19). Deletion of three residues in the glycine-rich
segment characteristic of
-MPP resulted in a drastic reduction in
affinity to the substrate
(20).2
Findings on functional amino acid residues both in precursor proteins
and MPP required for the processing reaction, especially for precursor
recognition by MPP, suggest that the two subunits of MPP cooperatively
form the substrate binding pocket and that they have several substrate
binding sites to cope with different structural elements in the
extension peptide. To elucidate the recognition mechanism that makes
feasible strict substrate specificity for MPP, it is necessary to
determine the structure of the presequence bound to the enzyme.
In the present study, fluorescence resonance energy transfer (FRET)
experiments provide the first evidence that the distal arginine and the
portion around the cleavage site of the presequence are located
at specific sites in the MPP molecule, irrespective of the position of
the distal arginine. An induced-fit mechanism of substrate recognition
of MPP seems likely.
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EXPERIMENTAL PROCEDURES |
Preparation of Fluorescence-labeled Peptides--
The
fluorescent dyes, 7-diethyl aminocoumarin-3-carbonic acid (DAC),
N,N'-dimethyl- N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), and
7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) were
purchased from Molecular Probes, Inc. (Eugene, OR). Peptide synthesis
and DAC labeling were done as described (9). IANBD amide labeling with
the cysteine residue of peptides for intra- and intermolecular FRET
experiments was done essentially the same as DAC labeling. Peptide
authenticity was identified by MALDI-TOF mass spectrometry (Voyager,
PerSeptive Biosystems). The concentrations of the fluorescence peptides
were calculated from the molar extinction coefficient of 45,000 (M
1
cm
1) for DAC or 23,500 (M
1
cm
1) for IANBD amide.
Preparation of Fluorescence-labeled MPP--
A
hexahistidine-tagged yeast
-MPP and yeast
/
E73Q complex were
purified as described (9). Purification and fluorescent labeling of a
hexahistidine-tagged yeast
E73Q were done as follows. The
supernatant from the BL21(DE3) strain carrying pET-
E73QHis was
loaded on a 5-ml Hi-trap chelating column (Amersham Pharmacia Biotech)
equilibrated with buffer A (20 mM Hepes-KOH, pH 7.4, containing 500 mM NaCl). The column was washed with 50 ml
of buffer A containing 50 mM imidazole. The
-MPP was
eluted with buffer A containing 200 mM imidazole. To the
fractions containing
-MPP were added 0.1 mM CPM, and
then the sample was left to react for 1 h at 25 °C. The
reaction was terminated using 1 mM cysteine for 30 min at
25 °C. The free dye was removed on a PD-10 desalting column
equilibrated with 20 mM Hepes-KOH, pH 7.4, containing 200 mM NaCl. Labeled
-MPP was then applied onto 2 ml of
Q-Sepharose FF (Amersham Pharmacia Biotech) equilibrated with 20 mM Hepes-KOH, pH 7.4, containing 20 mM NaCl.
The column was washed with 30 ml of the same buffer, and then the
protein was eluted with 20 mM Hepes-KOH, pH 7.4, containing
100 mM NaCl. The purity of CPM-labeled
-MPP was
confirmed by SDS-polyacrylamide gel electrophoresis followed by UV
transillumination and Coomassie Blue staining. The labeling efficiency
was calculated from a molar extinction coefficient of 33,000 (M
1
cm
1) for CPM. The labeling procedure resulted
in the incorporation of 0.11 ± 0.01 mol of CPM/mol of
-MPP.
The CPM-labeled
-MPP was mixed with an equal mol amount of
the purified
-MPP, and intermolecular FRET measurements were made.
Fluorescence Measurements--
Fluorescence was measured at
25 °C using a Hitachi F-4500 fluorescence spectrophotometer.
Steady-state fluorescence anisotropy measurements were performed with a
Hitachi F-4500 fluorescence spectrophotometer equipped with automatic
fluorescencce polarization system. In the intramolecular FRET
measurement of the double-labeled peptide, excitation of DAC was
measured at 390 nm and the emission intensity at 470 nm. MPP (0.5 µM) was diluted into 20 mM Hepes-KOH, pH 7.4, containing 30% glycerol, and then various concentrations of the
double-labeled peptides were added. The emission spectra were taken
after each sample and the blank had been thoroughly mixed and
allowed to equilibrate for 1-2 min. In the intermolecular FRET, the
excitation of CPM was measured at 390 nm and the emission intensity at
480 nm. To the IANBD-labeled peptides (0.5 µM),
CPM-labeled MPP was added at the indicated concentrations, and then the
emission spectra were taken, and the fluorescence intensity at 480 nm
was read.
The dissociation constant, Kd, was determined as
follows: F = (Fmax[Enz])/(Kd + [Enz]),
where F and Fmax are the measured and
maximal fluorescence intensity of the peptides, respectively, and
[Enz] represents the concentrations of the enzyme. A plot of
[Enz]/F versus [Enz] yields a linear function with a slope of 1/F and an ordinate intercept of
Kd.
From the decrease in fluorescence of donor DAC or CPM, induced in the
presence of acceptor IANBD, the energy transfer efficiency E
was calculated from
|
(Eq. 1)
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where QD stands for the unquenched
quantum yield of the donor and QDA is the
quantum yield in the presence of the acceptor. Quantum yield was
substituted for emission maximum intensity F. From the
energy transfer efficiency results, the distance between donor and
acceptor was calculated according to the Förster theory (21),
|
(Eq. 2)
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where R is the calculated distance and
R0 is the distance at which 50% energy transfer
would occur between the donor-acceptor pair; it is given in angstroms,
as shown in Equation 3,
|
(Eq. 3)
|
where J, the overlap integral, is the degree of
spectral overlap of donor emission FD(
) and
acceptor absorbance
A(
), as defined by Equation 4.
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(Eq. 4)
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2 is assumed to be 2/3. The refractive
index of the solvent, n, is used at a value of 1.4. QD, the quantum yield for the donor, was given
as
|
(Eq. 5)
|
where QR is the quantum yield for the
reference dye, FD and FR
are the fluorescence intensities for the donor and reference dye,
respectively, and AD and
AR are the fluorescence intensity for the donor
and reference dye, respectively. Fluorescein was used as the reference
dye and was assumed to have a quantum yield of 0.92 in 0.1 N NaOH.
Although
2 was taken as 2/3 for the
calculation of distances, the maximum and minimum values of
2 were estimated according to the method of
Dale et al. (21).
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(Eq. 6)
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(Eq. 7)
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where dD = (rD/0.4)1/2,
dA = (rA/0.4)1/2;
rD and rA are the
limiting anisotropies of the donor and acceptor, respectively. Using these values for the orientation factor, the maximum and minimum distances between probes were calculated and were regarded as the
probable error limits of the distance (R-limits).
 |
RESULTS |
Distance between the N-terminal End and the C-terminal Portion of
the Peptide Substrates Bound to MPP--
Sequence data on the
presequence of mitochodrial protein precursors show that position of
the distal basic acid is not so fixed among the extension peptides and
is located
7 to
17 from the cleavage site. To elucidate the
structure of the substrate peptide bound to the enzyme, it is vital to
determine whether the distal basic amino acids at various positions
interact with the same amino acid(s) in the enzyme, because the
proximal arginine must be fixed with certain amino acid residues near
the active center in
-MPP. In attempting this elucidation, we
measured the distance between the distal arginine and the residue
around the cleavage site, using intramolecular FRET. We synthesized a
series of peptides with different lengths of intervening sequence
between the proximal and distal arginines, labeling them at the
N-terminal end with fluorescent donor DAC and at position +4 cysteine
with the acceptor IANBD amide (Table I).
These sites were two and five amino acid residues away from the distal
and proximal arginines, respectively, to avoid interference by probes
in the interaction between the arginines and other recognition elements
of the peptide and MPP. In MDH-14A, arginine at position
3 (position
14 from the N-terminal end) was replaced with alanine in the original sequence of rat MDH, as it is not necessary for the residue at this
position to be arginine (10). This peptide has a distal arginine at
position
10 and is used as a standard peptide in the present work. In
MDH-
AAL, three intervening amino acid residues between proximal and
distal arginines were deleted so that the distal arginine was at
position
7. This peptide was found to have the minimum length for
effective cleavage by the MPP (13). MDH-AdAR and MDH-AdRA have the
distal arginine residue at position
14 and
17, respectively. In
these peptides, the linker sequence in the presequence of bovine
adrenodoxin precursor was introduced between the proximal and distal
arginines instead of the original one of the MDH presequence.
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Table I
Amino acid sequences of the fluorescence-labeled peptides for
intramolecular FRET measurements
Model peptide substrates were double-labeled with the fluorescence dyes
DAC and IANBD. The hyphens indicate the cleavage sites by MPP. The
proximal and distal arginine residues are indicated by single and
double underlines, respectively.
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The emission maximum of DAC is at 470 nm (Fig.
1). The absorption spectrum of IANBD,
which gives the peak at 498 nm, has an excellent overlap with the
emission spectrum of DAC (data not shown). The spectral overlap,
J, between these spectra is calculated to be 9.31 × 10
14 M
1
cm
1nm4. The spectral
characteristics of all the peptides were essentially the same. For the
intermolecular FRET measurements, fixed concentrations (0.5 µM) of the DAC or DAC/IANBD-labeled peptides were added
to various concentrations (usually 0-3 µM) of the
purified
/
E73Q, in which
-MPP is an inactive mutant with
glutamine substituting for the glutamate residue of the active center
(18) (Fig. 2). As demonstrated in our
previous study (9), the fluorescence of DAC introduced to the peptide
substrate bound to MPP increases with environmental change around the
dye (Fig. 1). Titration of the DAC-labeled peptides gave the
dissociation constant, Kd, of peptides for binding
to MPP (Fig. 2). All of the peptides bound to MPP with a high affinity
to the same extent.

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Fig. 1.
Spectral change of the emission of DAC- and
DAC/IANBD-labeled peptides with the addition of MPP. The emission
spectra of DAC-labeled (A) and DAC/IANBD-labeled
(B) MDH14A (0.5 µM, respectively) were taken
in the presence of various concentrations (0-3 µM) of
yeast MPP ( / E73Q). The excitation wavelength of DAC was 390 nm.
Note the differences in the range of Fluorescence. The
spectra changed from the spectrum 1 to the spectrum 2 with the addition
of MPP. a.u., arbitrary units.
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Fig. 2.
Titration of DAC- and DAC/IANBD-labeled
peptides with the MPP. The single- or double-labeled MDH- AAL
(A), MDH14A (B), MDH-AdAR (C), and
MDH-AdRA (D) (all at 0.5 µM) were
titrated with yeast MPP (0-3 µM). The fluorescence
intensities at 470 nm of DAC- and DAC/IANBD-labeled peptides
(circles and squares, respectively) were plotted
as a function of the concentration of MPP. The solid lines
are nonlinear least-squares fits of the plots to the equation,
F = (Fmax × [Enz])/(Kd + [Enz]), where F,
Fmax, and [Enz] represent increased
fluorescence intensity, the calculated maximum of F, and the
enzyme concentration, respectively. The excitation wavelength of DAC
was 390 nm. a.u., arbitrary units.
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Introduction of IANBD into the peptides led to a drastic suppression of
increase in DAC fluorescence through FRET in all of the peptides
studied (Fig. 2). In the titration of DAC- or DAC/IANBD-labeled peptides with MPP, the fitting curves showed a biphasic nature (Fig.
2). Because fluorescence anisotropy change of the peptides was
saturated at a stoichiometric amount of the enzyme (data not shown),
the biphasic fluorescence change in the titration experiments might be
due to increased scattering by increasing the concentration of
the enzyme. For calculation of FRET efficiency, E, the
fluorescence intensities, FD and
FDA for DAC- and DAC/IANBD-labeled peptides, respectively, were taken by extrapolating the second phase curve to the
ordinate. The calculated FRET efficiencies of all the peptides showed a
range of 80-85% (Table II). The quantum
yield, Q, of the fluorescence donor DAC showed a gradual
decrease with the increasing length of the peptides; this also
resulted in a decrease in the distance at which 50% FRET occurred
between the donor-acceptor pair, R0, suggesting
that the mobility of the N-terminal portion of the peptides bound to
MPP increases with length of the presequence. Taken together with the
parameters described above, the distances of the donor-acceptor pair,
R, were substantially the same among all of the peptides
measured and were calculated to be ~28 Å (Table II); this indicates
that the distance between the N-terminal end of the peptides and
cysteine residue at position +4 is fixed, irrespective of the length of
the intervening sequence between the proximal and distal arginines. Our
findings suggest that the distal basic amino acid interacts with a
specific site, probably the acidic residue(s), in the enzyme.
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Table II
Distance between DAC at the N terminus and IANBD at position +4 in the
various peptide substrates bound to MPP
The amino acid sequences of the peptides are shown in Table I.
Kd, dissociation constant; Q, quantum
yield; R0, distance at which 50% FRET would occur
between the donor-acceptor pair; E, FRET efficiency;
R, calculated distance between the donor-acceptor pair;
R-limits, probable error limits of the distance including
the orientation factor. See "Experimental Procedures" for the
calculation of these parameters.
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Location of Putative Sites in MPP Interacting with the N-terminal
and C-terminal Portions of the Substrate Peptide--
The similarity
of the calculated distances between donor-acceptor pairs on the
substrates might be because of compensation for changes in distance by
changes in orientation of the fluorescent dyes. To eliminate this
possibility, we next carried intermolecular FRET measurements between a
fluorescence donor in the
-MPP and the acceptor in the substrate
peptides bound to MPP. This measurement could also estimate the
location in the MPP of the N-terminal end of the peptide substrate and
the amino acid residue at +4 position from the cleavage site. We first
modified a single cysteine residue, Cys-252, in yeast
-MPP (
E73Q)
by CPM, as the fluorescent donor. The histidine-tagged
E73Q
partially purified by nickel-chelating column chromatography was
reacted with CPM (Fig. 3, lane
1). When CPM-labeled
E73Q was purified further with Q-Sepharose
chromatography (Fig. 3, lane 3), a single band was obtained
by both Coomasie Blue staining and UV transillumination. To confirm the
specificity of the labeling to the cysteine residue, the labeling with
CPM was done with a mutant enzyme,
E73Q/C252S, in which the single cysteine residue was substituted to serine. No visible band in UV
transillumination was obtained with the mutant enzyme (Fig. 3,
lanes 2 and 4), confirming the specific labeling
with CPM of Cys-252 in
-MPP.

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Fig. 3.
Specific modification of Cys-252 in
-MPP by CPM. A Coomassie Blue-stained gel
(left panel) and a UV-transilluminated (right
panel) SDS-polyacrylamide gel (8%) after electrophoresis are
shown. E73Q (lane 1) and E73Q/C252S (lane
2) following nickel-chelating column chromatography were reacted
with CPM. Then, each sample was finally purified by Q-Sepharose
chromatography. Lanes 3 and 4 represent the final
eluate of E73Q (6 µg) and E73Q/C252S (6 µg), respectively.
The purification and CPM labeling are described in detail under
"Experimental Procedures."
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A series of peptides labeled with IANBD amide at the N-terminal end or
at position +4 was synthesized (Table
III). For labeling with IANBD at the
N-terminal end of the peptides, a cysteine residue, instead of leucine,
was introduced at the N terminus. The
-amino group of the N-terminal
end of the peptides was acetylated to avoid the effect of the positive
charge of the
-amino group in the peptides. In intermolecular FRET
measurements, titration of the fixed concentration (0.5 µM) of CPM-labeled
/
E73Q was done with various
concentrations (typically 0-1.5 µM) of the
IANBD-labeled peptides. The emission spectrum of CPM, which has a
fluorescence maximum at 475 nm, is similar to that of DAC, and overlaps
the excitation spectrum of IANBD amide (J = 9.68 × 10
14
M
1
cm
1nm4). The quantum yield of CPM
was extremely high (0.90 ± 0.02) relative to that of DAC. The
addition of the acceptor-labeled peptides led to a decrease in the
fluorescence of CPM and a small increase in that of IANBD, which has a
peak at 545 nm (Fig. 4). Because FRET can
be detected when donor and acceptor molecules are in close proximity
(typically 10-100 Å) and fluorescence quenching by collision between
the donor-acceptor pair was negligible, under the conditions of this
measurement (data not shown), the observed decrease in fluorescence
indicates binding of the IANBD-labeled peptide to the CPM-labeled MPP
molecule. The absorption spectra of IANBD did not change with binding
to MPP. From the spectral parameters obtained, the Förster
distance, R0, was calculated to be 48.6 ± 0.3 Å. The titration curves were shown in Fig.
5 as a plot of 1
(FDA/FD)
versus [Pep], where [Pep] is the concentration of the
IANBD-labeled peptides. The Kd values calculated from the nonlinear least-squares fit of the data increased slightly relative to those of DAC-labeled peptides. In both cases of the peptides labeled at the N-terminal end and at position +4, the FRET
efficiency, E, which is calculated as the limitation value of 1
(FDA/FD),
showed essentially the same value among the peptides that have
distal arginine at different positions (Table
IV). Distances between CPM in
-MPP and
IANBD at position +4 of the peptides were calculated to be in the range
of 44 to 46 Å, whereas those between CPM in MPP and IANBD at the N
terminus of the peptides were in the range of 43 to 45 Å, irrespective
of the position of the distal arginine residue. The finding showing
that the distance between the cysteine residue in
-MPP and the N
terminus of the peptides is substantially the same among the peptides
with various lengths of intervening sequences between the
proximal and distal arginines confirmed that the distal basic amino
acid residue interacts with the specific site of the enzyme if the
distal basic residue is at least within
7 to
17 of the cleavage
site.
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Table III
Amino acid sequences of the fluorescence-labeled peptides for
intermolecular FRET measurements
Model peptide substrates were double-labeled with IANBD as the
fluorescent acceptor. The hyphen indicates the cleavage site by MPP.
The proximal and distal arginine residues are indicated by single and
double underlines, respectively. Ac indicates an acetyl group.
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Fig. 4.
Spectral change of the emission of
CPM-labeled MPP with the addition of IANBD-labeled MDH14A. The
emission spectra of CPM-labeled MPP (CPM- E73Q/ ) (0.5 µM) were taken in the presence of various
concentrations (0-1.5 µM) of IANBD-labeled MDH14A. The
excitation wavelength of CPM was 390 nm. The spectra changed from the
spectrum 1 to the spectrum 2 with the addition of the peptide.
a.u., arbitrary units.
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Fig. 5.
Fluorescence quenching of CPM attached to MPP
with the addition of IANBD-labeled peptides. Plots of the
fluorescence quenching of CPM attached to MPP, 1 (FDA/FD),
versus the concentration of the peptides labeled at position
+4 (A) or at the N terminus (B).
FDA and FD represent the
fluorescence of CPM in the presence or absence of IANBD-labeled
peptides, respectively. CPM-labeled MPP (0.5 µM) was
titrated with various concentrations (0-1.5 µM) of
IANBD-labeled MDH- AAL (squares), MDH-14A
(diamonds), MDH-AdAR (circles), and MDH-AdRA
(triangles). The excitation wavelength of CPM was 390 nm.
The fluorescence intensity at 480 nm of CPM-labeled MPP was read. The
solid lines are nonlinear least-squares fits of the plots to
the equation, 1 (FDA/FD) = (E × [L])/(Kd + [L]), where
E and [L] represent the calculated maximum of 1 (FDA/FD), with regard to
the FRET efficiency between the CPM-IANBD pair and the concentration of
the IANBD-labeled peptides, respectively. a.u., arbitrary
units.
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Table IV
Distance between CPM attached to -MPP and IANBD in the various
peptides bound to CPM-labeled MPP
The amino acid sequences of the peptides are shown in Table III. The
quantum yield of DAC, Q, and the distance at which 50% FRET
would occur between the donor-acceptor pair, R0,
were calculated to be 0.90 ± 0.02 and 48.6 ± 0.3 Å,
respectively. Kd, dissociation constant;
E, FRET efficiency; R, calculated distance
between donor-acceptor pair; R-limits, probable error limits
of the distance including the orientation factor. See "Experimental
Procedures" for the calculation of these parameters.
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 |
DISCUSSION |
We found that when mitochondrial protein precursors are bound to
MPP, distal basic amino acids in its presequences interact with the
specific site in the enzyme if the basic residues are present at
positions
7 to
17. This means that the intervening sequence between
the proximal arginine and the distal basic amino acid is flexible so
that the distal basic residue can fit into a specific binding site in
MPP. Thus, the present study is the first to propose the structure of
the presequence bound to MPP.
We generated an energy-minimized model of rat MPP (22) based on the
crystal structure of the core proteins in the bovine bc1
complex (23, 24). The two subunits form a ball with a crack leading to
the internal cavity. Functional amino acid residues predicted in our
previous works (17-19) are arranged around the cavity. In the
simulation model (Fig. 6), the distance
between Glu-73 in yeast
-MPP (corresponding to Glu-104 in rat
-MPP), which is a catalytic center (17, 18), and Glu-390/Asp-391 in
yeast
-MPP (corresponds to Glu-446/Asp-447 in rat
-MPP), which we
assumed to be residues interacting with the distal arginine in the
presequence (19), was calculated to be about 30 Å. The glycine-rich
loop in
-MPP, which is conserved among different organisms and has
been shown to be essential for MPP function, is close to the metal
binding active center in
-MPP and is located about 30 Å from
Glu-390/Asp-391 in
-MPP. These values are close to those (about 28 Å) obtained from FRET measurements between the N-terminal end and the
amino acid residue at position +4 of the peptides (Table II). On the
other hand, in the simulation model, Cys-252 in yeast
-MPP modified
with CPM seems to be located on the surface of the C-terminal domain of
-MPP and is about 50 and 40 Å away from Glu-390/Asp-391 and the
glycine-rich segment in
-MPP, respectively. Taking the uncertainty
of the predicted location of Cys-252 in the model of
-MPP into
consideration, these values are also similar to those obtained from
FRET measurement of about 45 Å from Cys-252 to the N-terminal
end and the amino acid residue at position +4 of the peptides. The
scissile bond of the presequence bound to MPP must be close to the
active center in
-MPP. Taken together, these findings lead one to
expect that the distal basic residue and the amino acid residue at
position +4 of the peptide interact with the acidic residue cluster and the glycine-rich region in
-MPP, respectively, and that the
substrate peptide in the cavity of MPP is needed to form the
loop structure, depending on the length of the intervening sequence
(Fig. 6).

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Fig. 6.
Predicted conformation of the peptide
substrate bound to MPP. Shown is the model of MPP, based on the
core proteins of the bovine bc1 complex, as described (24).
- and -MPP are shown in green and blue,
respectively. The bold line indicates the predicted
conformation of peptide substrate bound to MPP. N and
C indicate the amino and carboxyl terminus of the substrate,
respectively. The red arrow indicates the cleavage site of
substrate by MPP. Glu-390 in yeast -MPP, which corresponds to
Glu-446 in rat -MPP, and Glu-73 and Cys-252 in yeast -MPP, which
correspond to Glu-104 and Thr-280 in rat -MPP, respectively, are
shown in the space-filled presentation. The glycine-rich
segment in -MPP is shown in yellow. These residues and
segment are described in detail under "Discussion".
|
|
In the intervening sequence between the proximal arginine and the
distal basic amino acid in the presequence, several prolines and/or
glycines are usually present, and substitution of these residues for
alanine led to a reduction in cleavage efficiency (13).
Insertion of ethoxy linkage instead of peptide bonds, as a more
flexible linker, between the distal and proximal arginines led to a
2-fold increase in cleavage efficiency compared with that of the
control peptide (14). Studies using two-dimensional proton NMR combined
with circular dichroism on synthetic peptides corresponding to the
presequences of the precursor proteins have also demonstrated that
peptides to be cleaved by MPP have the potential ability to form a
helix-linker-helix structure, in which glycine and/or proline residues
serve as an
-helix-breaking linker (25-27). Taking those findings
together with the present results, one could surmise that the
intervening sequence forms a flexible loop structure and function to
aid structural elements, including the distal basic and proximal
arginine residues, in binding to multiple subsites in MPP.
During import into mitochondria, multiple proteins, including molecular
chaperones, receptors, and processing peptidases, recognize the
presequences of mitochondrial protein precursors. The formation of an
-helix of the presequence is required apparently for interaction
with these components (25-27). The NMR structure of the cytosolic
domain of Tom20, a component of the translocase complex in
mitochondrial outer membrane, with a synthetic peptide based on the
aldehyde dehydrogenase precursor has recently been resolved (28). The
peptide that forms an amphiphilic
-helix in a crack of the core
structure of Tom20 consists of four helices. The present results
indicate that structures required for targeting and processing differ
and that a flexible structure is required for the processing, although
basic residues in the presequences function as recognition signals for
both processes.
Structural convergence between MPP and thermolysin, a
Zn2+-peptidase with a typical metal-binding motif,
HExxH, has been discussed recently (29). Superimposition between
the N-terminal domain of core 1 protein of the bc1 complex
and the portion around the active site of thermolysin showed a similar
arrangement of secondary structural elements but with different
topological connections and in a reverse main chain orientation. This
structural architecture is based on four helices, which contain
metal ligands and the catalytic glutamate residue, and the neighboring
five strands of a
-sheet. A main chain of substrates of thermolysin
interacts with the edge of the
-sheet through hydrogen bonds. Like
thermolysin, the
-sheet structure around the active center of MPP
might interact with a nonhelical structure around the cleavage site of
the precursors through hydrogen bonding, to present the scissile bond
of the substrate to the active center in
-MPP. Further studies,
especially on the crystal structure of MPP, should reveal the precise
structure of the presequence bound to MPP and the mechanisms of
strict recognition and specific cleavage of precursor proteins by the enzyme.
 |
FOOTNOTES |
*
This work was supported in part by grants-in-aid for
Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (to A. I., T. O., and S. K.) and for Core Research for Evolutional Science and Technology in Japan (to A. I.).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.
To whom correspondence should be addressed. Tel. and Fax:
81-92-642-2530; E-mail: a.itoscc@mbox.nc.kyushu-u.ac.jp.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M003111200
2
In our previous works, the residues of MPP were
numbered from the N terminus of the mature protein reported in the data
base. In this studies, we numbered the residues according to the
full-length MPP precursors. For instance, His-101, Glu-104, and His-105
in rat
-MPP were represented as His-56, Glu-59, and His-60,
respectively, in our previous papers.
 |
ABBREVIATIONS |
The abbreviations used are:
MPP, mitochondrial
processing peptidase;
CPM, 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin;
DAC, 7-diethyl
aminocoumarin-3-carbonic acid;
FRET, fluorescence resonance
energy transfer;
IANBD, N,N'-dimethyl- N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine;
MDH, malate dehydrogenase;
- and
-MPP,
- and
-subunits,
respectively, of the mitochondrial processing peptidase.
 |
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