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J. Biol. Chem., Vol. 278, Issue 36, 34110-34113, September 5, 2003
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¶
From the
Departments of Surgery and Biochemistry
& Molecular Biology, Wayne State University School of Medicine, Detroit,
Michigan 48201 and Biophysics Research Division
and Department of Biological Chemistry, University of Michigan, Ann Arbor,
Michigan 48109
Received for publication, May 22, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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subunits of F1
(Wang, Z. G., and Ackerman, S. H. (2000) J. Biol. Chem. 275,
5767–5772). This binding action prevents the subunit from
associating with itself in non-productive complexes and fosters the formation
of a (
)3 hexamer. Following the premise that Atp11p
action is mediated primarily through a surface (as opposed to specific amino
acids, as in an enzyme active site), solving its three-dimensional structure
so that we may learn how the shape of the protein influences its function is a
high priority. Recombinant yeast Atp11p has proven refractory for such
analysis because of the presence of a disordered region in the protein. In
this article, we show that removal of 67 residues from the amino terminus of
recombinant Atp11p yields a subfragment of the protein (called
Atp11pTRNC) that retains molecular chaperone function as determined
in vitro with both a surrogate substrate (reduced insulin) and the
natural substrate (F1 ). Moreover, preliminary
15N-1H heteronuclear single quantum coherence spectra
obtained with Atp11pTRNC indicate that the truncated protein is
well ordered and amenable to structure determination by nuclear magnetic
resonance. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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33
oligomer
(2). In this structure, the
subunits and subunits are arranged, in alternate position, in a
hexamer that accounts for 
90% the mass of F1
(3,
4). The
33 structure is not observed to any
significant degree in mitochondria from atp11 mutants, instead, such
yeast accumulate F1 and subunits as large, insoluble
aggregates in the matrix of the organelle
(1). This particular phenotype
is observed also in yeast cells deficient for Atp12p, another protein of the
F1 biogenesis pathway
(1). Other instances of
F1 protein aggregation occur in yeast null mutants that are missing
either the or subunit, because such strains accumulate the lone
or subunit as aggregated proteins inside mitochondria
(1). In contrast, absence of
the subunit does not correlate with aggregation of and
subunits (5). Cumulatively,
these observations are consistent with the idea that assembles with
some sort of a soluble structure that is formed at an earlier
step in the pathway in a manner that is dependent on the actions of Atp11p and
Atp12p molecular chaperones.
Atp11p interacts with the nucleotide-binding domain of unassembled
subunits of F1 (6).
Information about the steric and chemical nature of surfaces in the protein
that mediate this binding interaction will be forthcoming once the
three-dimensional structure of Atp11p is solved. Progress in this area has
been hampered by our inability to crystallize Atp11p from preparations of the
recombinant protein. In this article, we report the purification and
characterization of an Atp11p subfragment that exhibits molecular chaperone
activities with model and natural substrates that are comparable with those of
the full-length protein. Atp11pTRNC prevents the aggregation of
insulin B-chains that are liberated when the disulfide bonds of the hormone
are reduced. In other work, the interaction of Atp11pTRNC with the
F1 subunit is revealed in protein affinity blotting studies.
Finally, we report that Atp11pTRNC shows great promise for
structure determination by nuclear magnetic resonance as indicated by
15N-1H
HSQC1 spectral data
that have been obtained with isotopically labeled protein.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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T) bp change (under-lined) in the codon for Ser107, which
introduced an XbaI restriction site unique to the mutant plasmid. The
PCR reaction (50 µl) contained 20 mM Tris-HCl, pH 8.8, 10
mM KCl, 10 mM
NH4(SO4)2, 2mM MgSO4,
0.1% Triton X-100, 0.1 mg/ml bovine serum albumin, 50 ng pPROEX/ATP11 plasmid
(DNA template), and 125 ng of each primer. After the reaction was incubated at
95 °C for 2 min and then allowed to slowly cool to room temperature, 3
µl of QuikSolution (Stratagene), 6 µl of 2 mM dTNP mixture,
and 1 µl of Pfu Turbo (2.5 units) were added to the tube, and the tube was
incubated at 68 °C for 5 min. Next, PCR was performed for 18 cycles (95
°C for 1 min, 55 °C for 1.5 min, 68 °C for 12 min). The resultant
mixture was digested with DpnI at 37 °C for 1 h to destroy the
original plasmid DNA template, after which 10 µl was used to transform
competent E. coli cells. Bacterial colonies harboring the mutant
plasmid (called pATP11/Xa) were identified by restriction analysis with
XbaI and tested in small-scale experiments for
isopropyl-1-thio--D-galactopyranoside-induced production of
recombinant protein. One such expressing clone was selected and used for
large-scale production/purification of the modified Atp11p protein
(His6Atp11pXa). DNA sequencing confirmed the correct
position of the inserted Factor Xa cleavage site and showed there were no
codon changes elsewhere in the gene for
His6Atp11pXa.
Purification of Recombinant Atp11p and Preparation of a Factor Xa
Cleavage Product, Atp11pTRNC—E. coli strain RRI served
as host for the production of unlabeled recombinant proteins. Unmodified
Atp11p was produced from the expression vector pTRC/ATP11
(8) and
His6Atp11pXa was produced from plasmid pATP11/Xa
(described above). Growth of bacterial cultures in 2x yeast tryptone
medium, induction of plasmid expression with
isopropyl-1-thio--D-galactopyranoside, cell breakage by sonic
irradiation, and purification of Atp11p proteins from clarified cell extracts
following sequential chromatographic steps through DEAE and CM Fast
Flow-Sepharose were as described by White and Ackerman
(8) with the following
modifications: the concentration of
isopropyl-1-thio--D-galactopyranoside used for induction was
0.6 mM; the buffer employed up to and including the DEAE column was
20 mM Tris-HCl, pH 7.5, 1 mM EDTA; lysozyme was omitted
from the cell-breaking step; the CM column was run with a continuous gradient
of phosphate buffer (50 to 500 mM KPO4, pH 7.5, 1
mM EDTA). Highly purified Atp11p or
His6Atp11pXa eluted from CM in 300 mM
KPO4, pH 7.5, 1 mM EDTA. Unmodified Atp11p was dialyzed
versus 20 mM Tris-HCl, pH 7.5, before being concentrated
(see below).
Atp11pTRNC was prepared from His6Atp11pXa
in the following manner. Factor Xa was added directly to the CM column eluate
at a protein/protein ratio of 1:2500 Factor
Xa/His6Atp11pXa and the solution was dialyzed against 4
liters of 20 mM Tris-HCl, pH 8.0, overnight at 4 °C. The
dialysate was adjusted to pH 8.0 (if necessary) and applied to a cobalt column
(1.5 x 5 cm; 9-ml bed volume) (TALON resin; Clontech) that had been
pre-equilibrated with 20 mM Tris-HCl, pH 8.0. The column was washed
with 60 ml of the equilibration buffer, and the flow-through was collected and
passed through a p-amino-benzamidine column (1 x 2.5 cm;
2-ml bed volume) (Sigma) to remove Factor Xa from the protein
preparation. Purified preparations of unmodified Atp11p and
Atp11pTRNC were concentrated to 10 to 15 mg/ml using a
Centricon-10, flash-frozen in liquid N2, and stored at –80
°C.
Preparation of 15N-Atp11p and
15N-Atp11TRNC—Bacterial strain BL21 was used
to propagate expression plasmids for preparation of recombinant Atp11p
proteins labeled with 15N. Transformed bacteria were grown
overnight at 37 °C in LB + ampicillin (40 µg/ml) and harvested the next
morning by centrifugation at 4000 x g for 10 min at 4 °C.
The cell pellet was resuspended with 2 ml of M9 media, inoculated into 500 ml
of M9 + ampicillin (40 µg/ml) medium, and the culture was grown at 37
°Ctoan A600 = 0.8. The cells were collected by
centrifugation and inoculated into 2 liters of M9 + ampicillin media that was
prepared with 15NH4Cl (Cambridge isotopes) in place of
unlabeled ammonium chloride. The labeled cultures were grown at 37 °C to
mid-log phase, at which point
isopropyl-1-thio--D-galactopyranoside (1 mM) was
added to induce production of 15N-labeled recombinant protein.
Purification of full-length 15N-Atp11p and the subfragment,
15N-Atp11pTRNC, from E. coli lysates followed
the methods described except that the final step involved dialysis of the
recombinant protein solutions against 10 mM
Na2PO4, pH 7.0, before concentration to 15 mg/ml in a
Centricon-10.
Nuclear Magnetic Resonance—Two-dimensional 15N-1H HSQC spectra and one-dimensional 15NT2 and 15NT1 experiments were collected on a Varian 800 MHz INOVA spectrometer at a sample temperature of 25 °C.
Protein Affinity Blots—The subunits of purified yeast F1 were resolved in an SDS-polyacrylamide gel as described previously (9) and transferred electrophoretically to nitrocellulose membrane. The blot was washed three times for 10 min each with 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1% Tween 20 (TBST) and then blocked for 30 min with a solution of TBST plus 1.5% milk. After removal of the blocking solution, the blot was overlaid with 10 ml of fresh TBST/milk that was supplemented with purified Atp11p protein (20 µg) and agitated on a rotary shaker at room temperature for 2 h. The protein solution was collected and the blot was washed with TBST three times for 10 min each. Next, polyclonal antibody against yeast Atp11p was added to the blot at 1:2000 dilution in 10 ml TBST/milk and allowed to react for 1 h. After this step, the blot was washed with TBST and then challenged with horseradish-conjugated goat anti-rabbit secondary antibody in TBST/milk for 30 min. After this reaction, the blot was washed with TBST, and immunologically reactive protein was detected with chemiluminescent reagents (Amersham Biosciences).
Miscellaneous Procedures—The light scattering assay for insulin B-chain aggregation was performed as described previously (10). Protein was estimated using the procedure of Lowry (11).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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A strategy based on known features of Atp11p was developed to allow the
preparation of a stable subfragment that might be more amenable to structure
analysis. The decision was made to remove the protease-hypersensitive NTD
(Fig. 1) from the protein (see
ref. 12). Complementation
studies have shown that the NTD is not required for the chaperone action of
Atp11p in yeast cells (12),
and the extreme hydrophilic character of this region was considered to be the
principle factor causing disorder of the protein structure. Efforts to produce
a truncated protein directly from an expression vector did not yield
detectable levels of the protein in bacterial cell lysates. This finding is
consistent with the observation that although N-terminally truncated forms of
Atp11p retain function, they are much less physically stable in vivo
relative to the full-length protein
(12). To overcome this
problem, recombinant Atp11p was engineered with a Factor Xa cleavage site
(IEGR 
) near the end of the NTD, in-between Lys106 and
Ser107 (Fig. 2A). The
protein was also modified with a His6-tag at the amino terminus. In
the purification scheme (see "Experimental Procedures"),
His6Atp11pXa is prepared from bacteria, and after
digestion with Factor Xa, truncated Atp11p (Atp11pTRNC, Ser-107
through Asn-318) is purified from the His6NTD and from any
undigested protein after passage of the digested mixture through a cobalt
column, which binds the His6 sequence
(Figs. 2, A and B). Subsequent
chromatography of Atp11pTRNC through p-amino benzamidine
agarose removes Factor Xa from the preparation and yields the highly purified
subfragment Atp11pTRNC (Fig.
2B, lane 3).
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Atp11pTRNC Prevents Aggregation of Reduced Insulin
B-chains—The molecular chaperone activity of Atp11p can be assessed
in vitro using reduced insulin as a surrogate substrate
(10). In this method, light
scattering is used to follow the progression of insulin B-chain aggregation
that occurs when the disulfide bonds in the hormone are reduced with
dithiothreitol (13).
Atp11pTRNC provides >60% protection against aggregation to
reduced insulin B-chains in this assay
(Fig. 3). This result can be
compared with the value of 70% protection that is obtained with full-length
recombinant Atp11p in similar studies
(10). Atp11pTRNC is
just five residues shorter at the amino terminus than
Atp11(
40–111)p
(12), a plasmid-borne variant
of Atp11p that has been shown to confer respiratory competence to a
atp11 yeast mutant in previous work. Hence, there is excellent
correlation of results obtained in vivo and in vitro to
support the argument that removal of the Atp11p NTD does not interfere
significantly with the molecular chaperone activity of the protein.
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Interactions between Atp11p and F1 Proteins
Studied by Affinity Blotting—Atp11p binds to the subunit of
F1 in yeast cells
(6) but not to a preparation of
recombinant mitochondrial subunit that is solubilized from bacteria
with alkaline buffer after export to the periplasmic face of the plasma
membrane.2 A likely
reason for this observation is that the alkaline-extracted protein poorly
reflects the character of the subunit that is presented to Atp11p
during F1 assembly. Protein affinity blotting has provided an
alternative method to study Atp11p interaction with its natural substrate
in vitro. In this work, purified yeast F1 was run in an
SDS gel under conditions that maximize resolution between and
subunits (9), after which the
proteins were transferred to nitrocellulose (NC) membrane
(Fig. 4). The NC was first
incubated in TBST (see "Experimental Procedures") and then treated
with TBST/milk to reduce nonspecific protein binding. Next, the blocked NC was
incubated with purified Atp11p (full-length or truncated subdomain) in
TBST/milk, and after the protein overlay solution was removed, it was
immunoblotted with antiserum against Atp11p. Atp11p (31 kDa) and
Atp11pTRNC (24 kDa) were detected with the antibody at the position
of F1 protein (55 kDa) in the blot
(Fig. 4, lanes 1 and
2). The Atp11p protein signals were not detected if immunoblotting
was done using pre-immune rabbit serum instead of anti-Atp11p serum
(Fig. 4, lane 3).
Moreover, under conditions in which the incubation step with purified Atp11p
was omitted from the experiment and the NC was instead exposed directly to
Atp11p antiserum, there was no immunoreactive signal
(Fig. 4, lane 4). Such
findings illustrate that anti-Atp11p does not recognize epitopes in the
F1 subunit; rather, the antibody lights up Atp11p bound to
the F1 subunit immobilized on the NC blot. The fact that the
subunit, which binds Atp11p in this assay, has been resolved from other
F1 subunits in an SDS gel is not inconsistent with our model for
Atp11p action, which suggests the chaperone recognizes a structural element in
its target protein (14). There
are examples in the literature of protein, transferred from an SDS gel to NC
paper, that retains or regains sufficient higher order structure to support an
interaction with nucleic acid
(15) or protein
(16,
17) applied as an overlay
solution to the blot. It is possible that either the F1
subunit is not completely denatured in the SDS gel or it renatures partially
after electrophoretic transfer from the denaturing gel and subsequent washing
of the NC membrane. A corollary finding of this analysis is that the
Atp11p:F1 binding interaction occurs in the absence of other
proteins or cofactors. The dependence of chaperone release on other factors
remains to be determined.
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Nuclear Magnetic Resonance Studies with Atp11pTRNC— 15N-labeled Atp11pTRNC (212 amino acids) yields a very well resolved 15N-1H HSQC spectrum at 25 °C, from which 200 strong main-chain cross peaks can be easily counted (Fig. 5). Their peak intensity and line-width is rather uniform. Lower level plots reveal another 10 resolved low intensity peaks. Together, the data are indicative of a structured protein, without mobile tails, and with few exchange broadened peaks. Using one-dimensional variants of standard 15N relaxation experiments, 15NT2 = 0.043 ± 0.005 s, and 15NT1 = 1.4 ± 0.2 s at 25 °C were found for resonances with a 1H chemical shift at 9 ppm (i.e. resonances belonging to the structured core of the protein). This yields a value of 14 ± 2 ns for the protein's rotational correlation time, corresponding to a molecular mass of 28 ± 4 kDa, which is near the value of 24 kDa calculated from the sequence of Atp11pTRNC. It may be inferred from the spectral data that the protein is mono-disperse at the concentrations (24 mg/ml) used in the experiment. The prospects for NMR assignment and solution structure determination of Atp11pTRNC, once uniformly triple-labeled with 15N, 2H, and 13C, are excellent. Having demonstrated here that the purified subfragment of Atp11p shows functional activities comparable with the full-length protein, there is every reason to believe that solving the solution structure of Atp11pTRNC will provide relevant information about the mechanism of this molecular chaperone.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: 421 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-8645; Fax: 313-577-7642; E-mail: sackerm{at}med.wayne.edu.
1 The abbreviations used are: HSQC, heteronuclear single quantum coherence;
TBST, Tris-buffered saline/Tween 20; NTD, N-terminal do-main; NC,
nitrocellulose.
2 D. Sheluho and S. H. Ackerman, unpublished observations.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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