Identification of Hsc70 binding sites in mitochondrial aspartate aminotransferase

Archives of Biochemistry and Biophysics 450 (2006) 30–38 www.elsevier.com/locate/yabbi

IdentiWcation of Hsc70 binding sites in mitochondrial aspartate aminotransferase Antonio Artigues 1, Ana Iriarte, Marino Martinez-Carrion ¤ Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri—Kansas City, Kansas City, MO 64110-2499, USA Received 27 January 2006, and in revised form 21 March 2006 Available online 5 April 2006

Abstract Hsc70 binds acid-unfolded mitochondrial aspartate aminotransferase (mAAT), forming either soluble or insoluble complexes depending on the relative concentrations of the proteins. Using partial proteolysis of Hsc70–mAAT complexes in combination with MALDITOF mass spectrometry, we have identiWed several potential Hsc70-binding regions in the mAAT polypeptide. Only one mAAT peptide was found bound to Hsc70 in the insoluble complexes while nine peptides arising from eight sequence regions of mAAT were found associated with Hsc70 in the soluble complexes. Most of these binding sites map to secondary structure elements, particularly -helix, that are partly exposed on the surface of the folded structure. These results suggest that these peptide regions must not only be exposed but still in a Xexible extended conformation in the mAAT folding intermediates recognized by Hsc70. Thus, for mAAT the discrimination between native and non-native structures by Hsc70 may rely more on the level of structure of the binding sites than on their degree of exposure to the solvent in the native structure. © 2006 Elsevier Inc. All rights reserved. Keywords: Chaperones; Hsc70; Binding sites; Aminotransferase; Mass spectrometry

The 70 kDa heat shock proteins (Hsp702) perform a large variety of functions in cells related to protein folding both under normal and stress conditions, assembly and disassembly of protein complexes, protein translocation across intracellular membranes, and protein degradation [1–4]. Hsp70 appears to behave also as an anti-apoptotic factor [5]. In all instances, the chaperone action of Hsp70 relies on the ATP-dependent transient association with its substrates [2]. Biochemical and structural studies have shown that Hsp70s consist of two main domains, an N-terminal *

Corresponding author. Fax: +1 816 235 5595. E-mail address: [email protected] (M. Martinez-Carrion). 1 Present address: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA. 2 Abbreviations used: Hsp70, 70-kDa heat shock protein; Hsc70, constitutively expressed Hsp70; AAT, aspartate aminotransferase; mAAT, mitochondrial aspartate aminotransferase; pmAAT, precursor to mitochondrial aspartate aminotransferase; PAGE, polyacrylamide gel electrophoresis; MALDI-TOF, matrix assisted laser absorption desorption-time-of-Xight mass spectrometry; PSD, post source decay. 0003-9861/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.03.021

44 kDa ATPase domain [6–8] and a C-terminal 30 kDa domain containing the substrate binding site [9]. Binding and hydrolysis of ATP at the N-terminal domain allosterically modulates the aYnity and kinetics of substrate binding to the peptide-binding domain [10,11]. The recent determination of the crystal structure of an intact Hsc70 has provided a better understanding of the interdomain interactions critical for chaperone function [12]. In addition, the chaperone activity of Hsp70 is regulated by a number of co-chaperones [4]. Mitochondrial aspartate aminotransferase (mAAT) is a dimeric enzyme located in the matrix of mitochondria. Like the majority of mitochondrial proteins, mAAT is synthesized in the cytoplasm as a larger precursor form (pmAAT) containing an amino-terminal presequence peptide of 29 amino acid residues [13], which is essential for the targeting and translocation of the protein into mitochondria. Protein import into mitochondria involves the participation of a number of molecular chaperones, including members of the Hsp70 family in both sides of the mitochondrial membrane.

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Mitochondrial Hsp70 is thought to act as an import motor, contributing to the pulling of the translocated protein into the matrix [14]. On the other hand, to be imported, mitochondrial precursors must be presented to mitochondria in a non-native state and therefore their folding in the cytosol must be prevented or at least delayed. In fact, the folding of the mAAT precursor (pmAAT) synthesized in a cell-free extract is slowed down by interactions with proteins present in the extract [15]. These interactions may also protect the incompletely folded polypeptide against undesirable reactions such as aggregation and proteolysis. Cytosolic Hsp70s, in particular the constitutively expressed Hsc70, appear to play a prominent role in this chaperoning of mitochondrial precursors in the cytosol [16–18] including pmAAT [19]. Hsc70 forms complexes with nascent pmAAT synthesized in cell-free lysates although the interaction is transient and the complexes dissociate as the protein slowly folds and loses its ability to be imported by mitochondria [19]. PuriWed bovine Hsc70 also binds pmAAT during refolding in vitro from its acid-unfolded state [20], and more importantly, the pmAAT in these complexes is import-competent and can be taken up by mitochondria [21]. Thus, it is clear that the formation of Hsc70–pmAAT complexes is of great biological signiWcance. The elucidation of the speciWc peptide-binding properties of Hsp70 has turned out to be a fairly challenging problem. Early on it was suggested that the main requirement for binding to Hsp70 is that a potential protein substrate exposes hydrophobic amino acid residues normally hidden in its native structure [22,23]. Yet, even though Hsp70 proteins interact rather promiscuously with many unfolded proteins, we have shown that bovine brain Hsc70 is able to discriminate between the closely related mitochondrial and cytosolic isozymes of AAT [20], binding exclusively to the mitochondrial form. The apparent selectivity of Hsc70 toward homologous proteins according to their cellular destination may play a role in the segregation of the AAT isozymes to their respective locations following synthesis in the cytoplasm. Some of the Hsp70s are also able to bind certain folded proteins with high speciWcity [24,25]. Studies using pools of random synthetic peptides or phage peptide display libraries have shown that Hsp70s preferentially interact with heptameric peptides containing large hydrophobic and basic residues but few, if any, acidic residues [26,27]. However, the members of the Hsp70 family diVer somewhat in their sequence speciWcities. The binding motif identiWed for BiP, the endoplasmic reticulum Hsp70, is the most hydrophobic (Hy(W/X)-HyXHyXHy, where Hy is a large hydrophobic residue and X is any amino acid except charged residues) [28] while the presence of basic residues increases the aYnity for Hsc70 and DnaK, the bacterial Hsp70 homologue [29,30]. Subsequently, the substrate speciWcity of DnaK was elucidated as consisting of a core of four or Wve hydrophobic residues Xanked on both sides by regions enriched in basic residues [31]. No such consensus sequence has been proposed for Hsc70, but screening of phage display peptide libraries with bovine Hsc70 identiWed

31

peptides that frequently contained 2–4 basic residues [32,33]. The problem with most of these studies is that very few of them were aimed at identifying Hsp70-binding sites in biologically relevant full-length protein substrates [31,34,35]. Perhaps for this reason we still do not fully understand the molecular basis for substrate recognition by Hsc70, in particular in intact polypeptide substrates known to bind Hsc70 in the cell. In a previous report, we described the identiWcation of seven putative Hsc70-binding regions in the pmAAT polypeptide by measuring the ability of a collection of synthetic peptides spanning the sequence of pmAAT to inhibit the formation of complexes between Hsc70 and denatured mAAT [36]. One of the binding sites was in the presequence peptide but the other six mapped to regions of the mature portion of the protein having the lowest similarity score with the cytosolic form of AAT. Peptides corresponding to analogous positions of cytosolic AAT, which is not a substrate of Hsc70, did not bind to the chaperone. We concluded that diVerences in the primary structure are likely responsible for the selective recognition of mAAT by Hsc70. However, because of the size of the synthetic peptides (14 residues long, about twice the minimum sequence length required for binding) and the fact that consecutive peptides shared only a four-residue overlap, we were unable to pinpoint the putative heptameric-binding sequence within the peptides that Hsc70 bound. Furthermore, some potential-binding sites could have been spliced between two consecutive peptides thereby escaping detection in our competition assay. Lastly, several synthetic peptides could not be tested because of solubility problems. More importantly, from these results we could not deduce which, if any, of the putative recognition sites was involved in the binding of Hsc70 to full-length mAAT. In this study we address this question directly by analyzing the binding interactions in complexes of bovine Hsc70 and full-length unfolded rat mAAT. We have identiWed eight peptide regions that remain associated with Hsc70 following mild proteolysis of the preformed complexes. Remarkably, Wve of these regions match peptides identiWed in our previous screening study [36] and two of the new regions correspond to synthetic peptides that could not be tested because of their limited solubility in water. To our knowledge, this is the Wrst report of the elucidation of potential-binding sites directly in an intact biologically relevant substrate of Hsc70. Materials and methods Materials and protein puriWcation Aspartic acid, cysteine sulWnic acid, and -ketoglutarate were purchased from Sigma. All other reagents were of the highest purity available. Published procedures were followed for the puriWcation of pmAAT and for the preparation of mature mAAT [15]. Protein concentrations were estimated from the absorbance at 356 nm of the pyridoxal 5⬘-phosphate cofactor using an extinction coeYcient

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 D 8500 M¡1 cm¡1 and Mr D 47,315 for pmAAT and 44,600 for mAAT. Hsc70 was puriWed from bovine brain following published procedures [37] with minor modiWcations [21]. The Hsc70 preparation was over 95% homogeneous according to SDS–PAGE analysis. The functional state of Hsc70 was assessed by testing its ability to arrest refolding of mAAT [20]. Protein concentration was measured using 280nm D 47,800 M¡1 cm¡1 [10]. Stock solutions of Hsc70 were kept at 4 °C in 20 mM Tris–HCl, 10 mM -mercaptoethanol, pH 7.5, and remained active for at least 12 months. Formation of Hsc70–mAAT complexes Acid unfolding of mAAT was performed according to published procedures [38]. BrieXy, a stock solution of the enzyme in 2 mM Tris–HCl, pH 7.5, was denatured by addition of enough diluted HCl to reach pH 2.0. The Wnal protein concentration was t8.7 M. The samples were then incubated for 90 min at 25 °C, conditions that were established previously to achieve maximum unfolding [38]. To form Hsc70–mAAT complexes, aliquots of acid-unfolded mAAT were diluted with ice-cold refolding buVer (40 mM Hepes, 0.1 mM EDTA, and 1 M dithiothreitol, pH 7.5) containing Hsc70 followed by incubation for 60 min at 10 °C. The Wnal protein concentrations were 1.8 M mAAT and 1.8 M or 270 M Hsc70 for the formation of insoluble or soluble complexes, respectively. Under the conditions used to prepare insoluble complexes, 80% of the total mAAT added was recovered in the pellet following centrifugation at 20,000 rpm on a microcentrifuge [20,21]. The 20% remaining in the supernatant represents predominantly free mAAT since it can refold and recover catalytic activity [20]. Only the mAAT in the pellet was treated with trypsin to identify the peptides bound to Hsc70 in the insoluble complexes. Limited proteolysis and puriWcation of Hsc70-bound peptides For digestion of free non-native mAAT, the acidunfolded protein was diluted in refolding buVer containing 0.13 M TPCK-trypsin at 10 °C. For Hsc70-bound mAAT, 0.13 M trypsin was added to the preformed soluble or insoluble complexes prepared as described above. In both cases, trypsin digestion was performed for 30 min at 4 °C [21]. The extent of proteolysis of both chaperone and mAAT was monitored by SDS–PAGE analysis. Samples of the digested complexes were chromatographed on a Sepharose 12 size-exclusion column, using 10 mM potassium phosphate, pH 7.5 as the elution buVer at a Xow rate of 0.2 ml/min, to isolate the mAAT tryptic peptides that remain bound to Hsc70 and therefore coelute with the chaperone. For MALDI-TOF analysis, samples were diluted 20£ in 200 mM potassium phosphate, pH 2.0, and batch-extracted using POROS R2 reverse-phase media (Perseptive Biosystems) (100 l, 1 mg/ml) in 2% acetonitrile, 0.1% TFA. After washing the resin with 2% acetonitrile,

0.1% TFA three times to remove salts, the peptides were eluted with 100 l of 75% acetonitrile, 0.1% TFA. For some samples, peptides were fractionated from the POROS R2 media using increasing concentrations of acetonitrile (2, 25, 50, 75, and 90%) in 0.1% TFA. After Speed-Vac concentration, samples were analyzed by MALDI-TOF as indicated below. MALDI-TOF mass spectrometric analysis MALDI-TOF mass spectra were acquired on a Voyager DE PRO time-of-Xight mass spectrometer (Applied Biosystems) operating in reXectron mode, using -cyano-4hydroxycinnamicacid or sinapinic acid (10 mg/ml) as the matrix [39,40] for small mass range (500–10,000 m/z) or large mass range (10,000–45,000 m/z) analysis, respectively. Laser intensity was set at 3600–3800, depending on the sample, 250–500 laser shots were averaged for each spectrum, and 175 ns delay time was used previously to extraction of the ions in the Weld-free region of the mass spectrometer. The spectra were baseline-corrected and calibrated using as external close standards a set of peptides in mass range of 800–1000 (0.05% mass accuracy). Processing of the MALDI-TOF data was done using the software provided by the manufacturer (Data Explorer). For peak detection, we used a 10% cut-oV ion intensity relative to the most abundant ion in the mass spectrum and a minimum signal to noise ratio of 100. Monoisotopic masses were obtained utilizing the deisotoping algorithm included in the software. These masses were searched against the protein sequences of rat liver mAAT and bovine brain Hsc70, using a 0.05% error window. Peptide hits were Wltered for trypsin cleavage at lysyl and arginyl residues and therefore peptides arising from cleavage at other locations were discarded. Post source decay (PSD) analysis of selected ions was performed on a Voyager DE PRO MALDI-TOF mass spectrometer, using a build-in collision cell at the entrance of the Weld-free region. The PSD mass spectrum was divided in 12 segments, for each segment the laser power, guide wire voltage, and input band were varied to optimize fragmentation and data collection. A total of 5500 spectra were manually collected and data processing was done using the software provided by the manufacturer (Voyager 5.1) with the default parameters and calibration. Results The aim of this work was to detect sequence regions of mAAT which are directly bound to Hsc70 in the complexes formed by diluting the acid-unfolded protein in refolding buVer containing a certain amount of Hsc70. Two types of complexes were analyzed: insoluble complexes obtained by incubating equal molar concentrations of Hsc70 and mAAT, and soluble complexes prepared in the presence of an excess of Hsc70 over mAAT [21]. The approach we used was to digest the Hsc70–mAAT complexes with trypsin and subsequently identify which mAAT fragments remained

A. Artigues et al. / Archives of Biochemistry and Biophysics 450 (2006) 30–38

bound to Hsc70. This approach was viable because, in contrast to the extreme protease resistance of the native protein, mAAT complexed to Hsc70 is highly susceptible to proteolysis [21], and the binding of peptide substrates to the peptide-binding domain of Hsc70 is essentially irreversible in the absence of ATP [11]. The conditions of proteolysis were selected such that over 90% of Hsc70 remained intact whereas all of mAAT was digested (data not shown). To develop suitable experimental conditions we Wrst obtained the total tryptic peptide mass Wngerprint of denatured mAAT in the absence of Hsc70 (i.e., acid-unfolded mAAT diluted in refolding buVer containing trypsin), which includes many peptide ions in the 500–3500 m/z range and a few low abundant large peptides (78000 Da). Sequence assignment of each peptide ion was based exclusively on the exact mass measurement and on the speciWcity of the protease. We identiWed a total of 48 tryptic peptides in the 500–3500 m/z range and 8 larger peptides. For clarity of presentation we are showing only 28 of them in Fig. 1. We were able to identify small tryptic peptides from almost

33

every region of the protein sequence, except a small section in the middle of the chain spanning residues 155 to about 200. Some of the larger peptides identiWed included this sequence region (data not shown). Overall the peptides identiWed covered about 71% of the polypeptide sequence. These results indicate that the majority of the potential trypsin sites are accessible to the protease in free refolding mAAT, which is consistent with a largely unstructured conformation of the population of early folding intermediates in the sample, as we reported earlier [41]. To identify those peptides that remained bound to Hsc70 after proteolysis of the Hsc70–mAAT complexes, the digested samples of both insoluble and soluble complexes were subjected to size exclusion chromatography. The fractions containing Hsc70 (elution volume 14–15 ml) were pooled and analyzed for the presence of mAAT peptides by MALDI-TOF following extraction with a reversephase medium and 75% acetonitrile in 0.1% TFA, as described in Materials and methods. Under these extraction conditions, Hsc70 remains bound to the reverse-phase

Fig. 1. Peptide mass Wngerprint of free mAAT. (A) Mass spectrum of a tryptic digest. Trypsin hydrolysis of refolding mAAT was performed by diluting acid-unfolded protein in refolding buVer containing trypsin (0.13 M) to a Wnal protein concentration of 1.8 M. After incubation for 30 min at 4 °C, trypsin digestion was stopped by a 1:20 dilution of the sample with 200 mM potassium phosphate, pH 2.0. The resulting peptides were concentrated using a C18 ZipTip (Millipore) and extracted with a 50% saturated solution of -cyano-4-hydroxycinnmaic acid in 70% acetonitrile, 0.1% TFA. Mass spectra were recorded on a Voyager DE PRO MALDI-TOF. The instrument was operated in reXectron mode with the following experimental conditions: accelerating voltage, 20,000; grid voltage 75%; guide wire 0.01; and extraction delayed time 150 ns. The numbers on top of the single charged ions indicates the position of the peptide in the mAAT sequence [13]. Sequence assignment was done based exclusively on the exact mass measurement and the speciWcity of trypsin. The large peak at m/z of about 550 is a background ion also observed in blank samples containing no protein. (B) The relative position of the mapped tryptic peptides within the amino acid sequence of mAAT are indicated with black bars placed above the central bar representing the mAAT polypeptide (402 amino acid residues). The potential trypsin cleavage sites in the sequence are indicated by inverted triangles. In the central bar, the gray portion represents the N-terminal segment bridging the two subunits in native mAAT (residues 3–14), the black region corresponds to the sequence segments that make up the small domain (residues 15–47 and 326–410), and the white area indicates the large domain (residues 48–325) [48].

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matrix and any peptides present are recovered in the eluate. The eluate was analyzed by MALDI-TOF for the presence of small (500–10,000 m/z) and large (10,000–45,000 m/z) fragments, but peptides were detected only for the m/z range of 700–1100 (Fig. 2). The detection of very small peptide ions (6500 m/z) by MALDI-TOF, although theoretically possible, is usually not feasible because of interference from the large excess of matrix ions present. Only six potential tryptic peptides of mAAT—Wve tripeptides and one tetrapeptide—have a mass of less than 500 Da. Considering that a minimum of Wve residues from the bound peptide are buried in the Hsc70 peptide-binding channel [8], it is unlikely that trypsinolysis of Hsc70-bound mAAT will generate peptides of less than Wve residues.

Fig. 2. IdentiWcation of mAAT peptide regions bound to Hsc70. Formation of insoluble (A) or soluble (B) Hsc70–mAAT complexes, treatment with trypsin and isolation of the Hsc70-bound peptides were as described under Materials and methods. MALDI-TOF mass spectra of the tryptic digests were acquired on a Voyager DE PRO mass spectrometer as described in Fig. 1. The peptide ions identiWed as arising from mAAT are included in boxes, whereas those originating from Hsc70 are in italics. The numbers refer to the position of the peptide residues in the sequence of the respective protein. When unequivocal assignments were not possible at the resolution of the analysis (0.05%) because more than one quasi-isobaric peptide fragment can be produced by trypsin hydrolysis in diVerent regions of the mAAT sequence or in the Hsc70 polypeptide, all the possible matching sequences are included. For some peptides, several species were identiWed as a consequence of alternative hydrolysis by trypsin at consecutive lysine residues and they have been connected with dotted lines to illustrate this point.

Based on the exact mass measurement and the speciWcity of trypsin, several peptide ions detected were identiWed as originating from mAAT (see Table 1). Interestingly, only one mAAT peptide fragment was found associated with Hsc70 in the insoluble complexes (Fig. 2A). This peptide corresponds to the sequence 283VESQLK288 in the mAAT polypeptide. Thus, the insoluble complexes prepared in the presence of approximately equal amounts of Hsc70 and mAAT contain a single Hsc70 attached exclusively to the 283 Val-Lys288 region. This same peptide plus eight other mAAT peptide ions were detected associated with Hsc70 after proteolysis and chromatography of samples of soluble complexes (Fig. 2B and Table 1). This Wnding agrees remarkably well with our previous estimate that in the soluble complexes between 5 and 7 molecules of Hsc70 are bound to each mAAT polypeptide [21]. Because the assignments were based solely on the exact mass of the peptide ions and the speciWcity of trypsin, there is a certain level of uncertainty in some of them. For example, for several peptide ions (those with m/z values of 719.54, 877.06, and 1003.91, Fig. 2B) an unequivocal assignment based on the exact mass measurement was not possible at the resolution of the analysis (0.05%) because more than one quasi-isobaric peptide fragment can be produced by trypsin hydrolysis in diVerent regions of the mAAT sequence or even in one case in the Hsc70 polypeptide. For other peptide regions (207EMAAVVKKK215 and 55 KAEAQIAGK63) several putative species were identiWed as a consequence of hydrolysis by trypsin at alternative cleavage sites (Fig. 2B). One way to conWrm the identity of those peptides whose assignment was uncertain according to the mass of the ion is by analysis of the peptide fragmentation spectrum following PSD. Unfortunately, this analysis could be performed only for the peptide ion with m/z of 719.54. The low intensity and low eYciency of fragmentation of the other two ions (m/z of 877.06 and 1003.91) precluded such an analysis. Also, because the deviation of the observed mass for the 719.54 ion from the calculated mass of the closest match in the mAAT sequence (1.22 Da or 0.16%, Table 1) was the greatest of all the assignments, we decided to conWrm it with sequencing data. For the 719.54 m/z ion, the PSD spectrum shows the presence of imonium ions for most of the amino acids present in the parental ion, with the only exception of serine. Most distinctly, a complete y series of ions (y1, y2, y3, y4, and y5) permits the reading of the entire peptide sequence. In addition, several a, b, and z ions were also detected. A complete list of all identiWed fragments is shown in Fig. 3. According to these sequence data, the correct assignment of the 719.52 m/z ion is to the peptide 26RDTNSK31 in the N-terminal region of the mAAT sequence. Thus, we have identiWed without ambiguity six-binding sites for Hsc70 in the mAAT polypeptide (26Arg-Lys31, 55 Lys-Lys63, 122Phe-Arg127, 207Glu-Lys213, 259Asn-Lys266, and 283Val-Lys288, labeled as binding regions R-1, R-2, R-4, R-5, R-6, and R-7 in Table 1). The identity of two other

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Table 1 IdentiWcation of mAAT tryptic peptides bound to Hsc70 by MALDI-TOF mass spectrometric analysis Monoisotopic mass (Da) Observed 704.1 719.54 747.52 787.41 831.59 877.06 916.5 941.46 1003.91

Peptide sequencea

Binding region code

Calculated 703.81 720.76 747.93 787.89 831.99 876.11 878.02 916.06 940.07 1004.28 1003.09

283

288

VESQLK RDTNSK31 207 EMAAVVK213 56 AEAQIAGK63 122 FFKFSR127 207 EMAAVVKK214 114 VGASFLQR121 55 KAEAQIAGK63 259 NMGLYGER266 207 EMAAVVKKK215 376 EFSVYMTK383 26

R-7 R-1 R-5 R-2 R-4 R-5 R-3 R-2 R-6 R-5 R-8

Observed vs. calculated mass diVerence (Da) Absolute

%

0.29 ¡1.22 ¡0.41 ¡0.48 ¡0.4 0.95 ¡0.96 0.44 ¡1.39 ¡0.37 0.82

0.04 0.16 0.05 0.06 0.05 0.11 0.11 0.05 0.14 0.04 0.08

a

The numbering of the residues is according to the numbering of the sequence of pig cytosolic aspartate aminotransferase (412 residues) [49] inserting gaps in the sequence of mAAT (402 residues) where necessary to maximize homology. The sequences within these peptides identiWed previously as Hsc70binding regions by screening a synthetic peptide library [36] are underlined.

peptide segments (114Val-Arg121 and 376Glu-Lys383, binding regions R-3 and R-8 in Table 1) could not be established with certainty. For the 877.06 ion the two possible assignments have the same relatively high deviation between observed and calculated mass (0.11%, Table 1). For the 1003.91 ion, the calculated mass of one of the possible peptide sequences (207Glu-Lys215, calculated mass 1004.28 or 0.04% diVerence) is closer to the observed mass than the other (376Glu-Lys383, calculated mass 1003.9 or 0.08% diVerence). However, this diVerence is not enough evidence to select one assignment versus the other. As we indicated above, the deviation between the observed and calculated mass of the only assignment that we conWrmed by analysis of its PSD fragmentation spectrum was 0.16% of the peptide mass, the greatest of any assignment reported in this study. Nevertheless, it is very likely that these two sequence regions are also involved in the interaction of mAAT with

Hsc70 for several reasons. First, these two mAAT sequences were identiWed previously as Hsc70-binding sites by screening a synthetic peptide library covering the mAAT sequence [36]. Furthermore, one of them (114Val-Arg121) is contiguous to the peptide 122Phe-Arg127 whose identity was established without uncertainty. The other peptide ions detected in the Hsc70-containing fractions from insoluble complexes (Fig. 2A) could not be mapped to any mAAT sequence region but rather to several sections of the Hsc70 protein. They are indicated in Fig. 2 in italics. One of three possible matches for the 1003.91 ion detected bound to Hsc70 in the soluble complexes is also an Hsc70 peptide (262Arg-Arg269, Fig. 2B). Coincidentally, this peptide corresponds to the same Hsc70 region as one of the peptides found in the insoluble complexes (265Thr-Arg269, Fig. 2A). Even under the mild digestion conditions used in these experiments, approximately

Fig. 3. PSD mass spectrum of the 719.5 m/z peptide ion. The ion at m/z 719.5 could be assigned to two possible mAAT sequences, 26Arg-Lys31 or 236AspArg241. The correct assignment of this ion to the 26Arg-Lys31 peptide region in the mAAT sequence was established by analysis of the peptide fragmentation spectrum following PSD. PSD spectra were obtained on a Voyager DE PRO mass spectrometer. A total of 5500 spectra were obtained and all segments were stitched using the software provided by the manufacturer. The assignment of ions to each peptide fragment is indicated by the appropriate letters above each ion and in the peptide sequence. [M + H]+1, un-fragmented parent peptide.

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10% of Hsc70 is at least partially hydrolyzed by trypsin. It appears that some of these peptide products have aYnity for Hsc70 and as a consequence they coelute with the chaperone in the size exclusion chromatographic step. This interaction probably lacks biological relevance and it may just reXect the well-known promiscuity of this chaperone regarding acceptable peptide substrates. Actually, in the solution structures of fragments of the substrate binding domain, the peptide-binding sites of both bacterial DnaK [42] and mammalian Hsc70 [43] have been found to bind residues from the C-terminal domain of the protein by intramolecular interactions. An alternative explanation for the origin of Hsc70 peptides bound to the chaperone could be the self-association of Hsc70 molecules. However, this self-association appears to be mediated by interactions through the -helical regions of the C-terminal peptidebinding domain [44,45]. The peptides we found attached to Hsc70 are located in the N-terminal ATPase domain. Discussion The ability to discriminate between native and nonnative structures of proteins is a distinctive characteristic of molecular chaperones. In this study, we present an analysis of the entire mAAT molecule for putative-binding sites for Hsc70 in an attempt to understand better the molecular basis of substrate recognition by this chaperone. By studying complexes between Hsc70 and mAAT we have identiWed eight potential-binding regions for Hsc70 in mAAT (Table 1). Five of these regions (R-3, R-4, R-5, R-7, and R8) have extensive overlap with peptides previously identiWed as possible Hsc70-binding sites by screening a library of synthetic peptides spanning the sequence of mAAT [36] (Table 1). At the time we could only speculate that those potential-binding sites within the mAAT sequence could be responsible for the observed interaction of Hsc70 with the full-length polypeptide. The data presented herein conWrm that indeed that is the case. Furthermore, because the synthetic 14-mer peptides were twice the apparent length of the Hsc70-binding motif (seven residues) [26], the exact binding sequence within a synthetic peptide could not be determined. The Hsc70-bound peptides identiWed in this work are only 6–9 amino acid residues long and therefore they allow for a much more precise deWnition of the Hsc70-binding sites. We should point out that the two new binding regions detected in this work (R-2 and R-6) map to synthetic peptides (pm-9 and pm-29) that could not be tested in our previous screening of the peptide library because of their low solubility in aqueous solutions. Thus, the present approach overcomes some of the shortcomings noted above about the library screening method. The majority of the binding regions identiWed in mAAT share some of the features thought to favor peptide binding to Hsc70 such as the presence of aliphatic or aromatic residues in the interior of the peptide Xanked on one side or the other by basic residues [29,33], but overall they are less hydrophobic than the consensus motif proposed for bind-

ing to DnaK which consists of a core of 4–5 hydrophobic residues Xanked by basic residues [31]. As shown in Table 1, most of the peptides contain one or more hydrophobic residues in the central portion of the sequence and all of them include a basic residue as C-terminus, as expected for peptides generated by trypsin digestion. Some of them also contain acidic residues, which are generally disfavored in Hsp70-binding sites, even in the Xanking regions [31]. For the most part, these acidic residues are at the margins of the peptide sequence (for example, the glutamate residue in R5, R-2, or R-8) and therefore they may not participate directly in binding to the chaperone. Finally, R-1 clearly deviates from the general pattern of Hsc70-binding sites. The assignment of this peptide was conWrmed by additional analysis of its PSD fragmentation spectrum and therefore there is no ambiguity about its identity. R-1 (26RDTNSK31) is composed exclusively of hydrophilic and charged residues, both basic and acidic. An Hsc70-binding site with similar features has been identiWed in the tumor suppressor protein p53 [35]. This binding epitope (280GRDRRTE286) has no bulky hydrophobic residues and includes two acidic residues as well as the consensus basic residues. We can speculate that depending on the chaperoning activity, Hsc70 may be able to recognize diVerent types of binding sites. When the function is to prevent aggregation and assist in the folding of nascent chains or misfolded proteins, Hsc70 may transiently bind hydrophobic sequences typically sequestered in the core of folded proteins. However, when performing a diVerent role such as helping in the translocation of proteins from the cytoplasm to intracellular organelles, Hsc70 may interact with diVerent amino acid motifs, including surface exposed sequences containing basic residues but not particularly hydrophobic such as mitochondrial presequence peptides [46,47]. Relatively stable association of Hsc70 with several sites may be necessary to maintain newly synthesized mitochondrial precursors such as the mAAT precursor in a partially folded and translocation-competent conformation long enough to allow it to engage the translocation machinery on the outer mitochondrial membrane. The location of the majority of Hsc70-binding sites close to the surface of the folded mAAT and at least partially exposed lends some support to this proposal. mAAT is an /  protein composed of two identical subunits. Each of the subunits in turn consists of two domains and an extended Nterminal arm that forms a bridge between the two subunits. Each domain contains a central sheet of  strands (seven in the large domain and four in the small domain) connected by  helices packing on both sides [48]. The potential Hsc70binding sites we identiWed are spread over the entire sequence at intervals of approximately 50 or 100 amino acid residues in both domains (Fig. 4C). Six of the eight proposed binding sites involve residues that in the native protein are folded into regular secondary structure elements, primarily -helix (R-2, R-3, R-5, and R-7). Perhaps most surprising is the scarcity of binding sites in the  sheets. Three residues of R-8 (Ser378 to Tyr380) form a short  strand (strand B2) in the small domain

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37

Fig. 4. Structure of dimeric mAAT and location of Hsc70-binding sites within the amino acid sequence of mAAT. (A) Diagram of the mAAT molecule viewed along the twofold axis with the peptide backbone of the upper subunit (subunit a) shown as ribbons and a portion of the lower subunit (subunit b) drawn as a backbone trace (orange). The ribbon diagram of the upper subunit highlights the position in the native structure of the Hsc70-binding regions R1 to R-8 as deWned in Table 1. In this view, the small domain of the upper subunit is at the upper left corner. The N-terminal (N) and C-terminal (C) ends are also labeled. (B) Close-up view to emphasize the interaction of residues from binding regions R-4 (magenta) and R-7 (green) with residues from the Nterminal end of the other subunit (orange). The diagrams were generated using PyMol (DeLano, W.L. The PyMol Molecular Graphics System (DeLano ScientiWc, San Carlos, CA, 2002)) and the X-ray structure of chicken mAAT (PDB code 7AAT) [48]. (C) The position of the peptides identiWed in this work as Hsc70-binding sites are indicated by white boxes below the bar representing the mAAT polypeptide whereas the position of synthetic peptides previously identiWed as potential Hsc70-binding sites [36] are indicated by black boxes. The N-terminal segment bridging the two subunits in the native structure of mAAT (residues 3–14) is shown as a light gray bar, the sequence segments that make up the small domain (residues 15–47 and 326–410) as dark gray bars, and the large domain (residues 48–325) is shown as a white bar. The native secondary structural elements predominating in the binding regions are indicated below the bars as  ( helix),  ( sheet), and I ( turns) [48].

but none of the potential-binding sites involve residues that form the  sheet at the core of the compact large domain (Fig. 4A). This is in contrast with the binding sites proposed for Hsc70 [29] and DnaK [31] in a variety of proteins, most of which involve residues that form  strands packed together at the core of the folded proteins. It could be argued that because of the mechanics of the in vitro experiments described here, in which the unfolded full-length mAAT is transferred to native conditions in the presence of the chaperone, the rapid collapse of the unfolded chain into a compact folding intermediate might hide potential-binding sites in the central  sheet before Hsc70 has a chance to bind them. However, none of the binding sites identiWed in mAAT by screening a synthetic peptide library mapped to the  sheet core either [36]. For mAAT the discrimination between native and nonnative structures by Hsc70 may rely more on the secondary structure of the binding sites than on their relative exposure to the solvent in the native fold. We should point out that Hsp70s bind substrates in an extended conformation [26,27] and this binding is mediated not only by side chain interactions but also hydrogen bonding with the backbone of the peptide [8]. Thus, the chaperone may be prevented from binding to potential-binding sites exposed at the surface of native proteins if the peptide backbone is inaccessible to solvent because of the formation of stable secondary structure or other reasons. Some of the hydrophobic resi-

dues found in the potential mAAT-binding sites are located either at the subunit or domain interface regions of the native dimer and therefore may be accessible in partially folded monomers but become cryptic following dimerization. For instance, several residues from R-3 (Phe118 and Leu119) and R-4 (Phe122 to Phe125) are found at hydrophobic pockets on the surface of the large domain that bind residues Trp5 and Trp6 from the extended N-terminal arm of the other subunit (Fig. 4B). The reasons for the preferential binding of Hsc70 to R-7 in the insoluble complexes are unclear but might include an intrinsic higher aYnity for this sequence, a higher Xexibility, and/or accessibility of this peptide segment in the folding intermediates recognized by Hsc70 or, more likely, a combination of both. Preliminary structural analysis of early mAAT folding intermediates by deuterium exchange in combination with mass spectrometry indicates that less than 5% of the residues in R-7 contain amide hydrogens that are protected against exchange after 10 s of refolding. Furthermore, the rate of acquisition of native-like amide hydrogen protection for R-7 (0.3 min¡1)3 is identical to the rate at which refolding mAAT loses its ability to bind Hsc70 [20]. It appears that when present in limiting amounts, Hsc70 is unable to bind mAAT once the region 3 J.A. Oses, A. Artigues, A. Iriarte, and M. Martinez-Carrion, manuscript in preparation.

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containing R-7 is properly folded. In the native dimer, R-7 maps to the central portion of an -helix on the surface of the large domain with several of its residues located at the subunit interface and in contact with the N-terminal arm from the other subunit (Fig. 4). Thus, binding of Hsc70 to this peptide region may prevent formation of the dimer and as a consequence the complete folding of the monomer. The Wndings presented in this work illustrate the diYculties encountered when trying to pin down the structural features at the basis of substrate recognition by Hsc70. No sequence consensus motif is apparent as the characteristics of the binding sites identiWed in mAAT diVer somewhat from those proposed in other proteins. It is likely that the still elusive characteristics that make a peptide segment a good substrate for Hsc70 may include its position within the context of the structure of a partially folded protein. We are currently analyzing early mAAT folding intermediates by a combination of deuterium exchange and mass spectrometry to explore a possible correlation between solvent accessibility and structural stability of discrete regions in the protein and binding to Hsc70. Acknowledgment We thank John Bollin for the puriWcation of mAAT and for technical assistance. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]

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