©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Solution Structure of the Acetylated and Noncleavable Mitochondrial Targeting Signal of Rat Chaperonin 10 (*)

(Received for publication, September 8, 1994; and in revised form, October 27, 1994)

Jackie A. Jarvis (1)(§) Michael T. Ryan(§) (2)(¶) Nicholas J. Hoogenraad (2) David J. Craik (1) Peter B. Høj (2)(**)

From the  (1)School of Pharmaceutical Chemistry, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia and the (2)School of Biochemistry, La Trobe University, Bundoora, Victoria, 3083, Australia

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Chaperonin 10 (Cpn10) is one of only a few mitochondrial matrix proteins synthesized without a cleavable targeting signal. Using a truncated form of Cpn10 and synthetic peptides in mitochondrial import assays, we show that the N-terminal region is both necessary and sufficient for organellar targeting in vitro. To elucidate the structural features of this topogenic signal, peptides representing residues 1-25 of rat Cpn10 were synthesized with and without the naturally occurring N-terminal acetylation. ^1H NMR spectroscopy in 20% CF(3)CH(2)OH, H(2)O showed that both peptides assume a stable helix-turn-helix motif and are highly amphiphilic in nature. Chemical shift and coupling constant data revealed that the N-terminal helix is stabilized by N-acetylation, whereas NOE and exchange studies were used to derive a three dimensional structure for the acetylated peptide. These findings are discussed with respect to a recent model predicting that targeting sequences forming a continuous alpha-helix of more than 11 residues cannot adopt a conformation necessary for proteolysis by the matrix located signal peptidases (Hammen, P. K., Gorenstein, D. G., and Weiner, H.(1994) Biochemistry 33, 8610-8617).


INTRODUCTION

Of the several hundred proteins found in mitochondria, only a small subset (13 in mammals) are encoded by mitochondrial DNA. The remaining nuclear encoded proteins are synthesized on cytosolic ribosomes and transported into their correct mitochondrial compartment because of specific topogenic signals (for review see Hartl et al., 1989). Signals for targeting to the matrix have been characterized in considerable detail. They are usually located at the N terminus of the protein and typically consist of 15-40 amino acids, which generally are proteolytically removed by matrix-located proteases. These transient topogenic signals (referred to as presequences) have been defined in a large number of mitochondrial precursors, and although no apparent sequence homologies have been established, they are rich in hydrophobic and basic residues. The absence of sequence homologies suggests that recognition of the topogenic signals by the mitochondrial import and processing apparatus is caused by a common structural feature, most likely an amphiphilic positively charged structure (von Heijne, 1986).

A small number of mitochondrial matrix proteins, namely rhodanese (Miller et al., 1991), 3-oxoacyl-CoA thiolase (Amaya et al., 1988), the beta-subunit of the human electron transfer flavoprotein (Finocchiaro et al., 1993), the mitochondrial ribosomal protein YmL8 (Matsushita and Isono, 1993) and chaperonin 10 (Rospert et al., 1993; Ryan et al., 1994) are unique because sequence data have established that the targeting signal remains in these proteins following import. Little is known about these topogenic signals, and although they are believed to reside within the N-terminal region, this has only been demonstrated experimentally for the 14-16 N-terminal residues of 3-oxoacyl-CoA thiolase (Arakawa et al., 1990).

We previously demonstrated that rat Cpn10 (^1)is synthesized without a cleavable presequence and yet is imported into mitochondria in a fashion characteristic of archetypical mitochondrial precursors (Ryan et al., 1994). We noted that the N-terminal sequence possesses a high amphiphilic potential between residues 1 and 15 and considered this to act as a mitochondrial targeting signal. It was further suggested that the in vivo acetylation of Ala^1 enhances the efficiency of this signal through an increase of the helical and amphiphilic potential, which in turn augments the ability of the protein to interact with and traverse the mitochondrial membrane.

In this study we demonstrate that the targeting information resides within the N-terminal region of mature Cpn10. To further characterize this topogenic signal, peptides representing the first 25 amino acids of Cpn10 were synthesized with (Ac-Cpn10) and without (Cpn10) N-terminal acetylation. The peptides inhibited mitochondrial import of pre-ornithine transcarbamylase (p-OTC), and their structures in aqueous trifluoroethanol (20% v/v) were determined using NMR spectroscopy. Both peptides adopted stable helix-turn-helix motifs. An increased N-terminal helix stability in the acetylated peptide (Ac-Cpn10) suggests that this post-translational modification assists mitochondrial import, although to only a subtle extent.


MATERIALS AND METHODS

Peptide Synthesis and Preparation

The peptide representing the 25-residue N-terminal sequence of rat chaperonin 10 (Cpn10) was synthesized by standard solid phase methods (Merrifield, 1963). The amino acid sequence, which included a cysteine at the C terminus to provide a linker mechanism for separate biochemical studies, was as follows (Ryan et al., 1994): ^1Ala-Gly-Gln-Ala-Phe-Arg-Lys-Phe-Leu-Pro-Leu-Phe-Asp-Arg-Val-Leu-Val-Glu-Arg-Ser-Ala-Ala-Glu-Thr-Val-Cys-NH(2).

The analogous peptide was also synthesized with N-acetylation (Ac-Cpn10). Both peptides were purified to homogeneity by reverse-phase HPLC, and their identity was confirmed by amino acid sequencing, amino acid analysis, and electrospray mass spectrometry ((M(r) Cpn10: 2923.4 ± -0.4; expected 2923.4) (M(r) Ac-Cpn10 2965.3 ± 0.2; expected 2965.4)).

Preparation of I-Cpn10 and I-Cpn10

In procedures to be reported elsewhere (^2)a protein mixture consisting of authentic Cpn10 (amino acids 1-101, Cpn10) and a form truncated at its N terminus (containing amino acids 31-101, Cpn10) was isolated from porcine liver. For radiolabeling, 20 µg of this mixture (in 20 µl of 100 mM Tris-Cl pH 7.5, 10 mM MgCl(2), 10 mM KCl) was mixed with 20 µl of 100 mM sodium phosphate, pH 7.4, and transferred into an Iodogen (Pierce) coated tube. Iodination was initiated by the addition of NaI (500 µCi in 5 µl, 15.2 mCi/µg, Amersham Corp.) and allowed to proceed for 10 min before the addition of 400 µl of 50 mM sodium phosphate, pH 7.0, and desalting on a Sephadex G-15 column (Pharmacia Biotech Inc.) equilibrated in the same buffer.

Mitochondrial Import Studies

S-p-OTC and ^3H-p-OTC were synthesized as per Lingelbach et al.(1986). Mitochondrial import reactions and associated manipulations were performed as described previously (Peralta et al., 1993).

For import competition studies using Ac-Cpn10 and Cpn10, peptides in 10 µl of 20% (v/v) isopropanol were mixed with freshly synthesized [S]p-OTC in 10 µl of rabbit reticulocyte lysate (Promega) and added to rat liver mitochondria (50 µg of protein) in 40 µl of import buffer. Import of [S]p-OTC proceeded at 30 °C for 15 min before mitochondria were pelleted and subjected to SDS-PAGE in 12% Tris-glycine gels (Fling and Gregerson, 1986) followed by PhosphorImage analysis (Molecular Dynamics).

Before import reactions, porcine I-Cpn10 and I-Cpn10 were denatured by the addition of saturating amounts of urea. Four µl (6000 cpm) were withdrawn and mixed simultaneously with ^3H-p-OTC in 20 µl of rabbit reticulocyte lysate plus rat liver mitochondria (100 µg of protein) in 76 µl of import buffer. Import proceeded at 30 °C for 60 min before electrophoresis in Tris-Tricine gels (Schägger and von Jagow, 1987) and quantitation by PhosphorImage analysis.

NMR Experiments

The Cpn10 peptides were made up for NMR experiments at a concentration of 3-4 mM in 20% deuterated trifluoroethanol (d(3)-TFE), 10% D(2)O, water (Cambridge Isotope Laboratories). For amide exchange studies, the peptides were dissolved in 20% d(3)-TFE, D(2)O. The pH values were in the range of 3.5-4.5.

^1H NMR spectra were recorded on Bruker AMX 300, 500, and 600 spectrometers. Two-dimensional NMR spectra were recorded in the phase-sensitive mode using time-proportional phase incrementation for quadrature detection in the f(1) dimension (Redfield and Kunz, 1975; Marion and Wüthrich, 1983). The water proton signal was suppressed by low power irradiation during the relaxation delay (1.8 s) and during the mixing time of NOESY experiments. Spectra were referenced to the residual proton signal of d(3)-TFE at 3.96 ppm, calibrated externally using 3-(trimethylsilyl)-1-propanesulfonic acid (TSP).

TOCSY experiments were recorded using an MLEV-17 mixing scheme (Bax and Davis, 1985) and with mixing times of 120 ms. Double quantum-filtered correlation spectroscopy experiments (Rance et al., 1983) were used to measure ^3J coupling constants and corrected for line width contributions of the dispersive peaks using a peak deconvolution routine (Marion and Wüthrich, 1983). Line widths were typically 5 Hz. NOESY experiments were acquired at 500 and 600 MHz with mixing times of 250 ms.

Two-dimensional experiments were collected over 4096 complex data points. Usually 512 increments of 48 scans were acquired over a spectral width corresponding to 11 ppm for both dimensions. Up to 64 scans/increment were acquired for less sensitive NOESY experiments, and only 8 scans/increment and 256 slices were required for diagnostic TOCSY experiments. Up to 800 increments were collected for improved resolution in the f(1) dimension in the correlation spectroscopy and NOESY experiments.

The data were processed on a Silicon Graphics (SGI 4D/30) computer using the UXNMR software package (version 1.1). The f(1) dimension was zero-filled to 4096 real data points, with both dimensions being multiplied by a QSINE function prior to Fourier transformation. A GM window function with a Gauss/Lorentz function coefficient of 0.3 and line broadening of 8 Hz was applied to some NOESY spectra to improve peak resolution for assignment purposes. Polynomial base-line correction was used to improve the appearance of the spectrum.

NOE cross-peak intensities were measured using the integration function within the Felix software package (Hare Research, Inc.). These were classified as strong, medium, and weak corresponding to interproton distance restraints of 1.8-2.7, 1.8-3.5, and 1.8-5.0 Å respectively (Clore et al., 1986). Methyl cross-peak intensities were divided by 3, and appropriate pseudoatom corrections were applied to nonstereo-specifically assigned methyl and methylene protons (Wüthrich et al., 1983).

Structure Calculations

Three-dimensional structures were determined using the experimentally observed NOE constraints in a simulated annealing and energy minimization protocol using the program XPLOR (version 3.0., Brünger, 1992) on an Iris 4D/30 workstation (Silicon Graphics Inc., Mountain View, CA). In the first stage of the calculation 107 inter- and 96 intra-NOE distance constraints were applied to a template structure with randomized Ø and angles and extended side-chains to generate a set of 30 structures (Nilges et al., 1988). For final refinement of the structures, a restrained Powell energy minimization was applied to each structure using a force field based on that of the program CHARMM (Brooks et al., 1983). Structure superpositions were accomplished using Insight (Biosym) on a 4D/220 GTX workstation.


RESULTS

The Mitochondrial Targeting Information Is Retained within the N-terminal Region of Cpn10

In a separate project we purified both authentic porcine Cpn10 (Cpn10) and a truncated form containing only amino acids 31-101 (Cpn10). This allowed us to test whether the mitochondrial targeting information of Cpn10 is contained within its N-terminal region as suggested from helical wheel projections (Ryan et al., 1994). Thus, I-Cpn10 and I-Cpn10 were prepared and subjected to mitochondrial import. As seen in Fig. 1, only the full-length Cpn10 species was imported into mitochondria and was inaccessible to proteinase K (lane 3). As expected, import requires a membrane potential since 2,4-dinitrophenol completely abolishes the process (lane 4). In contrast, not only did the truncated form of Cpn10 fail to be imported into a protease-protected compartment, but it also appeared to have lost its affinity for mitochondria (lane 2). The experiments shown in Fig. 1were performed in the presence of ^3H-p-OTC, the import of which was unaffected by the presence of the iodinated Cpn10 species (data not shown). It can therefore be concluded that the import apparatus was fully operational and that the lack of I-Cpn10 import was caused by the absence of a targeting signal rather than saturation of the import apparatus. Hence a region within amino acids 1-30 of Cpn10 contains information essential for targeting.


Figure 1: The N-terminal region of Cpn10 is required for import into mitochondria. A, A protein mixture consisting of porcine Cpn10 (Cpn) and amino-acids 31-101 of Cpn10 (Cpn) was radiolabeled with I (lane 1). The mixture was denatured in 8 M urea, mixed with rabbit reticulocyte lysate, and then incubated with mitochondria for 60 min at 30 °C (lane 2). The imported products were inaccessible to proteinase K (lane 3). 2,4-Dinitrophenol (DNP) treatment prior to and proteinase K digestion subsequent to the import reaction were also performed (lane 4). [^3H]p-OTC was used as a control protein in each import reaction (data not shown). Import reactions were analyzed by Tris-Tricine SDS-PAGE and PhosphorImaging (Molecular Dynamics). B, The radiolabeled bands shown in panel A were quantitated, and the percentage of starting material imported into mitochondria was calculated.



In order to confirm the targeting properties of the N-terminal region of Cpn10, a peptide corresponding to Ala^1-Val was synthesized with (Ac-Cpn10) and without N-terminal acetylation (Cpn10) and used in competitition studies with the mitochondrial precursor protein p-OTC. Both peptides were shown to inhibit the mitochondrial import of p-OTC and to a similar degree (Fig. 2). Whereas the concentrations of Cpn10 peptides required for effective competition were relatively high although not uncommon for mitochondrial targeting sequences (Hoyt et al., 1991; Pak and Weiner, 1990), this inhibition appears specific since 100 µM concentrations of an unrelated peptide (myosin light chain kinase substrate; KKRAARATSNVFA) had no effect (data not shown). Taken together, the evidence shows that the N-terminal region of Cpn10 is not only necessary but also sufficient for targeting to mitochondria.


Figure 2: Both Ac-Cpn10 and Cpn10 inhibit p-OTC import. A, 0-100 µM of Ac-Cpn10 or Cpn10 as indicated were incubated with mitochondria (100 µg of protein) in the presence of S-labeled p-OTC in a total volume of 100 µl. Import was performed for 15 min at 30 °C, and reactions were terminated by centrifugation and addition of SDS-PAGE sample buffer to the mitochondrial pellet. B, imported [S]p-OTC was quantitated by PhosphorImage analysis following Tris-glycine SDS-PAGE. 100% import refers to the level of import in the absence of competing peptides.



Recording of NMR Spectra and Their Assignment

The Cpn10 peptides contain many hydrophobic residues and have limited solubility in water. Spectra were therefore recorded in 20% TFE, water solutions, which provide a suitable mimic of the cellular environment (Rizo et al., 1993) and adequately solubilized the peptides. Spectra obtained at peptide concentrations between 0.5 and 4 mM displayed no line-broadening effects and no apparent chemical shift changes except for small downfield shifts for the alphaHs of Val or Val. The peptides did not show any tendency to aggregate under the experimental conditions. This observation is supported by preliminary analytical ultracentrifugation experiments, which indicate a monomeric state of these peptides in aqueous solution (data not shown).

Spectral Assignment

The spectra were assigned from examination of TOCSY and double quantum-filtered correlation spectroscopy spectra in which scalar coupling patterns were used to identify the amino acid residue side-chain patterns (Wüthrich et al., 1986). Fig. 3shows a region of the TOCSY spectrum for Ac-Cpn10 containing connectivities between alphaH signals and side-chain signals for each residue. Through space NOESY interactions allowed sequential connectivities to be determined from examination of both alphaH-NH and NH-NH spectral regions (Fig. 4). Signal dispersion was good at room temperature, and the only region containing overlap involved residues 22-24, for which the sequential connectivities in the NOESY spectrum were unclear.


Figure 3: Region of a 120-ms TOCSY NMR spectrum of Ac-Cpn10 (20% TFE, 120-ms mixing time, 25 °C, 500 MHz) showing the assignments of intraresidue side-chain connectivities.




Figure 4: NH-alphaH (upper panel) and NH-NH (lower panel) regions of a 250-ms NOESY NMR spectrum of Ac-Cpn10 (20% TFE, 250-ms mixing time, 25 °C, 500 MHz) showing intraresidue and sequential connectivities for each residue in the amino acid sequence.



Chemical Shift Values

The chemical shifts for all Ac-Cpn10 resonances in 20% TFE are listed in Table 1. The chemical shifts for the side-chain protons of the nonacetylated peptide were very similar, and only NH and alphaH proton shifts are reported. Fig. 5, A and B, illustrates the deviations from random coil shifts for the two peptides. A trend toward upfield shifts is clear in both peptides between residues 2 and 7 and also between residues 10 and 22. This is highly indicative of some population of turn-like or helical structures existing within these regions (Wüthrich, 1986; Wishart et al., 1991, 1992). In addition to structural effects, the upfield shift at the N-terminal residue and the downfield shift at the C-terminal residue reflect inductive effects of the end groups. The trend of upfield alphaH shift deviations is greatest for the latter of the two specified regions, suggestive of a high helical propensity. The alphaH shift deviations in the first region are generally not as large. Of particular interest, however, is the effect of N-acetylation on the conformation of Cpn10 in the first region. Fig. 5C shows the chemical shift changes brought about by acetylation. Whereas the N-terminal alphaH shift is moved in a downfield direction upon acetylation, as would be expected from the removal of the positively charged group, all other alphaH shifts between residues 2 and 10 are moved upfield. This difference is consistent with the N-terminal region showing greater helical or turn-like character in the acetylated derivative.




Figure 5: Deviation of Cpn10 (a) and Ac-Cpn10 (b) alphaH shifts from their ``random coil'' values (Wüthrich, 1986) in 20% TFE at 25 °C (-). The upfield trend between residues 3 and 7 and residues 10 and 22 is indicative of an alpha-helical conformation within these regions. Panel c shows the difference between the alphaH chemical shifts upon acetylation of Cpn10 (-).



^3J Couplings Constants

The ^3J coupling constants measured for Ac-Cpn10 and Cpn10 are listed in Table 2. These values help to define the secondary structural elements along the sequence (Kline et al., 1988; Dyson and Wright, 1991; Wüthrich, 1986). Most values are close to 6-7 Hz, indicating that a degree of conformational flexibility exists within these peptides. Slightly smaller coupling constants occur for all alanine residues, possibly reflecting local helix promotion conferred by this residue (Lyu et al., 1990; O'Neil and DeGrado, 1990). In both Ac-Cpn10 and Cpn10 there are also coupling constants of less than 6 Hz between residues 1 and 6. This could reflect an increased helical propensity in this region of the peptides. In both peptides a large coupling occurs at Phe^8, which is indicative of some type of turn being present, and interestingly, coincides with the termination of the first putative helix. There is no significant difference between the coupling constants of the acetylated and nonacetylated peptide.



NH Exchange

Upon transferring the peptides into 20% d(3)-TFE, D(2)O, one-dimensional, and two-dimensional NMR spectra were acquired to follow the exchange of the NH resonances at room temperature. A TOCSY spectrum run between 30 and 60 min after dissolution revealed that the three slowest exchanging NHs were those of residues 15, 16, and 17. These residues correspond to those showing large upfield shift deviations for their alphaH protons and coincide with the center of the second putative helix. Their position is consistent with a hydrogen bond-stabilized alpha-helix occurring in this region. The same three NHs were identified for both Ac-Cpn10 and Cpn10, suggesting that acetylation does not drastically alter the conformation of the Cpn10 peptide in this region.

Through Space NOE Connectivities

Fig. 6illustrates the relative sizes of the intra- and interresidue NOE connectivities for Cpn10 and Ac-Cpn. Most cross-peaks were well resolved, and their volumes could be readily categorized into strong, medium, and weak groupings. Despite this conservative classification, trends were apparent in the volume measurements that could be used to derive structural information. The observation of strong d(i, i) relative to d(i, i + 1) NOE connectivities is highly indicative of a helical conformation in which the intraresidue alphaH-NH distance is 2.4-2.6 Å and the sequential alphaH-NH distance is 3.5 Å (Dyson and Wright, 1991; Wüthrich, 1986). This is the case for both peptides between residues 13 and 24, in agreement with previous indications of helix in this region.


Figure 6: Summary of NOE connectivities measured for Cpn10 (a) and Ac-Cpn10 (b) in 20% TFE, 25 °C. The intensities of the NOE cross-peaks are indicated by the thickness of the line, grouped into strong, medium, and weak. Overlapping and, therefore, ambiguous cross-peaks are indicated by an asterisk. Connectivities not expected to be present because of the lack of the specified protons (i.e. Pro NH) are shaded. ^3J scalar coupling constants measured as greater than 8.0 Hz () or less than 6.0 Hz (down triangle) are indicated, as are amide protons identified as exchanging slowly with solvent (bullet).



In Ac-Cpn10, d(i, i) NOEs are also generally stronger than d(i, i + 1) NOEs in the stretch between residues 2 and 8, helping to confirm that a helix also exists in this region of the peptide. In the equivalent region of Cpn10, however, the d(i, i + 1) NOE connectivities are, on average, stronger, suggesting a significant population of random conformations is present (Dyson and Wright, 1988). It is therefore likely that a helix in Cpn10 between residues 2 and 8 is less stable than its counterpart in Ac-Cpn10. Medium strength connectivities between sequential NHs were observed along most of the length of both peptides. This is consistent with the peptides showing helical conformations.

The observation of medium range NOE connectivities, particularly the series of d(i, i + 3) and d(i, i + 3) NOEs, show conclusively that both peptides have a high propensity to form an alpha-helix between residues 11 and 22 (helix II) and another between residues 3 and 8 (helix I). d(i, i + 2), d(i, i + 4) and d(i, i + 2) NOE connectivities are also diagnostic of the presence of helical structures (Wüthrich, 1986). NOE signals from residues 22-24 fell in particularly overlapped regions of the spectra, and NOEs that may have been present were not observed. The extent of helix II is therefore not precisely determined. Also of note is the NOE data in light of the coupling information obtained for the two peptides. The large ^3J coupling constant, measured for residue 8 in both peptides, coincides with the apparent end of helix I. It is likely that a turn interrupts the structure at this point. The NOE information is consistent with this, although there is insufficient data to define the type of turn that may be present.

Simulated Annealing and Energy Minimization Using NOE Distance Constraints

The NMR data indicate that whereas the peptides' structures are likely to exist in a state of flux between well structured and disordered forms, there is a high propensity to form a helix-turn-helix motif under the adopted solution conditions. For Ac-Cpn10 in particular, for which there was evidence for greater stabilization of helix I, it was of interest to derive the three-dimensional structure that was consistent with all of the experimental NMR data, an aspect which has not been reported for any other mitochondrial targeting signal. It was rationalized that if the NOE distance restraints were inconsistent with a single predominant conformation, this would be apparent from the poor definition and convergence of the structures obtained.

A family of 30 structures was calculated using the NOE-based interproton distance restraints in a simulated annealing/energy minimization protocol. 107 inter- and 96 intraresidue constraints, as well as three hydrogen bond distance constraints were applied in these calculations to NHs of residues 15, 16, and 17. Torsion angle constraints were not applied since coupling constant measurements, while displaying a low trend at the N terminus, were neither below 5 Hz nor well above 8 Hz (Wagner et al., 1987). The derived structures were consistent with the experimental constraints, to within 0.1 Å for 93% of the NOE constraints in all 30 structures. No NOE constraint in any structure was violated by greater than 0.5 Å. Structural convergence was also assessed from the calculation of angular order parameters (AOPs) for each amino acid residue in the sequence. These are a measure of the correlation of the and angles as a score out of 1 (Detlefsen, 1991). Fig. 7shows these values calculated for the 10 derived Ac-Cpn10 structures with lowest NOE energies. The AOP is close to 1 between residues 11 and 20, indicating the good definition of the structure in the region of helix II. High AOPs are also observed for the region comprising residues 3-7, indicating a good convergence in the region of helix I. As expected for a proline residue, a high AOP is also observed for the angle of residue 10. AOPs are low toward the C terminus of the peptide, indicating a lack of structural constraint in this region. For residues 22-24, this may not be caused by a lack of structure but by a lack of NMR data for this region. The variation in structural definition may be seen by noting that the average pairwise root mean square deviation over backbone atoms for the helical regions are 1.38 and 0.89 Å, whereas it is 4.89 Å over the molecule as a whole.


Figure 7: Angular order parameters for backbone torsion angles of the 10 structures calculated for Ac-Cpm10 best satisfying the NOE distance constraints. For each residue, Ø and are shown as filled and empty bars, respectively. The convergence to a common backbone configuration is seen for the region between residues 3 and 8 and, particularly, between 10 and 21.



Fig. 8shows two superpositions of the 10 lowest NOE energy structures, one in which residues 2-7 are superimposed and the other in which residues 10-20 are superimposed. The two helices cannot be simultaneously overlaid because of the flexible linker region between residues 7 and 10. The set of structures confirms that the NOE distance constraints are consistent with the helix-turn-helix motif qualitatively observed and is a visual aid for better understanding the properties of this region of Cpn10 containing the mitochondrial targeting signal.


Figure 8: Superposition of backbone atoms of the 10 structures calculated for Ac-Cpn10 best satisfying the NOE distance constraints. The structures are superimposed over residues 2-7 (a) and 10-20 (b), corresponding to helices I and II. No structural restraint is observed for other regions of the molecule.




DISCUSSION

A small proportion of nuclear encoded mitochondrial proteins appear to be synthesized without a cleavable presequence. In this report we investigated one of these proteins, namely chaperonin 10 from rat liver, and showed that the mitochondrial targeting information resides within the first 30 amino acids of the protein (Fig. 1). Thus, like 3-oxoacyl-CoA thiolase (Arakawa et al., 1990) and most likely rhodanese (Zardeneta and Horwitz, 1992), the noncleavable mitochondrial targeting signal of Cpn10 is located at the N terminus.

To further characterize the topogenic signal of Cpn10, two peptides (Ac-Cpn10 and Cpn10) were synthesized and structurally analyzed by NMR in the hope that essential structural features required for mitochondrial targeting eventually can be identified from a data base of targeting sequence structures. Additionally comparison with cleavable targeting sequences may help delineate structural features, if any, required by processing peptidases.

The NMR studies showed that Cpn10 contains a helix-turn-helix motif in 20% TFE at 25 °C and that N-terminal acetylation increases the helical propensity of the first helix. The evidence, derived from alphaH chemical shifts, coupling constants, and NOE (^3J) measurements as well as the observation of slow exchange NHs suggests that the structure exists in a significant proportion of the peptide population. The structural stability of these peptides is particularly high as compared with other mitochondrial signal sequences, which have required much higher percentages of membrane mimetic agents, such as TFE and micelles, and low temperatures to promote helix formation (Endo et al., 1989; Karslake et al., 1990; Bruch and Hoyt, 1992; Hammen et al., 1994). This warranted the calculation of three-dimensional structures for the visualization of the peptide.

It can be seen from Fig. 9that helix I, spanning residues 2-7, is highly amphiphilic in nature (hydrophobic moment = 0.77). Thus our structural studies are consistent with and strengthen the current model for mitochondrial import in which the signal sequence is required to present a hydrophobic face to the import machinery (Roise et al., 1986). The evidence suggests that a classical alpha-helix is adopted and consists of approximately two turns. The turn that exists between residues 7 and 10, while defining the termination of helix I, is not sufficiently constrained for classification into a particular type. However, the lack of NOE or slow exchange data suggests that this region is fairly flexible.


Figure 9: View of the N-terminal region of a calculated structure of Ac-Cpn10, showing the amphiphilic nature of helix I. Side-chains are color-coded according to hydrophobicity with charged species in red or blue, uncharged hydrocarbons in green, and aromatics in brown as follows: Ala^1, green (acetyl group highlighted pink), Gln^3red; Ala^4, green; Phe^5, orange; Arg^6, dark blue; Lys^7, dark blue; Phe^8, orange; Leu^9, green; and pro, light blue.



Helix II, spanning residues 10-22, is of particularly high stability and may extend further, but because of the overlap of residue 22-24 resonances, NMR data was scant for this region of the peptide. The amphiphilic nature of helix II is not so strong (hydrophobic moment = 0.12), and it may not constitute a crucial part of the targeting signal, although presentation of a hydrophobic face is clearly possible at several regions along the signal sequence (Fig. 10). The relatively high stability of helix II may be caused by its length or by the presence of salt bridges. Whereas there is no direct NMR evidence for salt bridges in Ac-Cpn10, it could be seen that the two Arg-Glu pairs (14 and 18, 19 and 23) in helix II are held in close proximity (Fig. 10). In the absence of competing ionic interactions, it is highly likely that these bridges are contributing to its structural stability and serve to accommodate negative charges in mitochondrial targeting signals as predicted by Lemire et al.,(1989).


Figure 10: View of a calculated structure of Ac-Cpm10 showing the positions of the charged residues. Salt bridges between residues 14 and 18 and residues 19 and 23 may help to stabilize helix II. The color coding is as described in the legend to Fig. 9.



A particularly interesting feature of the Cpn10 signal sequence is the post-translational acetylation of the N terminus. In eukaryotic cells up to 80% of all proteins are N-acetylated (Brown and Roberts, 1976), but the biological significance of this modification is in most cases not clear. In the current study the structural properties of Cpn10 were compared with those of the N-acetylated analogue, and it was found that this modification clearly enhanced the helical propensity of helix I. It has been demonstrated that a stable N-terminal amphiphilic helix is a likely requirement for successful import (Roise et al., 1986; Wang and Weiner, 1993). It is therefore possible that N-acetylation assists import of Cpn10, but this cound not be concluded from the indirect analysis presented in Fig. 2in which the effect of Cpn10 and Ac-Cpn on the targeting of the mitochondrial precursor protein p-OTC was assessed.

The mechanism by which acetylation mediates helical stabilization is interesting to contemplate. It is well established that the alpha-helix, because of the orientation of the amide repeating unit, possesses a dipole, effecting a positive potential at the N terminus and a negative potential at the C terminus of the helix (Hol, 1985; DeGrado et al., 1989). This has been used to rationalize the observation that compensating charged groups at either end of the helix can help to stabilize the structures (Shoemaker et al., 1987). In particular, specific studies of N-group blockage by N-acetylation have been shown to increase the helicity of a peptide in solution (Venkatachalapathi et al., 1993). Our results showing that removal of the N-terminal positive charge results in an increased helix I stability further support these findings. Additionally, the removal of the charge on Ala^1 may assist membrane transport through the extension of the amphiphilic face of helix I. As can be seen in Fig. 9, acetylated Ala^1 is positioned on the hydrophobic side of the amphiphilic helix, where an N-terminal positive charge would otherwise reduce the hydrophobic moment. The N-acetylated helix may therefore have better membrane binding properties than the charged species.

The helix-turn-helix motif observed for Cpn10 is comparable with several other mitochondrial signal sequences also structurally analyzed (Fig. 11). Cytochrome c oxidase subunit IV (Endo et al., 1989); the beta-subunit of the F(1)-ATPase complex (Bruch and Hoyt, 1992), and rat liver aldehyde dehydrogenase (Karslake et al., 1990) all assume N-terminal amphiphilic helices of approximately two to three turns, followed by a flexible region and, in the case of the later two proteins, a second stretch of helix (Fig. 11). Of interest is the fact that these three signal sequences all undergo processing upon import, unlike the signal sequence of Cpn10. Other mitochondrial proteins possessing nonprocessed signal sequences include 3-oxoacyl-CoA thiolase and rhodanese, which have each been shown to possess long, continuous helices (Hammen et al., 1994). Cpn10 therefore is the first example of an imported protein containing a nonprocessed signal sequence with a helix-turn-helix motif. Our data therefore show that whereas a flexible region within a helical signal sequence may be required for productive interaction with the processing protease (Wang and Weiner, 1993; Hammen et al., 1994), it is not a sufficient criterion for proteolysis. The structural requirement for proteolysis therefore remains elusive although, as pointed out by Hammen et al.(1994), signal sequences with long helical structures appear less likely to undergo processing than signal sequences adopting only short regions of continuous helical structure. The presence of the long helix II in Ac-Cpn10 is consistent with this view, but mutational and further structural studies of the topogenic signals listed in Fig. 11are required to resolve these questions.


Figure 11: Schematic diagram showing structural details of mitochondrial signal sequences as determined by two-dimensional NMR spectroscopy. Helical and nonhelical regions are indicated as well as whether mitochondrial processing of the signal sequence occurs. COXIV, cytochrome c oxidase subunit IV (Endo et al., 1989); F(1)-ATPase beta, beta-subunit of the F(1)-ATPase complex (Bruch and Hoyt, 1992); ALDH, aldehyde dehydrogenase (Karslake et al., 1990); Cpn10, Chaperonin 10 (this study); ALDHDelta(11-13), ALDH mutant lacking a flexible linker comprising residues 11-13 (Thornton et al., 1993); RHODANESE and THIOLASE, 3-oxoacyl-CoA thiolase (Hammen et al., 1994).




FOOTNOTES

*
This work was supported in part by grants from the Australian Research Council and the National Health and Medical Research Council (to P. B. H. and N. J. H.) and from the Australian Research Council (to D. J. C. and J. A. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
J. A. J. and M. T. R. contributed equally.

Recipient of an Australian Research Scholarship.

**
To whom correspondence and reprint requests should be addressed. Tel.: 61-3-479-2l96; Fax: 61-3-479-2467.

(^1)
The abbreviations used are: Cpn10, chaperonin 10; Ac-Cpn10, synthetic peptide representing residues 1-25 of rat Cpn10 with N-terminal acetylation; Cpn10, as for Ac-Cpn10 but without N-terminal acetylation; Cpn10, truncated form of Cpn10 spanning residues 31-101; d(i, j), d(i, j), etc., intramolecular distance between the protons alphaH and NH, NH and NH, etc., on residues i and j; NOE, nuclear Overhauser enhancement; NOESY, NOE spectroscopy; p-OTC, the 39-kDa precursor of rat liver ornithine transcarbamylase; TFE, trifluoroethanol; TOCSY, total correlation spectroscopy; TSP, 3-(trimethylsilyl)-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.

(^2)
M. T. Ryan, D. J. Naylor, N. J. Hoogenraad, and P. B. Høj, unpublished data.


ACKNOWLEDGEMENTS

We thank R. Condron, I. Thomas, A. Kirkpatrick, and G. Howlett for their contributions to this work.


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