Tom20 recognizes mitochondrial presequences through dynamic equilibrium among multiple bound states

Abstract

Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The N-terminal presequences of mitochondrial-precursor proteins contain a diverse consensus motif (φχχφφ, φ is hydrophobic and χ is any amino acid), which is recognized by the Tom20 protein on the mitochondrial surface. To reveal the structural basis of the broad selectivity of Tom20, the Tom20–presequence complex was crystallized. Tethering a presequence peptide to Tom20 through a disulfide bond was essential for crystallization. Unexpectedly, the two crystals with different linker designs provided unique relative orientations of the presequence with respect to Tom20, and neither configuration could fully account for the hydrophobic preference at the three hydrophobic positions of the consensus motif. We propose the existence of a dynamic equilibrium in solution among multiple states including the two bound states. In accordance, NMR ¹⁵N relaxation analyses suggested motion on a sub-millisecond timescale at the Tom20–presequence interface. We suggest that the dynamic, multiple-mode interaction is the molecular mechanism facilitating the broadly selective specificity of the Tom20 receptor toward diverse mitochondrial presequences.

Introduction

Amino-acid sequences that cannot be defined as a simple consensus motif are often used as recognition motifs in protein transport systems (Schatz and Dobberstein, 1996). Protein import into mitochondria represents a good example. Tom20, a 20-kDa subunit of the translocase of outer mitochondrial membrane complex, interacts with the N-terminal sequences of the precursor proteins destined for the mitochondrial matrix and inner membrane, and thereby functions as a receptor for the mitochondrial preproteins synthesized in the cytosol (Sollner et al, 1989; Ramage et al, 1993; Pfanner, 2000; Pfanner and Geissler, 2001; Endo and Kohda, 2002; Endo et al, 2003; Rapaport, 2003). These N-terminal-cleavable segments are collectively referred to as mitochondrial presequences. Statistical analyses revealed that a presequence typically consists of 15–40 amino-acid residues, with an abundance of positively charged residues, and tends to form an amphiphilic helical conformation (von Heijne, 1986). The Tom20 protein is anchored to the mitochondrial outer membrane by the N-terminal hydrophobic segment, and exposes its C-terminal domain to the cytosol. Two types of Tom20 protein exist in animal genomes, including human, mouse, and rat (Likic et al, 2005). The type II Tom20 gene is expressed ubiquitously in various mouse tissues, but the type I gene is specifically expressed in testis. In contrast, only one type of Tom20 (type I) exists in fungal genomes, including Saccharomyces cerevisiae and Neurospora crassa. Thus, in the vast majority of cells, a single Tom20 protein distinguishes ca 1000 mitochondrial proteins from other non-mitochondrial proteins and sorts them into mitochondria. In other words, Tom20 recognizes the targeting signal encrypted in the diverse amino-acid sequences of mitochondrial presequences, and at the same time does not accept other amino-acid sequences in the N-terminal portion of many non-mitochondrial proteins.

The consensus motif for the Tom20 recognition has not been fully defined, but our previous NMR and peptide-library analyses revealed that the consensus motif was represented as φχχφφ (where φ is a hydrophobic amino acid, such as leucine, isoleucine, phenylalanine, tryptophan, valine, and tyrosine, and χ is any amino acid) for a subset of presequences (Muto et al, 2001; Obita et al, 2003). Recently, some presequences containing alanine mutations at the hydrophobic positions within the consensus motif have been reported to mediate the import into mitochondria, despite their reduced Tom20 binding (Mukhopadhyay et al, 2006). Since the deletion of Tom20 is not lethal in yeast (Lithgow et al, 1994), it is possible that the mutated preproteins bypass Tom20 and use an alternative import pathway. However, a more likely explanation is that the mutated presequences actually bind to Tom20 very weakly, at a level lower than the detection limit of the bacterial two-hybrid system, but still enough to support the import.

We previously determined the solution structure of the cytosolic domain (residues 51–145) of rat Tom20 (ubiquitous form, type II) complexed with a presequence peptide derived from rat mitochondrial aldehyde dehydrogenase (ALDH) by NMR spectroscopy (Abe et al, 2000). The ALDH presequence contains LSRLL as the Tom20 recognition motif. The core structure of Tom20 consists of four α-helices, with a fifth α-helix loosely attached to the core (Figure 1). The bound portion of the presequence peptide adopts an amphiphilic helical conformation. Since presequences alone exhibit very little secondary structure in aqueous solutions (Roise et al, 1988; Pak and Weiner, 1990), the helical formation is essential for keeping the three leucine residues close together, so they can interact with the hydrophobic groove of Tom20.

Figure 1.

Design of the Tom20–presequence complex. The solution structure of the cytosolic domain of Tom20, previously determined by NMR (Abe et al, 2000), consists of five α-helices, α1–α5 (gray), with flexible N- and C-terminal segments. The presequence associates with and dissociates from Tom20 rapidly in solution. The presequence from aldehyde dehydrogenase (ALDH) is shown in green. The presequence peptide has no tertiary structure in the free state, but assumes an α-helical conformation in the bound state. Note that the numbering starts at 12, since this sequence corresponds to the C-terminal half of the ALDH presequence (Farres et al, 1988). A cysteine residue (yellow) was attached to the C terminus after the three-residue linker (red). The intermolecular disulfide bond was formed with the single cysteine residue of Tom20 (Cys100, yellow circle), and the mobile α5 and two flexible termini were removed, to prepare the Tom20–presequence complexes for crystallization and the NMR relaxation study.

The NMR structure, however, did not provide more detailed information on the bound presequence peptide due to the insufficient number of distance constraints within the peptide and between the peptide and Tom20 even at saturated peptide concentrations (Abe et al, 2000). This implied that the peptide still had some residual mobility in the Tom20 groove. Consequently, although the structure of Tom20 itself was well determined, the position and structure of the peptide were not sufficiently defined for an in-depth discussion about the recognition mechanism of Tom20.

In the present study, we designed a fragment corresponding to the core structure of Tom20 for X-ray crystallography. Consideration of the NMR structure prompted us to remove the flexible N- and C-terminal segments, which may interfere with crystallization (Figure 1). Our first attempt to cocrystallize the Tom20 fragment with the ALDH presequence failed, probably due to the weak affinity of the presequence peptide for Tom20. Therefore, we tethered the presequence peptide to Tom20 via an intermolecular disulfide bond. This tethering method was successfully used to analyze the presequence recognition of Tom20 by a novel peptide-library approach (Obita et al, 2003); the reasonable consensus pattern thus obtained indicates that this strategy works well to fix the presequence peptide in the binding groove of Tom20 in an unbiased manner. We successfully obtained two forms of crystals suitable for data collection. The three-dimensional structures of the complex of Tom20 and the ALDH presequence peptide were determined at 1.9- and 2.1-Å resolutions. The comparison of the two crystal structures implied that a dynamic equilibrium exists among two or more bound states of the presequence peptide on Tom20 in solution. In accord with this model, an NMR relaxation study revealed motion on the sub-millisecond timescale at the interface between Tom20 and the presequence peptide. We propose a dynamic, multiple mode of recognition that explains the structural basis of the broadly selective specificity of Tom20 toward diverse mitochondrial presequences.

Results

Crystal structure determination

We prepared a 68-residue fragment that corresponds to the core structure (residues 59–126) of Tom20. This fragment contains a single cysteine residue (Figure 1). The presequence peptide (GPRLSRLLSXAGC) consists of a sequence derived from mitochondrial pALDH (bold), a three-residue linker (italic), and a C-terminal cysteine residue. We used two different linker designs: an alanine residue at the X position (A linker) and a tyrosine residue at the same position (Y linker). The length of the linker was previously optimized by a novel peptide-library approach (Obita et al, 2003). The presequence peptide was incubated with Tom20 to prepare the disulfide-bond-linked complex by air oxidation (Figure 1). The results from a gel filtration analysis were consistent with a monomeric form of the Tom20–presequence complex in solution. The complex containing the Y-linker peptide eluted at the same volume as the complex containing the A-linker peptide, which corresponds to the monomeric molecular weight (data not shown). The resulting complex was purified by reverse-phase chromatography, concentrated, and crystallized (Igura et al, 2005). The three-dimensional structures of the two complexes with the two different linker designs were solved at resolutions of 1.9 and 2.1 Å (Table I).

Table 1.

Data collection and refinement statistics^a

	Tom20–SS-pALDH(A) (A-linker crystal)	Tom20–SS-pALDH(Y) (Y-linker crystal)
Data collection
Space group	P21	C2
Cell dimensions, a, b, c (Å), β (deg)	33.6, 27.6, 71.0, 103.0	151.8, 64.1, 68.0, 94.7
No. of molecules in AU	2	7
Resolution (Å)	50–1.90 (1.98–1.90)	20–2.05 (2.12–2.05)
Rmergeb	0.057 (0.193)	0.067 (0.302)
Mean I/σ(I)	24.4 (8.4)	16.7 (2.5)
Completeness (%)	98.9 (93.2)	95.8 (77.7)
Redundancy	3.1 (2.6)	5.2 (3.4)
Refinement
Resolution (Å)	50–1.90 (1.98–1.90)	20–2.05 (2.11–2.05)
No. of unique reflections	8906 (639)	34 455 (1696)
Rworkc/Rfreed	0.1781/0.2396	0.2481/0.3084
No. of atoms
Protein	1091	3271
Peptide	177	584
Water	168	378
B-factors (average) (Å2)
Protein	19.1	53.6
Peptide	21.5	57.1
Water	35.1	61.9
R.m.s. deviations
Bond lengths (Å)	0.012	0.010
Bond angles (deg)	1.42	1.226
PDB entry	2V1T	2V1S
Values in the parentheses are for the highest resolution shell.
aStatistics for data collection of the SeMet derivative of Tom20–SS-pALDH(Y) was summarized in the previous report (Igura et al, 2005).
bRmerge=∑h∑j∣〈I〉h−Ih,j∣/∑h∑jIh,j, where 〈I〉h is the mean intensity of symmetry-equivalent reflections.
cRwork=∑h∣∣Fo∣−∣Fc∣∣/∑h∣Fo∣, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively, for reflection h.
dRfree is equivalent to Rwork for a 9% test set of reflections.

There are two complex molecules in the asymmetric unit of the crystal containing the A-linker peptide, and seven complex molecules in that of the crystal containing the Y-linker peptide (Supplementary Figure 1). The presequence peptide binds to the Tom20 molecule as a monomeric form in the crystal containing the A-linker peptide. In contrast, all of complex molecules in the crystal containing the Y-linker peptide exist as intertwined dimers, due to the exchange of the presequence peptides between adjacent Tom20 molecules. There are many examples of intertwined dimer formation in protein crystal structures. Although such intertwining phenomenon is proposed to be a mechanism for the emergence of oligomeric proteins and for protein misfolding and aggregation (Rousseau et al, 2003), in many cases, the proteins exist as a monomer in solution, as well as the disulfide-tethered Tom20–presequence complex. There are no contacts between the two Tom20 molecules in the intertwined dimers, whereas the two presequence peptides contact each other. Because non-essential serine residues are mainly involved in the peptide–peptide interaction, we consider that the intertwined dimer formation in the crystal is not biologically relevant. We reconstituted the monomeric forms by swapping the coordinates of the presequence peptides in the intertwined pairs. No special treatment was performed for the atoms near the disulfide bond, thus the two sulfur atoms are not in the bond distance. The reconstituted monomeric forms were used in the following discussion. The superposition of the two crystal structures is shown in Figures 2A and B.

Figure 2.

Comparison of the crystal and solution structures of the Tom20–presequence complex. Superposition of (A) two A-linker crystal structures (disulfide-linked Tom20–pALDH complex containing the A linker), (B) five Y-linker crystal structures (disulfide-linked Tom20–pALDH complex containing the Y linker), and (C) 20 solution structures determined by NMR (PDB entry, 1OM2) (Abe et al, 2000). The Cα atoms of residues 65–121 of Tom20 were used for the superposition. The backbone atoms of the Tom20 protein are shown in gray, and those of the presequence peptide are cyan, orange, or green. The C-terminal cysteine residues of the peptide are depicted as yellow spheres. (D) Stereo views of the α-helical conformation of the presequence peptide in the A-linker structure (two structures, cyan) and in the Y-linker structure (five structures, orange). Residues 12, 13, 23, and 24 are omitted for clarity. (E) Relative accessible surface area (ASA) of the side chains of the presequence peptide in the complex. The relative ASA of the total of the side-chain atoms for each residue was calculated by the program NACESS, and the average values are plotted as a function of the residue number of the peptide.

Overall structures of the Tom20–presequence complexes

The core structure of the cytosolic domain of Tom20 consists of four α-helices, and the presequence peptide is accommodated in the groove formed between α1 and α3 of Tom20 (Figures 2A and B). Overall, the crystal structures fully agree with the NMR structure, which was determined in solution without the disulfide-bond tether (Figure 2C). Thus, neither the introduction of the intermolecular disulfide bond nor the crystal contacts had any deleterious effects.

Conformations of the presequence peptides in the bound states

The electron density maps demonstrated that the structures of the presequence peptides, including the linker regions, were well defined in both crystals (Supplementary Figure 2). Our previous NMR structure determination suggested that the bound peptide assumes an α-helical conformation, but a detailed analysis was not possible. In the present study, the crystal structures confirmed that the presequence peptide adopts an α-helical conformation (Figure 2D). The α-helix comprises not only the peptide region corresponding to the consensus motif (Leu15′–Leu19′, presequence numbering is marked by prime) but also the region close to the C terminus (Ser20′–Ala22′).

The covalent trapping could change the position of the presequence peptide and strain its conformation. Despite the concern, the helical regions of the peptides are well superimposed on each other, whereas the N-terminal region (Gly12′–Pro13′) and the C-terminal linker region (Gly23′–Cys24′), which are not in contact with Tom20, have no preferred positions, as compared to the region in contact with Tom20 (Figures 2A and B). Furthermore, we attempted to reduce the tethering disulfide bond in situ by soaking the crystals in a DTT-containing mother liquor. The crystal structure determination revealed that the disulfide bond was successfully cleaved in the A-linker crystal, but the overall structure was not changed except that the C-terminal cysteine residue of the peptide became disordered and invisible (to be published elsewhere). The attempts using the Y-linker crystal are now in progress. These results argue against the position and its conformation being forced by its covalent attachment.

Comparison of the two structures

The differences between the two crystal structures were analyzed with the program DynDom (Hayward and Lee, 2002), which detects domain movement by comparing two conformations of the same protein. The Tom20 structure consists of two helix–turn–helix domains, α1–α2 and α3–α4. Cys100, which was used as a tethering point, actually acts as the hinge of the domain rearrangement. In the Y-linker structure, the α1–α2 domain moves upward relative to α3–α4, with a 10 deg rotation around an axis as compared to the A-linker structure (Figure 3). In a coordinated manner, the presequence helix tilts its C terminus upward in the Y-linker structure as compared to the A-linker structure.

Figure 3.

Detailed comparison of the two crystal structures. (A) Conformational differences between the two Tom20 structures. The two crystal structures were compared using the program DynDom (Hayward and Lee, 2002). The Tom20 structure consists of two domains, α1–α2 (residues 59–99) and α3–α4 (residues 101–126). The α3–α4 domain (white) is superimposed. The α1–α2 domain of the Y-linker structure (green) rotates 10 deg around the axis and moves 0.2 Å along the axis relative to the A-linker structure (blue). The hinge residue was identified as Cys100 (orange). The bound presequence peptide of the A-linker structure is magenta, and that of the Y-linker structure is yellow. (B) Open-book representations of the contact residues at the Tom20–presequence interface. The surface of Tom20 that contacts one of the three leucine residues of the presequence peptide is displayed in the same color. Two atoms are defined as being in contact if the distance is less than 4.0 Å. The buried surface areas are similar: 305±2 Ų for the A-linker structure versus 338±32 Ų for the Y-linker structure.

The nearly parallel arrangement of the α1 and α3 helices forms a shallow groove, which serves as the binding site for the presequence. The contacting residues at the Tom20–peptide interface were examined by calculations of the inter-atom distances (Figure 3B). The three leucine residues of the presequence peptide are involved in the contacts. The side chain of Leu18′ in the presequence contacts the side chains of Val109 and Thr113 of Tom20 in both crystal structures. The side chain of Leu18′ is partially buried and rests against the rim formed by the methyl groups of Val109 and Thr113. The side chain of Leu19′ in the presequence is accommodated in a hydrophobic pocket created by Ile74, Glu78, Leu106, and Leu110 of Tom20 in the A-linker structure. To our surprise, the pocket formed by the same residues holds the side chain of Leu15′ in the presequence in the Y-linker structure. That is, the leucine residues at different positions in the presequence are recognized by the same hydrophobic pocket. The interaction is tight, since the relative accessible surface area (ASA) of Leu19′ in the A-linker structure and that of Leu15′ in the Y-linker structure are virtually 0% (Figure 2E). Moreover, the side chain of the third leucine residue, Leu15′ of the A-linker structure and Leu19′ of the Y-linker structure, has few contacts with the Tom20 surface (Figure 3B). In summary, Tom20 is equipped with only two hydrophobic subsites (subsite 1: the rim formed by residues 109 and 113; subsite 2: the pocket formed by residues 74, 78, 106, and 110) for the recognition of the three hydrophobic side chains in the φχχφφ motif.

The amino-acid residues of Tom20 involved in the two hydrophobic subsites are evolutionally conserved (Supplementary Figure 3). It is notable that the residue at position 78 is Glu or Asp, and that at position 113 is Ser or Thr. Thus, the side chains of these conserved non-hydrophobic residues seem to be functionally important. The functional groups, carboxylate and hydroxyl, are involved in hydrogen bond networks including bound water molecules (Supplementary Figure 4). The replacement of Glu78 by Ala, however, only slightly reduced the preprotein binding (Abe et al, 2000).

Mutational analysis of the amino-acid residues involved in the hydrophobic subsites

We examined the effects of mutations of the two hydrophobic subsites. Val109 and Thr113 from the subsite 1 and Ile74 from the subsite 2 were selected and changed to Ala or Ser. Figure 4 summarizes the results of the NMR titration experiments with the pALDH presequence peptide. The wild-type Tom20 used in this study (residues 59–126) bound to the pALDH presequence (residues 12–22) with the dissociation constant of 0.4 mM. Our previous NMR titration experiment using a longer version of Tom20 (residues 51–145) with a longer pALDH (residues 1–22) reported a dissociation constant of 0.02 mM (Abe et al, 2000). The present higher dissociation constant may be explained by the different lengths of Tom20 and pALDH used in this study. The substitutions of Ile74 and Val109 to an Ala residue did not change the extent and pattern of the chemical shift changes, as compared to the wild-type Tom20, suggesting that the Ala residues at positions 74 and 109 maintained the hydrophobic interactions with the Leu side chains of the presequence peptide. In contrast, the substitutions of Ile74 and Val109 to a Ser residue resulted in the substantial reduction of the chemical shift changes. This indicates that the hydrophobicity contributes to the presequence binding at both the subsites 1 and 2. The substitution of Thr113 to Ser did not change the interaction, which is consistent with the group conservation of Thr and Ser at this position (Supplementary Figure 3). Finally, the substitution of Thr113 to Ala increased the chemical shift changes. The mutation apparently strengthened the interaction, but the underlying explanation is unclear. A detailed study, for example a systematic mutation at position 113, is necessary in future.

Figure 4.

Chemical shift changes of backbone amides of the wild-type and mutated Tom20s upon binding to the pALDH presequence peptide at the molar ratio of 1:1. The chemical shift change of each backbone amide in [¹H, ¹⁵N]HSQC spectra was calculated according to the equation, [Δδ(¹H)²+(Δδ(¹⁵N)/5)²]^1/2. The positions of proline residues are indicated with the letter ‘p' in the top panel.

NMR relaxation analyses

We carried out NMR ¹⁵N relaxation experiments using the disulfide-tethered Tom20–presequence complex to analyze the protein dynamics in solution. We used the A-linker for the detailed relaxation study, but the choice of the linker was not critical, because a preliminary study on the complex with the Y-linker provided consistent results. Two labeling schemes were used to minimize the spectral overlaps. In one preparation, the presequence peptide was ¹⁵N-labeled, and in the other preparation, Tom20 was labeled (Supplementary Figure 5).

We used two methods to estimate the exchange-induced spin-relaxation rate, R_ex. First, we used the model-free analysis under the isotropic rotational diffusion model, using the measured R₁, R₂, and {¹H} ¹⁵N steady-state heteronuclear NOE values for ¹H–¹⁵N (Lipari and Szabo, 1982). The overall rotational diffusion time, τ_m, was calculated independently for the [¹⁵N]peptide and [¹⁵N]Tom20 in the complex. The good agreement (5.4 versus 5.6 ns) shows the validity of the model-free analysis (Supplementary Figure 6). As a second approach, we measured the transverse CSA/dipolar cross-relaxation rate (η_xy) for ¹H–¹⁵N, which is independent of chemical exchange processes (Tjandra et al, 1996; Kroenke et al, 1998). The R_ex values were estimated from a simple approximate equation, R_ex=R₂−aη_xy (see Materials and methods). The R_ex values obtained by the two methods agree well, which demonstrates the reliability of the determined R_ex values (Figure 5A). This cautious approach was taken because each method has its own drawbacks in approximation and simplification. We also measured the relaxation dispersion curves by a Carr–Purcell–Meiboom–Gill experiment (Supplementary Figure 7). No obvious changes were observed in the τ_cp range from 1 to 65 ms, indicating that the motion is not occurring in this time range. We estimate that the time range of the motion is on the order of the sub-millisecond timescale (see details in Materials and methods). For comparison, we estimated the R_ex rates of the [¹⁵N]Tom20 in the free form (Figures 5C and Supplementary Figure 6).

Figure 5.

Distribution of slow motions. (A) Plot of R_ex as a function of residue number of the disulfide-bond-tethered complex of Tom20 and the presequence. The yellow bar represents the R_ex values estimated from the model-free analysis, and the green line with the closed circles is R_ex from the measurement of transverse CSA/dipolar cross-relaxation rates (η_xy). The broken line indicates the threshold for the R_ex mapping on the three-dimensional structures. The positions of proline residues are indicated with the letter ‘p'. (B) Open-book representations of the spatial distributions of amide ¹⁵N spins with an R_ex value larger than 1 s⁻¹ from the η_xy measurement in the two crystal structures. The amide nitrogen atoms, shown as blue spheres, of Ser16′, Leu19′, and Ser20′ of the presequence peptide and of Ile74 and Gln75 of Tom20 are located at the hydrophobic contact surface (red patches on the ribbons). The amide nitrogen atoms, shown as cyan spheres, of Gly23′, Ile97, and Leu126 probably correspond to the conformational changes accompanying the motion of the presequence peptide. Finally, the yellow sphere represents the amide nitrogen atom of Gly101, which shows exceptional discrepancy between the two R_ex values. This could arise from the variation in the R_ex value calculated from η_xy due to the variation in magnitude and orientation of the CSA tensor. Note that the large values of the three ¹⁵N spins at positions 16, 19, and 20 of the presequence suggest a very interesting rule: an amide ¹⁵N spin in an α-helical conformation monitors the side-chain motion of the preceding residue, in the present case, Leu15′, Leu18′, and Leu19′. (C) Plot of R_ex as a function of residue number of the free form of Tom20. The yellow bar represents the R_ex values estimated from the model-free analysis, and the green line with the closed circles represents R_ex from the η_xy measurement. The broken line indicates the threshold for the R_ex mapping on the three-dimensional structure. The positions of proline residues are indicated with the letter ‘p'. (D) The spatial distribution of amide ¹⁵N spins with an R_ex value larger than 1 s⁻¹ from the η_xy measurement. The Tom20 structure in the A-linker crystal structure was used instead of the free structure. The amide nitrogen atoms with large R_ex values, shown as green spheres, are widely distributed in the Tom20 structure.

Discussion

We successfully obtained the high-resolution crystal structures of a Tom20–presequence complex. Our success was a consequence of the sophisticated design of the complex, with reference to our previous NMR structure: (i) The flexible N-terminal 8 residues and C-terminal 19 residues were omitted, to produce a shorter fragment of Tom20 that corresponds to the core structure. (ii) The single cysteine residue in the Tom20 fragment was used as an anchoring point. This cysteine residue is located at the optimal position for anchoring. (iii) A cysteine residue was added to the C terminus of the presequence peptide after the linker sequence. The optimal length of the linker (three residues) was previously determined in our peptide-library experiment (Obita et al, 2003). (iv) An intermolecular disulfide bond was formed to tether the presequence peptide onto the Tom20 protein.

Necessity of the intermolecular disulfide tethering

The fixation of the presequence peptide to Tom20 and the reduction of the residual mobility of the peptide in the bound state, by the introduction of the disulfide tether, were essential for the successful crystallization. In fact, no crystals have ever been obtained by simply mixing Tom20 with the presequence peptide. Previously, the local concentration of the peptide ligand was increased by attaching it as an N- or C-terminal extension, with a flexible polypeptide linker sequence, in crystallization and NMR studies (Fremont et al, 1996; Hennecke et al, 2000; Pellegrini et al, 2002; Freund et al, 2003). We also attempted this strategy: the ALDH presequence was fused to the N terminus of the Tom20 fragment (residues 64–126) via a 9- or 14-residue Gly-rich linker sequence. However, these constructs did not produce any crystals. We consider that the increase in the local concentration was not sufficient for the crystallization of the Tom20–presequence complex. As the previous NMR study suggested (Abe et al, 2000), the peptide has substantial mobility in the bound state. The long linker allows the multiple conformations to exist, thus disturbing the crystallization. In contrast, the short linker effectively traps one of the possible conformations in a crystal lattice, i.e. a state conformationally locked by the crystal contacts (Supplementary Figure 1). A linker as short as three residues is only possible by using disulfide bond tethering, in the case of Tom20.

Note that the short linker itself has sufficient flexibility in solution, since the peptide-library experiment using the three-residue linker produced a reasonable consensus motif (Obita et al, 2003). This should be accompanied by the (partial) melting of the α-helical conformation of the linker region (Ser20′–Ala22′), to increase the conformational degrees of freedom in solution. No steric hindrance imposed by the short linker assures the use of the disulfide-tethered Tom20–presequence complex for the NMR relaxation studies. It is possible to use the ¹⁵N-labeled peptide without tethering, at a saturated concentration in the presence of Tom20, but the fast exchange involving the free state would obscure the effects of the exchange among the multiple bound states on the relaxation.

A multiple-mode interaction

Two crystal structures with different linker designs were determined. It is striking that in each structure, one of the three leucine residues of the presequence has few contacts with Tom20 (Figure 3B). In fact, the mutagenesis study revealed that these three leucine residues are all necessary for binding to Tom20 (Abe et al, 2000). In contrast, Tom20 is equipped with only two hydrophobic subsites. The mutagenesis study showed that both the two hydrophobic subsites are important for the interactions of Tom20 with the presequence (Figure 4). Thus, neither of the crystal structures alone can explain the need for the three hydrophobic leucine side chains of the ALDH presequence. We consider both crystal structures to represent a snapshot of the solution states of the Tom20–presequence complex. The sequence, L₁S₂R₃L₄L₅, in the ALDH presequence is recognized as a rigid α-helix by the two states of Tom20 (Figure 6A).

Figure 6.

Comparison of the recognition mode of Tom20 with that of the nuclear receptor. (A) Close-up view of the binding interface of Tom20 with the presequence peptide, and (B) that of the nuclear receptor with a coactivator peptide containing the LXXLL motif (estrogen receptor α complex (Shiau et al, 1998); PDB entry 3ERD). The three leucine side chains of the peptide ligands, at positions 1, 4, and 5, are colored cyan, green, and orange, respectively. The molecular surface contacting the leucine residue at position 4 is blue, and that contacting the leucine residue at position 1 or 5 is red. Schematic representation of the binding modes (C) of Tom20 and (D) of the nuclear receptor. The peptide adopts an α-helical conformation in both the bound states. The helices are drawn as cylinders to imply the firmness of the helical structure. The side chain at position 4 lies along the edge of the interaction surface (blue disks). In the case of Tom20, the two states exist in a rapid equilibrium and the side chain at position 4 functions as a fulcrum. In each state, one of the side chains at positions 1 and 5 is recognized by the single hydrophobic subsite of Tom20 (red disk). It is likely that the third or more bound states exist, and they are in equilibrium with the A and Y states. In the nuclear receptor, both the side chains at positions 1 and 5 are buried in the two hydrophobic subsites of the nuclear receptor (red disks). The charge clamp residues, Glu and Lys, position the helix and determine its length.

We propose that the presequence peptide has mobility within the binding groove of Tom20 without releasing the peptide. There are at least two different modes of interaction, represented by the A- and Y-linker structures (Figure 6C). L₁ and L₄ are recognized in the ‘Y-state' configuration in a given moment, and L₄ and L₅ are recognized in the ‘A-state' configuration in another moment. In other words, L₄ is a molecular fulcrum held by two supporting residues (φ₄ subsite: Val109 and Thr113) of Tom20. L₁ or L₅ of the presequence is accommodated in the same hydrophobic pocket (φ₁/φ₅ subsite: Ile74, Glu78, Leu106, and Leu110) in each state of Tom20 (Figure 3B). This is concertedly accomplished by the combination of the tilting of the presequence helix, the rotation of the side chains of L₁ and L₅, and the conformational change of Tom20 (Figure 3A).

To prove the hypothetical motion of the presequence peptide on the Tom20 molecule, we measured the ¹⁵N relaxation of the disulfide-tethered Tom20–presequence complex. The tethering of the presequence peptide to Tom20 suppresses the rapid exchange between the free and bound states of the peptide, by increasing the local concentration. The exchange-induced spin-relaxation rate, R_ex, was estimated for each amide ¹⁵N spin (Figure 5A). The two independent methods provided consistent R_ex values, which demonstrate the reliability of our R_ex determination. The amide ¹⁵N spins with a large R_ex value (>1 s⁻¹, from the η_xy measurement) are mapped onto the two crystal structures (Figure 5B). The model-free R_ex data (yellow bars) are not used in the following analysis, because the inclusion of the R_ex parameter for a given residue is dependent on the model selection from the five possible sets of model-free parameters (Mandel et al, 1995). The amide nitrogen atoms of Ser16′, Leu19′, and Ser20′ of the presequence peptide, and those of Ile74 and Gln75 of Tom20 are located at the hydrophobic contact surface. This suggests molecular motion of the presequence peptide in the binding groove of Tom20. In this respect, our previous NMR structure (Abe et al, 2000) should be considered as the average of the structures corresponding to the two or more modes of interaction. The three other amide nitrogen atoms, located in Tom20 and the linker of the presequence peptide, probably correspond to the conformational change accompanying with the motion of the presequence peptide (Figure 3A).

We also estimated the R_ex values using the [¹⁵N]Tom20 in the absence of the presequence peptide (Figure 5C). Unexpectedly, relatively large R_ex values were obtained over the entire structure, indicating pervasive distribution of conformational fluctuations on sub-millisecond timescales (Figure 5D). This suggests that, in the free form, the core structure of Tom20 is regarded as a loosely packed four-helix bundle. Upon binding to a presequence, the motions converge to a small number of states that are necessary for the recognition of presequences. The motions seen in the free form are unrelated to the collective motions in the bound form, since the amide nitrogen spins with a large R_ex value are totally different between the complex and the free forms (Figures 5B and D).

LXXLL-motif recognition by nuclear receptors

Nuclear receptors are transcription factors that are activated by the binding of small hydrophobic ligands, such as steroid hormones (Savkur and Burris, 2004; Plevin et al, 2005). The hormone-bound nuclear receptors undergo a conformational change and gain the ability to bind to a variety of proteins called coactivators. The LXXLL motifs (L is leucine and X is any amino acid, also designated as the NR box), which are commonly found in the coactivators, mediate the interaction between the coactivators and their cognate nuclear receptors. The requirement of the leucine side chains is strict: the replacement with a valine residue was not tolerated, for example, in the case of the RIP-140 coactivator (Heery et al, 1997). The structural basis of the strict specificity toward the three leucine residues was established by several crystal structures (Darimont et al, 1998; Nolte et al, 1998; Shiau et al, 1998). Figure 6B shows a close-up view of the interface of the estrogen receptor α–LXXLL peptide complex (Shiau et al, 1998). The LXXLL portion of the 13-residue peptide (KHKILHRLLQDSS) adopts an α-helical conformation, and the side chains of the three leucine residues are recognized simultaneously by three hydrophobic subsites. The position and the length (two turns) of the short α-helix are specified by two helix-capping interactions (Savkur and Burris, 2004). The ‘charge clamp' comprises two highly conserved, charged residues of the nuclear receptors: the γ-carboxylate group of glutamate and the ɛ-amino group of lysine form hydrogen bonds with the N-terminal backbone amide group and the C-terminal backbone carbonyl group of the LXXLL α-helix, respectively. The complementary interface and the helix-capping interaction facilitate the strict recognition of the LXXLL motifs by a conventional key-and-lock mechanism.

The paxillin LD motif, LDXLLXXL (where L is leucine, D is aspartate, and X is any amino acid), is another example of a short motif containing leucine residues (Hurley, 2003). It should be emphasized that the structural basis of the interaction of the LD motif and the focal adhesion targeting domain shares many common features with that of the LXXLL motif and the nuclear receptors (Hoellerer et al, 2003).

A dynamic equilibrium among multiple bound states

A structural comparison with the nuclear receptor highlights the dynamic features of the presequence recognition by Tom20 (Figure 6). Even though the peptide sequences are very similar to each other (LSRLL for Tom20 versus LHRLL for estrogen receptor α), the recognition modes of the two systems differ considerably. Our previous peptide-library experiment revealed that Tom20 accepted Leu, Phe, Trp, and Ile at position 1, Leu, Ile, Trp, Phe, and Met at position 4, and Leu, Phe, Val, Trp, and Ile at position 5 of the ALDH presequence (Obita et al, 2003). In fact, Tom20 must recognize more diverse five-residue sequences in other protein presequences. The experimentally deduced examples include WKRCM of the yeast F₁β (F₁-ATPase β-subunit) presequence and LRRAY of the yeast mitochondrial heat shock protein 60 presequence (Muto et al, 2001). To achieve such broadly selective specificity, a static mode of recognition, like that of nuclear receptors, seems to be inadequate even if an induced fit mechanism is considered, because Tom20 needs to adapt simultaneously to the two side chains at positions 1 and 5, in addition to the third side chain at position 4. In contrast, dynamic recognition is an attractive mechanism. One state just recognizes two of the three hydrophobic residues, which increases the chance of adapting to various hydrophobic side chains by an induced fit mechanism. The change in the relative orientation of the two helix–turn–helix units (Figure 3A) is likely to be the induced fit mechanism of Tom20.

The dynamic equilibrium between the two states revealed by the two crystal structures are apparently enough to explain the five-residue consensus motif of Tom20. The fact that a preliminary S-linker structure (complex containing the pALDH presequence peptide with the Ser-linker) is almost identical to that of the A-linker structure supports this notion. However, it is quite likely that the real motion of the presequence peptide in the binding groove is far more complicated dynamic equilibrium among multiple bound states than simple exchange between the two states. A series of crystal structures with different linker designs are desired to prove or reject the existence of the third or more bound states. Finally, we suggest that the multiple-mode dynamic recognition of presequences by Tom20 could be necessary for the transfer of presequences to the next receptor, Tom22, as it is likely that Tom20 and Tom22 recognize opposite sides of the same presequence; Tom20 recognizing the hydrophobic surface and Tom22 the hydrophilic surface (Pfanner, 2000).

Not all presequences contain a consensus motif that exactly matches the pattern of φχχφφ; e.g. the yeast cytochrome c oxidase subunit IV) presequence and the rat ornithine transcarbamylase presequence do not (Muto et al, 2001). Thus, Tom20 may recognize similar, but different, patterns from the φχχφφ motif. The pervasive conformational fluctuations seen in the free form of Tom20 (Figure 5D), in combination with the multiple-mode recognition, might explain the outstanding specificity of Tom20.

Concluding remarks

We designed the Tom20–presequence complex for crystallography with reference to the NMR structure. This is a good example of NMR-assisted crystallography. Unexpectedly, two different structures were obtained, and neither could fully account for the hydrophobic preference at the three hydrophobic positions of the presequence. NMR relaxation analyses suggested motion on a sub-millisecond timescale at the Tom20–presequence interface. In view of the high-resolution crystal structures supplemented with the NMR dynamics data in solution, we propose a multiple-mode recognition model: two or more modes of interaction exist in dynamic equilibrium, and each mode recognizes a different feature of the short, but diverse, consensus motif. In this context, it is interesting to ask whether the plant Tom20, which convergently evolved from a distinct ancestral gene, shares a similar dynamic mechanism for the recognition of plant mitochondrial presequences (Perry et al, 2006).

Materials and methods

Sample preparation

The core structure of the cytosolic domain of Tom20, encompassing Asp59–Leu126 from Rattus norvegicus (accession no. Q62760), was produced and purified, as described (Igura et al, 2005). The ¹⁵N-labeled protein was prepared as described (Abe et al, 2000). The peptide sequence is Gly¹²–Pro¹³–Arg¹⁴–Leu¹⁵–Ser¹⁶–Arg¹⁷–Leu¹⁸–Leu¹⁹–Ser²⁰–X²¹–Ala²²–Gly²³–Cys²⁴. The numbering starts at 12, because the sequence in bold is derived from the C-terminal half of the presequence of mitochondrial ALDH from R. norvegicus (pALDH, Accession no. P11884) (Farres et al, 1988). The linker region contains an alanine residue (A-linker peptide) or a tyrosine residue (Y-linker peptide) at the position of X. The peptides used for crystallization were custom-made with an amidated C terminus by Operon Biotechnologies (Tokyo, Japan). The ¹⁵N-labeled peptides were produced as a GST–SUMO–fusion protein in E. coli BL21(DE3)pLysS cells. The transformed E. coli cells were cultured in M9 minimum medium containing ¹⁵NH₄Cl. After purification by glutathione Sepharose 4B resin (Amersham), each fusion protein was digested with SENP2 protease (100:1, w/w). The resulting mixture was fractionated on a reverse-phase HPLC column (Cosmosil 5C18-AR-II) to purify the peptide. The disulfide-bond-tethered complex with a presequence peptide was prepared by air oxidation, as described (Igura et al, 2005).

Gel filtration analysis

The gel filtration experiment was performed on a TSK-GEL super SW2000 column (Tosoh, Tokyo, Japan), preequilibrated with 0.1 M sodium phosphate buffer, pH 7.0, containing 0.5 M NaCl, at a flow rate of 0.5 ml min⁻¹. The following protein standards were used: bovine serum albumin (67 kDa), ovalbumin (43 kDa), and lysozyme (14 kDa). The injected protein concentration was 0.33 mg ml⁻¹ and the eluted protein concentration was estimated to be about 0.05 mg ml⁻¹.

Data collection and structure determination

The disulfide-bond-tethered complexes were crystallized as described (Igura et al, 2005). Native and SeMet (selenomethionyl) derivative data sets were collected at 100 K at beamlines BL40B2 and BL41XU, SPring-8, Harima, Japan, as described (Igura et al, 2005). All diffraction data sets were processed and scaled with the DENZO/Scalepack package (Otwinowski and Minor, 1997). A starting model was obtained from the data set of the SeMet Y-linker complex; the initial phase calculations were performed by the MAD method using SOLVE (Terwilliger and Berendzen, 1999), and, subsequently, a solvent-flattening procedure was carried out using RESOLVE (Terwilliger, 2000). The model building was carried out manually with O (Jones et al, 1991) with reference to the NMR structure (PDB entry, 1OM2). The subsequent molecular dynamics refinement was performed using CNS (Brünger et al, 1998) and Refmac (CCP4, 1994) with translation libration screw parameters, up to 2.05-Å resolution, with the native data set of the Y-linker complex. The final model of the Y-linker structure comprises six complex molecules and 355 water molecules. In the later stages of the refinement, parts of the seventh complex molecule (chains G and N) could be built, but their electron densities were not clear enough to build all of the residues of both the protein chain and peptide chain. The final model of the Y-linker structure, comprising seven complex molecules and 378 water molecules, was refined to an R factor of 24.8% and an R_free factor of 30.8%. This model was used to solve the structure of the A-linker complex by the molecular replacement procedure, using MOLREP in the CCP4 package (CCP4, 1994). The final structure of the A-linker complex, comprising two complex molecules and 168 water molecules, was refined to an R factor of 17.8% and an R_free factor of 24.0% with the translation libration screw parameters. Statistics for data collection and refinement is summarized in Table I.

Structural analysis

The stereochemical quality analysis and the secondary structure assignment were carried out with PROCHECK (Laskowski et al, 1993). The Ramachandran plot of the backbone angles of the non-glycine and non-proline residues of the A-linker structure gave 95.6% in the most favored region and 4.4% in the additionally allowed region. The Ramachandran plot of the Y-linker structure gave 92.6% in the most favored region, 5.9% in the additionally allowed region, 1.2% in the generously allowed region, and 0.2% in the disallowed region. The distribution of these five residues in the generously allowed region and one residue in the disallowed region was not directly correlated with the disulfide bond formation, intertwined dimer formation, or Tom20–peptide interaction. The ASA was calculated with NACESS (http://wolf.bi.umist.ac.uk/naccess/) with a probe radius of 1.4 Å. The contribution of the linker region was omitted in the calculations of ASA. Domain rearrangement and hinge-bending motion were analyzed with DynDom (Hayward and Lee, 2002). The average structures of the A-linker structures and that of the Y-linker structures were used as input with the parameters of window length=5 (default), minimum domain size=15, and ratio of inter- to intradomain displacement=1.0 (default). The figures were prepared with PyMOL (DeLano Scientific; http://pymol.sourceforge.net/).

NMR spectroscopy

NMR spectra were recorded at 298 K on Avance600 spectrometer equipped with a TXI cryoprobe. The NMR samples contained 0.4 mM (spectral assignment and relaxation study) or 0.1 mM (titration study) ¹⁵N- and/or ¹³C-labeled proteins in 90% ¹H₂O/10% ²H₂O, containing 20 mM MOPS-NaOH, pH 7.0, and 50 mM NaCl. The spectral assignments of the wild-type and mutated Tom20s, and the disulfide-tethered complexes were made as described (Abe et al, 2000).

Single amino-acid-substituted Tom20 proteins were ¹⁵N labeled, and titrated with the pALDH peptide, Ac-GPRLSRLLSYA-NH₂, to monitor the binding, at the molar ratio of 0:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, and 3:1. DTT was added to the sample solutions at the final concentration of 5 mM to prevent the formation of Tom20 dimers during the titration. The titration curves were analyzed with the program xcrvfit ver 4.0.9, developed by R Boyko and BD Sykes (University of Alberta; http://www.bionmr.ualberta.ca/bds/software/xcrvfit).

Spectra for ¹⁵N longitudinal relaxation rates, R₁, ¹⁵N transverse relaxation rates, R₂, and {¹H} ¹⁵N steady-state heteronuclear NOE values were acquired at 298 K using [¹⁵N]Tom20–SS-presequence(A-linker), Tom20–SS-[¹⁵N]presequence(A-linker), and [¹⁵N]Tom20C100S samples at 600 MHz ¹H frequency. The cysteine residue was replaced by serine for the prevention of dimer formation during the prolonged period of experimental time. The pulse sequences used were the gradient sensitivity enhancement version (Farrow et al, 1994). R₁ was determined from the data with relaxation delays of 30, 80, 150, 300, 500, and 800 ms, and R₂ from the data with relaxation delays of 57.6, 86.4, 115.2, 144.0, 172.8, and 201.6 ms. In the heteronuclear NOE experiment, ¹H saturation for 3.0 s during the relaxation delay was applied for NOE enhancement. The model-free analysis was performed (Lipari and Szabo, 1982) with the programs MODELFREE-4.10 (Mandel et al, 1995) and TENSOR2 (Dosset et al, 2000). The model-free parameters for each amide ¹⁵N spin were selected, as described in the MODELFREE manual. We used the isotropic diffusion model for the model-free analysis, but the axially symmetric diffusion model and the fully anisotropic diffusion model, combined with the two crystal coordinates, provided essentially the same results. The overall shapes of the Tom20–SS-presequence complexes and the free form of Tom20 are both regarded as a prolate ellipsoid with the ratio D_∣∣/D_⊥=1.2, and thus a sphere shape is a good approximation. The transverse CSA/dipolar cross-relaxation rate (η_xy) for ¹H–¹⁵N was measured as described (Tjandra et al, 1996; Kroenke et al, 1998). The R_ex values were estimated from a simple approximate equation R_ex=R₂−aη_xy (a=1.3 at 600 MHz ¹H frequency) (Fushman et al, 1998). The relaxation dispersion curves were obtained using the relaxation-compensated Carr–Purcell–Meiboom–Gill experiment, as described (Millet et al, 2000). The R₂ value was determined from the data with τ_cp of 1.0, 2.0, 4.0, 6.6, 10.8, 21.6, and 64.8 ms.

We estimate the rate constant of the motion of the peptide in the binding groove. We assume exchange between two states for calculation. The relaxation dispersion experiment suggested that the motion occurs in the fast-exchange regime (i.e. k_ex≫Δω, Supplementary Figure 7), and so the following expression approximates the effect of two-site chemical exchange: R_ex=p_Ap_B(Δω)²/k_ex, where p_A and p_B are the populations of the two bound states (p_A+p_B=1), Δω is the difference in chemical shift of the ¹⁵N spins in the two bound states, and k_ex is the chemical exchange rate constant (k_ex=k₁+k₋₁, k₁ is the forward exchange rate and k₋₁ is the reverse exchange rate). It is assumed that the two states are equally represented in solution, i.e. p_A=p_B=0.5. The R_ex values are found in the range of 1–4 s⁻¹ (Figure 5A). Assuming a reasonable value of 1–4 ppm (60–240 Hz at 60 MHz ¹⁵N frequency) for Δω, we estimate the rate constant of the motion to be on the order of 10 000s⁻¹.

Supplementary Material

Supplementary Figure 1

Supplementary Figure 2

Supplementary Figure 3

Supplementary Figure 4

Supplementary Figure 5

Supplementary Figure 6

Supplementary Figure 7