The peroxisomal receptor Pex19p forms a helical mPTS recognition domain
2010; Springer Nature; Volume: 29; Issue: 15 Linguagem: Inglês
10.1038/emboj.2010.115
ISSN1460-2075
AutoresNicole Schueller, Simon J. Holton, Krisztián Fodor, M. Milewski, Petr V. Konarev, Will A. Stanley, Janina Wolf, Ralf Erdmann, Wolfgang Schliebs, Young‐Hwa Song, Matthias Wilmanns,
Tópico(s)Metal-Catalyzed Oxygenation Mechanisms
ResumoArticle8 June 2010free access The peroxisomal receptor Pex19p forms a helical mPTS recognition domain Nicole Schueller Nicole Schueller EMBL c/o DESY, Notkestrasse 85, Hamburg, Germany Search for more papers by this author Simon J Holton Simon J Holton EMBL c/o DESY, Notkestrasse 85, Hamburg, Germany Search for more papers by this author Krisztian Fodor Krisztian Fodor EMBL c/o DESY, Notkestrasse 85, Hamburg, Germany Search for more papers by this author Morlin Milewski Morlin Milewski EMBL c/o DESY, Notkestrasse 85, Hamburg, Germany Search for more papers by this author Petr Konarev Petr Konarev EMBL c/o DESY, Notkestrasse 85, Hamburg, Germany Search for more papers by this author Will A Stanley Will A Stanley EMBL c/o DESY, Notkestrasse 85, Hamburg, GermanyPresent address: ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, 35 Stirling Highway, Crawley 6009, Western Australia, Australia Search for more papers by this author Janina Wolf Janina Wolf Department of Systems Biology, Faculty of Medicine, Institute for Physiological Chemistry, Ruhr University of Bochum, Bochum, Germany Search for more papers by this author Ralf Erdmann Ralf Erdmann Department of Systems Biology, Faculty of Medicine, Institute for Physiological Chemistry, Ruhr University of Bochum, Bochum, Germany Search for more papers by this author Wolfgang Schliebs Wolfgang Schliebs Department of Systems Biology, Faculty of Medicine, Institute for Physiological Chemistry, Ruhr University of Bochum, Bochum, Germany Search for more papers by this author Young-Hwa Song Young-Hwa Song EMBL c/o DESY, Notkestrasse 85, Hamburg, Germany Search for more papers by this author Matthias Wilmanns Corresponding Author Matthias Wilmanns EMBL c/o DESY, Notkestrasse 85, Hamburg, Germany Search for more papers by this author Nicole Schueller Nicole Schueller EMBL c/o DESY, Notkestrasse 85, Hamburg, Germany Search for more papers by this author Simon J Holton Simon J Holton EMBL c/o DESY, Notkestrasse 85, Hamburg, Germany Search for more papers by this author Krisztian Fodor Krisztian Fodor EMBL c/o DESY, Notkestrasse 85, Hamburg, Germany Search for more papers by this author Morlin Milewski Morlin Milewski EMBL c/o DESY, Notkestrasse 85, Hamburg, Germany Search for more papers by this author Petr Konarev Petr Konarev EMBL c/o DESY, Notkestrasse 85, Hamburg, Germany Search for more papers by this author Will A Stanley Will A Stanley EMBL c/o DESY, Notkestrasse 85, Hamburg, GermanyPresent address: ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, 35 Stirling Highway, Crawley 6009, Western Australia, Australia Search for more papers by this author Janina Wolf Janina Wolf Department of Systems Biology, Faculty of Medicine, Institute for Physiological Chemistry, Ruhr University of Bochum, Bochum, Germany Search for more papers by this author Ralf Erdmann Ralf Erdmann Department of Systems Biology, Faculty of Medicine, Institute for Physiological Chemistry, Ruhr University of Bochum, Bochum, Germany Search for more papers by this author Wolfgang Schliebs Wolfgang Schliebs Department of Systems Biology, Faculty of Medicine, Institute for Physiological Chemistry, Ruhr University of Bochum, Bochum, Germany Search for more papers by this author Young-Hwa Song Young-Hwa Song EMBL c/o DESY, Notkestrasse 85, Hamburg, Germany Search for more papers by this author Matthias Wilmanns Corresponding Author Matthias Wilmanns EMBL c/o DESY, Notkestrasse 85, Hamburg, Germany Search for more papers by this author Author Information Nicole Schueller1,‡, Simon J Holton1,‡, Krisztian Fodor1, Morlin Milewski1, Petr Konarev1, Will A Stanley1, Janina Wolf2, Ralf Erdmann2, Wolfgang Schliebs2, Young-Hwa Song1 and Matthias Wilmanns 1 1EMBL c/o DESY, Notkestrasse 85, Hamburg, Germany 2Department of Systems Biology, Faculty of Medicine, Institute for Physiological Chemistry, Ruhr University of Bochum, Bochum, Germany ‡These authors contributed equally to this work *Corresponding author. EMBL-Hamburg, Notkestrasse 85, Hamburg 22603, Germany. Tel.: +49 40 89902 126; Fax: +49 40 89902 149; E-mail: [email protected] The EMBO Journal (2010)29:2491-2500https://doi.org/10.1038/emboj.2010.115 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The protein Pex19p functions as a receptor and chaperone of peroxisomal membrane proteins (PMPs). The crystal structure of the folded C-terminal part of the receptor reveals a globular domain that displays a bundle of three long helices in an antiparallel arrangement. Complementary functional experiments, using a range of truncated Pex19p constructs, show that the structured α-helical domain binds PMP-targeting signal (mPTS) sequences with about 10 μM affinity. Removal of a conserved N-terminal helical segment from the mPTS recognition domain impairs the ability for mPTS binding, indicating that it forms part of the mPTS-binding site. Pex19p variants with mutations in the same sequence segment abolish correct cargo import. Our data indicate a divided N-terminal and C-terminal structural arrangement in Pex19p, which is reminiscent of a similar division in the Pex5p receptor, to allow separation of cargo-targeting signal recognition and additional functions. Introduction Peroxisomes are single-membrane organelles with distinct translocation pathways for peroxisomal matrix proteins and peroxisomal membrane proteins (PMPs). Most matrix proteins include a short peroxisomal-targeting signal (PTS) motif. Two different motifs, categorized as type 1 (PTS1) and type 2 (PTS2), are recognized by the specific receptors, Pex5p and Pex7p, respectively. Structures of the PTS1 receptor Pex5p in the absence and presence of a cargo have unravelled how the conformation of the receptor adapts to recognize peroxisomal matrix proteins through their C-terminal PTS1 motif (Gatto et al, 2000; Stanley et al, 2006). In contrast, several PMPs have one or more membrane protein-targeting signal (mPTS) motifs with a cluster of basic residues in a predicted α-helical conformation (Fransen et al, 2001; Jones et al, 2001; Rottensteiner et al, 2004). This binding site is generally flanked by one or two transmembrane segments. Correct topogenesis of most of these targets, categorized as class-I mPTS, within the peroxisomal membrane, however, requires two peroxins: Pex3p and Pex19p. These findings have led to diverse arguments that consider Pex19p as a PMP receptor, an assembly factor, a PMP chaperone or a factor combining more than a single function (Eckert and Erdmann, 2003; Jones et al, 2004; Halbach et al, 2006). Recent data have reconciled earlier hypotheses by indicating complementary tasks for Pex3p and Pex19p in PMP topogenesis (Pinto et al, 2006). In this model, Pexp19p recognizes newly translated PMPs in the cytosol, thus functioning as a soluble PMP receptor. In a second step, the PMP cargo is directed to the peroxisomal membrane by the binding of Pex19p to membrane-bound Pex3p, assigning Pex3p the function of a Pex19p co-receptor (Heiland and Erdmann, 2005; Matsuzono et al, 2006; Matsuzaki and Fujiki, 2008). The first step, in which Pex19p shields the hydrophobic PMP Pex19p-binding site, may also be considered as a chaperone-like activity. Furthermore, there is evidence that Pex19p binds to the docking and assembly complex Pex13p/Pex14p, indicating a functional role in the formation of a multi-component importomer complex (Fransen et al, 2004; Neufeld et al, 2009). In vivo, Pex19p has been found to be associated with the peroxisomal membrane (Gotte et al, 1998; Matsuzono et al, 1999) and with the ER (Hoepfner et al, 2005) showing its dynamic nature and involvement in shuttling processes. The importance of correct peroxisome function is highlighted by the existence of fatal human genetic peroxisomal biogenesis disorders. For example, a frame-shift mutation to the Pex19p coding region (Met255) is one of the underlying causes of Zellweger syndrome (Matsuzono et al, 1999). Pex19p is a soluble 299-residue protein with a conserved C-terminal farnesylation site (Kammerer et al, 1997; Matsuzono et al, 1999). However, little is known about its structural organization. On the basis of an analysis of splice variants and available binding data, a three-domain organization of Pex19p was proposed (Mayerhofer et al, 2002). Limited proteolysis and biophysical data indicated that Pex19p consists of a folded C-terminal part, preceded by a flexible N-terminal sequence (Shibata et al, 2004). Mutagenesis data have mapped the primary PMP-binding site to the C-terminal Pex19p domain (Fransen et al, 2005). Other in vitro data indicate that only full-length Pex19p is capable of PMP binding, thus indicating a contribution from the N-terminal part of Pex19p in PMP recognition (Shibata et al, 2004). In contrast, the N-terminal part of Pex19p is sufficient to bind both Pex3p, which establishes the Pex3p/Pex19p PMP receptor complex, and the peroxisomal assembly factor Pex14p (Muntau et al, 2003; Fransen et al, 2004; Jones et al, 2004; Shibata et al, 2004; Neufeld et al, 2009). The importance of the very C-terminus of Pex19p varies for different PMP interactions. A splice variant of human Pex19p, lacking residues 273–299, is able to bind Pex3p and the PMPs ALDP, ALDRP and PMP70 (Mayerhofer et al, 2002). To date, there are conflicting reports about the biological impact of Pex19p farnesylation (Vastiau et al, 2006). Removing the C-terminal CAAX (A=aliphatic, X=any amino acid) motif, and thus the farnesylation site, from Pex19p, affects its ability to bind several PMPs (Fransen et al, 2002; Rucktaschel et al, 2009). However, in some investigations, the absence of farnesylation of Pex19p in Saccharomyces cerevisiae has no effect on the yeast cell viability (Fransen et al, 2001; Vastiau et al, 2006). To unravel the molecular basis of the unusual Pex19p function as both a PMP-docking factor and receptor, we have investigated the natively folded part of Pex19p that comprises the C-terminal segment of the protein sequence. The high-resolution crystal structure of the C-terminal Pex19p domain reveals an α-helical bundle with an overall arrangement that is without precedence in other available protein structures. Quantitative-peptide-binding experiments and complementary functional data show that this domain is a functional mPTS-binding module in Pex19p. Results Selection of Pex19p constructs for functional/structural characterization As a prerequisite for structural analysis of the human Pex19p PMP receptor, we searched for suitable fragments of the protein by combining earlier published data with our own findings. In an earlier investigation, residue 156 was identified as a main proteolytic cleavage site, allowing separation of the N- and C-terminal parts of the protein (Shibata et al, 2004). By using a modified proteolysis protocol, we were able to reveal two additional prominent cleavage sites at residue positions 281 and 283 of the Pex19p sequence (Figure 1). On the basis of these data, we selected either residue 283 or the native C-terminus as the protein fragment boundary. Figure 1.Pex19p sequence/structure relationships. (A) Pex19p fragments used for investigation; the sequence segment, whose 3D structure we determined in this study, is highlighted in yellow. The C-termini and N-termini of each protein fragment are listed. (B) Multiple sequence alignment of the C-terminal part of Pex19p, showing sequence/structure relationships. A representative set of Pex19p sequences is shown, including H. sapiens (UNIPROT code P40855), M. musculus (Q8VCI5), C. griseus (Q60415), R. rattus (Q9QYU1), C. elegans (P34453), H. polymorpha (Q96WN7), P. pastoris (Q9Y8C5), S. cerevisiae (Q07418), Y. lipolytica (Q96W74) and A. thaliana (Q9SRQ3). The sequences are quite divergent, thus allowing reliable multiple alignments for restricted sequence segments only (orange boxes). Invariant, highly conserved (maximum one deviating amino acid) and conserved (maximum three deviating amino acids) residue positions are indicated by '*', ':' and '.' symbols, respectively. The sequence numbers refer to the H. sapiens Pex19p sequence. The Pex19p sequence segment for which the 3D structure has been determined is indicated by a line above the alignment. Cylinders indicate the positions of α-helices. The mPTS-binding helix α1 is coloured in green. Those residues that are involved in the formation of the central Pex19p cavity (cf. Supplementary Figure S4) are indicated by 'Δ' symbols. Cys296, which presents the C-terminal farnesylation site, is shown in yellow characters and black background. Surface exposed polar amino acids with conserved residue properties are shown on magenta background (cf. Figure 3). Surface exposed hydrophobic amino acids with conserved residue properties are shown on green background (cf. Figure 3). The latter are clustered at the N-terminus of the Pex19p structure, covering helix α1. Sequences were aligned using CLUSTALW (Pearson, 1994), followed by manual adjustments. A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint For further functional and structural studies, we used Pex19p constructs with three different N-termini (residues 1, 161, 182). The rational for the choice of residue 161 as the N-terminus was to allow separated structural/functional analysis of the C-terminal part of Pex19p. The full-length version of protein was used as reference for comparison. The third construct with the N-terminus at residue 182 emerged from the structure of the Pex19p C-terminal domain (see below). All three constructs were expressed with two different C-termini (residues 283, 299), generating a total of six different Pex19p fragments for functional characterization (Figure 1A). Folding of all Pex19p constructs investigated was verified by circular dichroism (CD) (Supplementary Figure S1). Whereas the CD spectra of the two Pex19p constructs that include the complete N-terminal part (1–283, 1–299) indicate a significant unfolded protein content, in agreement with earlier biophysical data (Shibata et al, 2004), the spectra of all remaining wild-type constructs were basically indistinguishable and indicate that the C-terminal part of Pex19p is mostly α-helical. C-terminal part of Pex19p folds into an α-helical bundle domain We were able to crystallize the Pex19p(161–283) fragment (Figure 1A), which includes all conserved Pex19p sequence segments for which an unambiguous multiple sequence alignment is possible (Figure 1B, orange boxes). The X-ray structure of Pex19p(161–283) was determined at a resolution of 2.05 Å, using experimental phases from xenon-derivatized crystals of the protein (Table I). The final model of the structure includes residues 171–280; the remaining residues at the two termini lack interpretable electron density. Mass spectrometry confirmed that there was no further degradation of the crystallized protein (data not shown), indicating that these missing residues adopt flexible conformations within the crystal. Table 1. Crystallographic statistics Native Xenon derivatized Data collection Space group P212121 P212121 Cell dimensions (Å) a=67.1 a=67.6 b=91.0 b=91.3 c=122.3 c=127.7 Wavelength (Å) 0.98 1.50 Overall resolution range (Å) 73–2.05 55–2.87 Highest resolution range (Å) 2.16–2.05 3.10–2.87 Number of unique reflections 47 467 17 855 Multiplicity 6.2 20.3 Mean I/σ(I) 7.6 (2.0) 6.2 (2.7) Completeness (%) 99.6 (100) 99.6 (97.1) Anomalous completeness (%) — 99.1 (96.0) Rsyma 6.2 (45.2) 7.2 (26.2) Mosaicity (deg) 0.38 0.46 Phasing Figure of merit (acentric/centric) — 0.70/0.62 (0.34/0.29) Number of sites — 4 Refinement Protein atoms 3717 Other atoms 192 Rconvb/Rfreec 21.5/25.0 RMS deviations Bond lengths (Å) 0.015 Bond angles (deg) 1.574 a where Ih,j is the intensity of the jth observation of unique reflection h. b where Foh and Fch are the observed and calculated structure factor amplitudes for reflection h. c Rfree is equivalent to Rconv, but is calculated using a 5% disjoint set of reflections excluded from the maximum likelihood refinement stages. The structure of Pex19p(161–283) reveals that this part of the protein folds into a three-helical bundle domain α2–α3–α4, preceded by a highly exposed N-terminal helix α1 (Figure 2). The overall shape of the helical bundle is cylindrical, with approximate dimensions of 25 × 25 Å2 across and 50 Å along the cylindrical axis. The first helix of the bundle (α2) displays a 24° kink at the conserved Pro200, thus effectively generating two helical segments, α2′ (185–197) and α2″ (199–210). The two long 5-turn helices α3 (212–233) and α4 (240–261) form an antiparallel arrangement and pack against helix α2′ with a tilt angle of about 30°. Residues 262–283, constituting the C-terminal region of the Pex19p(161–283) construct, do not adopt any significant secondary structural motifs. In the crystal structure, this sequence segment associates with other parts of the structure in such a way that the hydrophobic core of the three-helical bundle domain is shielded from the solvent environment (Figure 2A). Figure 2.Crystal structure of the mPTS-binding domain of Pex19p(161–283). (A) Ribbon representation of the Pex19p(161–283) structure. The secondary structural elements and the termini of the visible part of the Pex19p(161–283) sequence are labelled. Those parts of the structure for which a reliable multiple sequence alignment exists (orange boxes, cf. Figure 1) are shown as orange ribbons. (B) Side chains of invariant and highly conserved residues (for definition see Figure 1 caption) of the Pex19p(161–283) structure are shown in orange and yellow, respectively, in stick presentation and are labelled. Oxygen and nitrogen atoms are shown in red and blue, respectively. The illustration reveals that the hydrophobic core of the Pex19p(161–283) helical bundle is most conserved. A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint Overall similarity among Pex19p sequences from different organisms is limited mostly to the helical bundle structure and the preceding helix α1 (Figure 1B, orange boxes), whereas the sequences of the connecting loops and the C-terminal part of the structure substantially differ and cannot be reliably aligned. Most of the conserved and invariant residues of Pex19p(161–283) are either involved in the formation of the Pex19p(161–283) helical bundle core or are surface exposed (Figure 2B, orange). The first N-terminal helix visible in the structure (α1, 172–183) does not pack against other parts of the structure and is highly exposed (Figure 3). The surface of this helix shows an extensive hydrophobic patch from several conserved amino acids or residues with conserved hydrophobic side chain properties (Figure 3, coloured in green). The side chains of these residues (Ile171, Met175, Ile178, Met179, Leu182, Val187) align with the face of helix α1, which is oriented towards the part of the cylindrical surface of the Pex19p(161–283) helical barrel, comprising the C-terminus of the structure. The only conserved polar residue from this segment (Gln180) is oriented towards the opposite face of helix α1. The nature and the pronounced exposure of this hydrophobic surface patch markedly differ from other surface parts of the structure, thus suggesting a potential function in Pex19p function (see below). A second distinct polar surface patch is formed by several conserved charged residues, most of which are located on helix 2α′ (Figure 3, coloured in magenta). One of the residues in this surface patch, Lys193, is invariant (Figures 1 and 2B). Figure 3.Pex19p(161–283) surface patches with conserved residue properties. (A) Worm-type ribbon representation of the Pex19p(161–283) structure; (B) semi-transparent surface and ribbon representation of the Pex19p(161–283) structure in two different orientations, in which the right panel is rotated around a vertical axis in the paper plane by 180° with respect to the left panel. The side chain contributions of polar surface residues with conserved amino-acid properties are shown in magenta. The side chain contributions of hydrophobic residues with conserved amino-acid properties are shown in green. All coloured residues are labelled. In the worm-type presentation of the Pex19p(161–283) structure, oxygen, nitrogen and sulphur atoms are shown in atom-specific colours: red, blue, yellow, respectively. A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint Interestingly, for all four protomers in the asymmetric unit, helix α1 forms an identical antiparallel coiled-coil interaction with the equivalent helix from either a crystallographic or non-crystallographic symmetry-related molecule (Supplementary Figure S2). However, none of our solution data support oligomerization of Pex19p(161–283) or any other Pex19p constructs studied in this contribution (data not shown), suggesting that the observed assembly is transient. Further structural features of Pex19p, including the observation of a large internal cavity, are described in the Supplementary data. C-terminal helical bundle constitutes the Pex19p mPTS-binding site In the absence of structural information for Pex19p in complex with PMP target proteins, we became interested in whether the C-terminal domain of Pex19p, as visualized in the X-ray structure, constitutes a functional mPTS-binding element. We, therefore, tested three different Pex19p fragments (1–299, 161–283, 161–299), using an earlier established methodology for PMP-peptide scans (Rottensteiner et al, 2004; Halbach et al, 2005). The analysis included seven mPTS-peptide motifs from six PMPs, which were scanned in seven two-residue steps each, using earlier published mPTS motifs (Halbach et al, 2005) (Figure 4A–D). The remaining four spots of each membrane were used for control experiments. All three Pex19p constructs showed a significant ability for specific binding of most of the selected mPTS peptides. The analysis revealed the strongest binding pattern for peptides from Pex11p, Pex13p, Pex16p, ALDP and the second mPTS motif of Pex26p. Figure 4.In vitro binding of mPTS-peptide motifs by Pex19p. Peptide blot data showing the binding profile of several Pex19p fragments to mPTS peptides. (A) Layout of the mPTS motifs (Halbach et al, 2005), consisting of 27 residues that were scanned over with 15-mer peptides in two-residue steps, indicated by Pex2p (residues 141–167, FV′IG′LL′KL′GG′LI′NFLIFLQRGKFATLT, spots 1–7), Pex11p-β (residues 180–206, GG′GL′PQ′LA′LK′LR′LQVLLLARVLRGHPP, spots 8–14), Pex13p (residues 171–197, LK′IH′FT′KV′FS′AF′ALVRTIRYLYRRLQR, spots 15–21), Pex16p (residues 109–135, RW′LV′IA′LI′QL′AK′AVLRMLLLLWFKAGL, spots 22–28), ALDP (residues 62–88, AA′KA′GM′NR′VF′LQ′RLLWLLRLLFPRVLC, spots 29–35), Pex26p motif-1 (residues 247–273, FF′SL′PF′KK′SL′LA′ALILCLLVVRFDPAS, spots 36–42), Pex26p motif-2 (residues 270–296, DP′AS′PS′SL′HF′LY′KLAQLFRWIRKAAFS, spots 43–49). In addition, the sequence segment covering helix α1 of Pex19p (residues 164–190, EG′DG′EG′NI′LP′IM′QSIMQNLLSKDVLYP, spots 50–56), the C-terminal PTS1 sequence of mSCP2 (residues 129–143, MKLQNLQLQPGNAKL, spot 57, negative control), a poly-histidine sequence (HHHHHH, spots 58–59, positive control) and an empty spot (60, negative control) were analysed. Spot numbers are indicated to the left and to the right. Experimental data are shown in (B) Pex19p(1–299), (C) Pex19p(161–299), (D) Pex19p(161–283) and (E) Pex19p(182–283). Download figure Download PowerPoint On the basis of these observations and peptide solubility tests, we selected two mPTS peptides, Pex13p and Pex11p, for quantitative binding affinity measurements using fluorescence polarization (FP) (Table II; Supplementary Figure S3). The C-terminal Pex19p(161–283) fragment used for X-ray structural analysis binds the two peptides with equilibrium dissociation constants of 8.5 and 23.4 μM, respectively. Interestingly, when adding the C-terminal tail (284–299) that comprises the Pex19p farnesylation site, the binding affinity decreases by about five-fold, suggesting a potentially inhibiting effect of the non-farnesylated tail. For comparison, Pex19p versions that include the N-terminal part of the sequence (1–160) show about the same or slightly increased binding affinity as Pex19p(161–283), regardless the presence or absence of the C-terminal tail. The data thus indicate that the C-terminal part of Pex19p is sufficient to recognize the mPTS motif, however, potentially complemented by a presently still uncharacterized, additional function of the N-terminal part of Pex19p in enhancing the mPTS-binding affinity. Table 2. Fluorescence polarization (FP) measurements Pex19p construct mPTS(Pex13p) mPTS(Pex11p) kD (μM) R2 a Relative 1/kD kD (μM) R2 a Relative 1/kD Pex19p(1–299) 8.4±0.9 0.99 1.01 11.8±1.5 0.99 1.98 Pex19p(1–283) 7.2±0.7 0.99 1.18 14.1±1.9 0.99 1.66 Pex19p(161–299) 38.3±5.7 0.99 0.22 85.8±25.4 0.97 0.27 Pex19p(161–283)b 8.5±1.4 0.98 1.00 23.4±7.1 0.96 1.00 Structure-based Pex19p mutants Pex19p(182–283) No No Pex19p(182–299) No No Pex19p(161–283, I178W) 70.1±5.6 0.99 0.12 Weakc Pex19p(161–283, I178P) Weakc No Pex19p(161–283, L182W) Weakc No Pex19p(161–283, L182P) 31.7±9.0 0.96 0.27 Weakc a Adjusted R2, expressing the goodness of fit, as defined in Origin version 8 software. b Reference, relative 1/KD=1.00. c Weak binding: KD>100 μM, quantitative calculation not possible. We were further interested in identifying the molecular features in the C-terminal helical bundle domain of Pex19p(161–283) that are responsible for mPTS-motif recognition. The high amount of sequence conservation of residues from helix α1 and the presence of an extensive hydrophobic patch generated by the structure of this helix (Figures 1B and 3, coloured in green) suggested that this region of Pex19p could be responsible for mPTS binding. To assess this hypothesis, we expressed and purified two additional versions of Pex19p that lack the corresponding sequence segment (182–283, 182–299). Comparative analysis of the CD spectra of these Pex19p fragments show that their fold content is virtually indistinguishable from other C-terminal Pex19p constructs (Supplementary Figure S1). Initial pepblot analysis revealed that the truncated Pex19p(182–283) construct basically has lost the ability to bind to any of the mPTS peptides used in this qualitative investigation (Figure 4E). Quantitative FP experiments confirmed that for both Pex19p fragments lacking the sequence segment that includes helix α1, Pex19p(182–283) and Pex19p(182–299) have entirely lost the ability to bind to both mPTS peptides (Table II), showing that the presence of helix α1 is essential for mPTS recognition. In an attempt to further locate residue-specific mPTS-binding sites, we mutated two conserved surface residues from helix α1, Ile178 and Leu182 (Figures 1B and 3), either into a tryptophan or proline, within the Pex19p(161–283) construct that was used for 3D structure determination. The rational of these mutations was to test for potential steric clashes and for structural interference of the formation of helix α1. As expected, the Pex19p(161–283, I178P) and Pex19p(161–283, L182P) variants show a slight but significant loss of secondary structural content, most likely because of disruption of helix α1 because of the insertion of a proline residue, whereas the spectra of the two equivalent tryptophan variants were basically indistinguishable with wild-type Pex19p(161–283) (Supplementary Figure S1, panels C and D). All Pex19p mutants led to a complete or almost complete loss of binding of the Pex11p mPTS peptide, whereas the observed effects on Pex13p binding were not as strong (Table II). Collectively, the data indicate that the binding modes of the two mPTS motifs are probably not identical, which may reflect the lack of specific, directed interactions within a Pex19p-mPTS interface that seems to be dominated by hydrophobic residues from the Pex19p helix α1 (Figure 3) and the two mPTS motifs. We also tested the importance of helix α1 in Pex19p for mPTS binding in vivo by monitoring Pex19p variants for their capacity to functionally complement a peroxisome biogenesis defect in PEX19-deficient fibroblasts. Functional complementation restores correct topogenesis of peroxisomal matrix and membrane proteins and formation of peroxisomes. Thus, only those cells in which functional Pex19p enables the correct assembly of peroxin complexes at the peroxisomal membrane exhibit a punctuate pattern for the PTS1-cargo EGFP-SCP, by co-localizing it with the endogenous PMP Pex14p (Figure 5). For this assay, we used the same single-residue Pex19p variants that were investigated for in vitro binding of mPTS peptides, however, in context of the full-length Pex19p sequence (Figure 3). In contrast to the tryptophan
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