Recombinant Human Peroxisomal Targeting Signal Receptor PEX5

Import of matrix proteins into peroxisomes requires two targeting signal-specific import receptors, Pex5p and Pex7p, and their binding partners at the peroxisomal membrane, Pex13p and Pex14p. Several constructs of human PEX5 have been overexpressed and purified by affinity chromatography in order to determine functionally important interactions and provide initial structural information. Sizing chromatography and electron microscopy suggest that the two isoforms of the human PTS1 receptor, PEX5L and PEX5S, form homotetramers. Surface plasmon resonance analysis indicates that PEX5 binds to the N-terminal fragment of PEX14-(1–78) with a very high affinity in the low nanomolar range. Stable complexes between recombinant PEX14-(1–78) and both the full-length and truncated versions of PEX5 were formed in vitro. Analysis of these complexes revealed that PEX5 possesses multiple binding sites for PEX14, which appear to be distributed throughout its N-terminal half. Coincidentally, this part of the molecule is also responsible for oligomerization, whereas the C-terminal half with its seven tetratricopeptide repeats has been reported to bind PTS1-proteins. A pentapeptide motif that is reiterated seven times in PEX5 is proposed as a determinant for the interaction with PEX14. Import of matrix proteins into peroxisomes requires two targeting signal-specific import receptors, Pex5p and Pex7p, and their binding partners at the peroxisomal membrane, Pex13p and Pex14p. Several constructs of human PEX5 have been overexpressed and purified by affinity chromatography in order to determine functionally important interactions and provide initial structural information. Sizing chromatography and electron microscopy suggest that the two isoforms of the human PTS1 receptor, PEX5L and PEX5S, form homotetramers. Surface plasmon resonance analysis indicates that PEX5 binds to the N-terminal fragment of PEX14-(1–78) with a very high affinity in the low nanomolar range. Stable complexes between recombinant PEX14-(1–78) and both the full-length and truncated versions of PEX5 were formed in vitro. Analysis of these complexes revealed that PEX5 possesses multiple binding sites for PEX14, which appear to be distributed throughout its N-terminal half. Coincidentally, this part of the molecule is also responsible for oligomerization, whereas the C-terminal half with its seven tetratricopeptide repeats has been reported to bind PTS1-proteins. A pentapeptide motif that is reiterated seven times in PEX5 is proposed as a determinant for the interaction with PEX14. peroxisomal targeting signal tetratricopeptide repeat glutathioneS-transferase dithiothreitol tobacco etch virus polyacrylamide gel electrophoresis response units nickel-nitrilotriacetic acid N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine Proteins residing inside organelles must be translocated across lipid bilayers to reach their final destination. It has been shown for some compartments that protein translocation across hydrophobic membranes occurs through proteinaceous complexes, which are evolutionary conserved (1Pohlschröder M. Prinz W.A. Hartmann E. Beckwith J. Cell. 1997; 91: 563-566Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). However, the initial notion that the translocation machineries and their mechanisms are also conserved among different organelles has turned out to be an oversimplification (2Schatz G. Dobberstein B. Science. 1996; 271: 1519-1526Crossref PubMed Scopus (914) Google Scholar). Combined classical and molecular genetic analyses of protein import into peroxisomes in lower and higher eukaryotes have identified a large number of genes (PEX genes), the protein products (peroxins) of which play essential roles in the different steps of transport from the cytoplasm to the peroxisomal matrix (for recent reviews, see Refs.3Subramani S. Physiol. Rev. 1998; 78: 171-188Crossref PubMed Scopus (281) Google Scholar, 4Erdmann R. Veenhuis M. Kunau W.-H. Trends Cell Biol. 1997; 7: 400-407Abstract Full Text PDF PubMed Scopus (115) Google Scholar, 5Elgersma Y. Tabak H.F. Biochim. Biophys. Acta. 1996; 1286: 269-283Crossref PubMed Scopus (70) Google Scholar, 6Waterham H.R. Cregg J.M. Bioessays. 1997; 19: 57-66Crossref PubMed Scopus (53) Google Scholar). In a number of cases it has been possible to link mutations in human orthologues to peroxisomal disorders, most being fatal (7Subramani S. Nat. Genet. 1997; 15: 331-333Crossref PubMed Scopus (82) Google Scholar,8Kunau W.-H. Curr. Opin. Microbiol. 1998; 1: 232-237Crossref PubMed Scopus (46) Google Scholar). Import of peroxisomal matrix proteins (for recent reviews, see Refs.3Subramani S. Physiol. Rev. 1998; 78: 171-188Crossref PubMed Scopus (281) Google Scholar, 4Erdmann R. Veenhuis M. Kunau W.-H. Trends Cell Biol. 1997; 7: 400-407Abstract Full Text PDF PubMed Scopus (115) Google Scholar, 5Elgersma Y. Tabak H.F. Biochim. Biophys. Acta. 1996; 1286: 269-283Crossref PubMed Scopus (70) Google Scholar, 6Waterham H.R. Cregg J.M. Bioessays. 1997; 19: 57-66Crossref PubMed Scopus (53) Google Scholar) depends on two well defined targeting signals, termed PTS1 and PTS2.1 Two import receptors, Pex5p and Pex7p, have been identified which specifically bind PTS1 and PTS2, respectively. Both receptor proteins contain repetitive sequence motifs, each belonging to established structural families. Pex5p possesses seven tetratricopeptide repeats (TPRs) (9Hirano T. Kinoshita N. Morikawa K. Yanagida M. Cell. 1990; 60: 319-328Abstract Full Text PDF PubMed Scopus (237) Google Scholar, 10Lamb J.R. Tugendreich S. Hieter P. Trends Biochem. Sci. 1995; 20: 257-259Abstract Full Text PDF PubMed Scopus (545) Google Scholar, 11Goebl M. Yanagida M. Trends Biochem. Sci. 1991; 16: 173-177Abstract Full Text PDF PubMed Scopus (372) Google Scholar), while Pex7p has six WD-40 motifs (12Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1280) Google Scholar). Pex13p and Pex14p, membrane-bound peroxins, have been demonstrated to bind the two PTS receptors. Both are components of a recently reported complex network of interacting peroxins (4Erdmann R. Veenhuis M. Kunau W.-H. Trends Cell Biol. 1997; 7: 400-407Abstract Full Text PDF PubMed Scopus (115) Google Scholar, 8Kunau W.-H. Curr. Opin. Microbiol. 1998; 1: 232-237Crossref PubMed Scopus (46) Google Scholar, 13Huhse B. Rehling P. Albertini M. Blank L. Meller K. Kunau W.H. J. Cell Biol. 1998; 140: 49-60Crossref PubMed Scopus (124) Google Scholar, 14Albertini M. Rehling P. Erdmann R. Girzalsky W. Kiel J.A.K.W. Veenhuis M. Kunau W.-H. Cell. 1997; 89: 83-92Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Pex13p binds the PTS1 receptor Pex5p with its cytoplasmic SH3 domain (15Gould S.J. Kalish J.E. Morrell J.C. Bjorkman J. Urquhart A.J. Crane D.I. J. Cell Biol. 1996; 135: 85-95Crossref PubMed Scopus (209) Google Scholar, 16Erdmann R. Blobel G. J. Cell Biol. 1996; 135: 111-121Crossref PubMed Scopus (184) Google Scholar, 17Elgersma Y. Kwast L. Klein A. Voorn-Brouwer T. van den Berg M. Metzig B. America T. Tabak H.F. Distel B. J. Cell Biol. 1996; 135: 97-109Crossref PubMed Scopus (183) Google Scholar). Pex14p interacts with both PTS-dependent receptors (14Albertini M. Rehling P. Erdmann R. Girzalsky W. Kiel J.A.K.W. Veenhuis M. Kunau W.-H. Cell. 1997; 89: 83-92Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 18Brocard C. Lametschwandtner G. Koudelka R. Hartig A. EMBO J. 1997; 16: 5491-5500Crossref PubMed Scopus (107) Google Scholar, 19Fransen M. Terlecky S.R. Subramani S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8087-8092Crossref PubMed Scopus (135) Google Scholar). Therefore, it was proposed that Pex14p may represent the point of convergence of both PTS-dependent import pathways (14Albertini M. Rehling P. Erdmann R. Girzalsky W. Kiel J.A.K.W. Veenhuis M. Kunau W.-H. Cell. 1997; 89: 83-92Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). The functional importance of Pex14p for peroxisome biogenesis is further supported by its interaction with Pex13p and with an additional membrane bound peroxin Pex17p (13Huhse B. Rehling P. Albertini M. Blank L. Meller K. Kunau W.H. J. Cell Biol. 1998; 140: 49-60Crossref PubMed Scopus (124) Google Scholar). An understanding of peroxisomal protein import at the molecular level requires knowledge about the structure of the peroxins and conformational changes resulting from their interactions. Herein, we report initial biochemical and biophysical studies of the human PTS1 receptor PEX5, overexpressed in Escherichia coli, purified to homogeneity, and its interaction with PEX14. Cloning experiments were performed withE. coli strain DH5α. The cDNA fragments coding for PEX5L, PEX5S, and the truncated versions PEX5L-(1–251) and PEX5L-(323–639) were from pcDNA3 (Invitrogen) derived plasmids. 2G. Dodt, D. Warren, T. Yahraus, M. Soukupova, E. Becker, P. Rehling, and S. J. Gould, manuscript in preparation. A polymerase chain reaction product corresponding to PEX5L-(214–639) was amplified from pGD106 (20Braverman N. Dodt G. Gould S.J. Valle D. Hum. Mol. Genet. 1998; 7: 1195-1205Crossref PubMed Scopus (148) Google Scholar) using the sense primer 5′-ATTGTCGACCATGGAGTTCCTGAAATTC-3′ containing aNcoI site (underlined) and a vector-specific antisense primer corresponding to the Sp6 promotor region (5′-TATTTAGGTGACACTATAG-3′). DNA fragments encoding full-length PEX5L, PEX5S, and the C-terminal fragments PEX5L-(214–639) and PEX5L-(323–639) were digested withNcoI/BglII. The resulting fragments, which also contained additional 218 base pairs from the 3′-noncoding region and anNcoI/NotI fragment corresponding to PEX5L-(1–251), were subcloned into expression plasmids kindly provided by G. Stier (EMBL, Heidelberg). These plasmids were derived from pET9d (Novagen) by replacing the unique NcoI site with a DNA fragment encoding a hexahistidinyl (His6) tag and a TEV (tobacco etch virus) protease cleavage site and containing several unique endonuclease recognition sites. The PEX5 coding fragments were ligated with NcoI/BamHI orNcoI/NotI digested vectors, thus, fusing a peptide with the sequence MKHHHHHHPMSDYDIPTTENLYFQGAM to the N termini of the PEX5 proteins. A DNA fragment encoding GST-PEX14-(1–78) was amplified by polymerase chain reaction using pGEX-PEX14-(1–134) 3Will, G., Soukupova, M., Hong, X., Erdmann, K. S., Kiel, J. A. K. W., Dodt, G., Kunau, W.-H., and Erdmann, R., in press. as a template and the primers 5′-GCAGTGGTCTCTCATGTCCCCTATACTAGGTT-3′ (sense,BsaI recognition site underlined) and 5′-CCAAGCTTAGTCGACCGAAGGCTCATCGGCAGC-3′ (antisense,HindIII recognition site underlined), digested withBsaI (creating a NcoI-compatible 5′-overhang) andHindIII and subcloned intoNcoI/HindIII digested pET21d plasmid (Novagen). The DNA constructs were verified by restriction analysis and partial DNA sequence analysis using an ABI automated sequencer (Applied Biosystems). A nucleotide exchange was found in the coding region for PEX5L-(1–251), resulting in an amino acid substitution of glutamic acid to aspartic acid at position 33. Expression of His6-tagged PEX5 forms and GST-PEX14-(1–78) was carried out in E. coli strain BL21(DE3). Fresh transformants were grown in Luria Broth medium supplemented with 30 mg/liter kanamycin or 100 mg/liter ampicillin. Cells were induced in the mid-log phase with 0.4 mmisopropyl-β-d-thiogalactopyranoside and grown for another 4–6 h at a temperature of 37 °C. Cells were harvested by centrifugation and were stored at −80 °C. Cell pellets were thawed in buffer A containing 50 mm mm and mm dithiothreitol and with a in or two Cell and were by centrifugation The containing the proteins was on a or a with buffer A. PEX5L-(323–639) was purified from on as in the PEX5 proteins were from with mm GST-PEX14-(1–78) bound to a with mm was affinity steps were carried out at and by cleavage was performed at 37 for was at a of of purified whereas units of TEV protease of PEX5 proteins the The was with mm for at 37 °C. A exchange with mm mm was to and and to the recombinant proteins. that PEX14-(1–78) was with a between and mm whereas for recombinant PEX5 from to mm were chromatography was performed on a or a with buffer A at a of at °C. were and respectively. were to Biochem. PubMed Scopus Google Scholar) using as a proteins were by Nature. PubMed Scopus Google Scholar) or to the of and G. Biochem. PubMed Scopus Google Scholar) and by or J. R. 1: Scopus Google Scholar). were to of and for were with of buffer and with a of for 30 was by The and were in a at on the interaction between the PEX5 proteins and GST-PEX14-(1–78) were performed by plasmon resonance using a binding to as the is on a and the interaction with an in the is in the on the are to changes in the of the is by using the of plasmon resonance and in response units whereas to a in of about of E. B. C. J. Sci. 1991; Scopus Google Scholar). number were to the to a response of by the GST-PEX14-(1–78) at a of was to an level of about to a interaction experiments were carried out in a buffer containing 50 mm and mm at a of 30 PEX5L, PEX5S, PEX5L-(214–639) were in from to binding was using an for each changes to buffer were by using each interaction the were by two with mm or with the by with and phase were both for The resulting were with the The were from the binding for the of the PEX5 The from to were to were with the and of to the order A using in The binding were from the to In these the are is the response at the initial is the response at a is the binding is the of the a are the and and is the binding It has been reported that the PTS1 receptor Pex5p of and higher eukaryotes bind to the two peroxins Pex13p (15Gould S.J. Kalish J.E. Morrell J.C. Bjorkman J. Urquhart A.J. Crane D.I. J. Cell Biol. 1996; 135: 85-95Crossref PubMed Scopus (209) Google Scholar, 16Erdmann R. Blobel G. J. Cell Biol. 1996; 135: 111-121Crossref PubMed Scopus (184) Google Scholar, 17Elgersma Y. Kwast L. Klein A. Voorn-Brouwer T. van den Berg M. Metzig B. America T. Tabak H.F. Distel B. J. Cell Biol. 1996; 135: 97-109Crossref PubMed Scopus (183) Google Scholar) Pex14p (14Albertini M. Rehling P. Erdmann R. Girzalsky W. Kiel J.A.K.W. Veenhuis M. Kunau W.-H. Cell. 1997; 89: 83-92Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 18Brocard C. Lametschwandtner G. Koudelka R. Hartig A. EMBO J. 1997; 16: 5491-5500Crossref PubMed Scopus (107) Google Scholar, 19Fransen M. Terlecky S.R. Subramani S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8087-8092Crossref PubMed Scopus (135) Google Scholar) at the cytoplasmic of the peroxisomal In higher eukaryotes two forms of PEX5 have been reported which in an of 37 amino as a of (20Braverman N. Dodt G. Gould S.J. Valle D. Hum. Mol. Genet. 1998; 7: 1195-1205Crossref PubMed Scopus (148) Google K. K. Y. E. M. T. T. K. Y. Mol. Cell. Biol. 1998; PubMed Google Scholar, G. N. C. A. P. Valle D. Gould S.J. Nat. Genet. 1995; PubMed Scopus Google Scholar). into the structural of the interactions of Pex5p with peroxins, large of protein were this we His6-tagged versions of the and the form of human PEX5 PEX5L and and in E. coli by using a expression A TEV protease cleavage site was between the and the PEX5 the of the affinity Expression and were by both recombinant PEX5 forms we high of expression both proteins with their molecular of and with an molecular of as been for G. N. C. A. P. Valle D. Gould S.J. Nat. Genet. 1995; PubMed Scopus Google Scholar, U. Subramani S. J. Cell Biol. 1995; PubMed Scopus Google Scholar). In studies truncated versions of PEX5 were also A between and molecular was for an N-terminal fragment for C-terminal fragments that the is to the high of the N-terminal half. Both PEX5L and PEX5S, were and be purified to in using affinity chromatography The tag was by TEV The and were by exchange of protein be from experiments have been chromatography a molecular of about for both PEX5 forms a of The of PEX5 was supported by from electron of recombinant with a of about These have the of a with a and appear to of It is important to that the recombinant PEX5 also to form which were in the It is that their number and is in low that their is by interactions. of 100 have been for another containing protein (9Hirano T. Kinoshita N. Morikawa K. Yanagida M. Cell. 1990; 60: 319-328Abstract Full Text PDF PubMed Scopus (237) Google microscopy of recombinant human of purified in mm mm and mm are by Two of are shown at higher purified the buffer the number of is the number and of is The 100 are to be in interactions. the binding of the PTS1 to its receptor Pex5p is by the G. N. C. A. P. Valle D. Gould S.J. Nat. Genet. 1995; PubMed Scopus Google Scholar, C. T. Hartig A. Biochem. Biophys. 1994; PubMed Scopus Google Scholar, S.R. D. E. Subramani S. EMBO J. 1995; PubMed Scopus Google Scholar). are also to with each J.R. Tugendreich S. Hieter P. Trends Biochem. Sci. 1995; 20: 257-259Abstract Full Text PDF PubMed Scopus (545) Google Scholar, M. Mol. Cell. Biol. 1996; 16: PubMed Scopus Google Scholar). In order to the responsible for and of PEX5, truncated His6-tagged PEX5L-(1–251), and PEX5L-(323–639) were coli purified by affinity chromatography and to the two proteins were the the seven motifs was and be purified 100 amino which PEX5L-(214–639) and are to and the protein analyses an molecular of for PEX5L-(1–251), whereas PEX5L-(214–639) a protein of These are with a structure for PEX5L-(1–251), which the N-terminal half of PEX5, and a structure for PEX5L-(214–639) the C-terminal of of PEX5L-(1–251) very in in to found in of full-length PEX5 However, in to full-length PEX5 were A of the for the PEX5 PEX5L, PEX5S, PEX5L-(1–251), and revealed that the of the between amino acid and to the to form this structural is on a determinant the amino in to be to the seven interaction of Pex5p and Pex14p was by the to the that Pex14p be a for the protein of the PTS1 receptor at the peroxisomal membrane (14Albertini M. Rehling P. Erdmann R. Girzalsky W. Kiel J.A.K.W. Veenhuis M. Kunau W.-H. Cell. 1997; 89: 83-92Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 18Brocard C. Lametschwandtner G. Koudelka R. Hartig A. EMBO J. 1997; 16: 5491-5500Crossref PubMed Scopus (107) Google Scholar). analysis the binding site for to the amino acid of and W.-H. Kunau, manuscript in preparation. the human of was identified and also was shown to with PEX5 M. Terlecky S.R. Subramani S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8087-8092Crossref PubMed Scopus (135) Google Scholar). of Pex14p orthologues that the amino acid of to the amino acid of human we a protein of a cleavage and the amino acid of human to the binding of PEX5 and in vitro. The protein was in E. coli and be purified in high of GST-PEX14-(1–78) or in were and into two was through a while the was to in in both cases the complex of and GST-PEX14-(1–78) be specifically by and respectively. The binding of PEX5 to the N-terminal fragment of was expression of of GST-PEX14-(1–78) to the that PEX5 binds PEX14. was supported by another in binding with purified GST-PEX14-(1–78) and The two proteins were in a of and by that the of the two proteins contained an which is each protein was high molecular complex of GST-PEX14-(1–78) and as by analysis of in formed complexes between and analysis of complexes by and affinity chromatography and by chromatography Proteins were in polyacrylamide and with The of complexes was carried out in mm mm and mm the affinity of the complexes by and by of E. coli or GST-PEX14-(1–78) were and for h at with chromatography on a was performed with a of both affinity purified proteins for at the gel for purified and GST-PEX14-(1–78) and for the of both The of complexes a high affinity binding between PEX5 and The of interaction between PEX5 and the peroxisomal protein were using plasmon resonance Both recombinant PEX5 PEX5L and PEX5S, bind to the GST-PEX14-(1–78) with a whereas the is very The binding are in the low nanomolar were also by analysis and found to be in with by the experiments with His6-tagged PEX5 on a as and GST-PEX14-(1–78) as the high affinity binding and binding of the interaction of GST-PEX14-(1–78) and PEX5 and binding were from plasmon resonance using GST-PEX14-(1–78) to an as the and the PEX5 forms as in a and binding were from plasmon resonance using GST-PEX14-(1–78) to an as the and the PEX5 forms as In order to determine the part of GST-PEX14-(1–78) the binding with PEX5, the protein was with to a fragment PEX14-(1–78) in binding experiments with different forms of the form PEX5L, the form PEX5S, the N-terminal fragment PEX5L-(1–251), and the C-terminal fragment this the components were and by the In binding experiments PEX14-(1–78) was in of the with the PEX5 proteins in the or of PEX14-(1–78) revealed that the PEX5 proteins PEX14-(1–78) was the of analyses of the corresponding the of both PEX14-(1–78) and the PEX5 whereas the PEX14-(1–78) was found as a in is shown for PEX5L in was that forms of PEX5 were to with binding is with the from plasmon resonance analysis that both truncated versions PEX5L-(1–251) and PEX5L-(214–639) bind to GST-PEX14-(1–78) with affinity as the and the form of PEX5 and the molecular of of the different PEX5 and of their complexes, we the of the complexes to and The and forms of PEX5 to bind seven and six PEX14-(1–78) fragments that the acid of the form binding The number of binding sites was by analysis of the complexes formed by the two truncated PEX5 proteins. The N-terminal fragment PEX5L-(1–251) to bind fragments of PEX14-(1–78) whereas the C-terminal fragment PEX5L-(214–639) binds or the region of these two forms of PEX5 contained the with binding their binding sites to six or of the of molecular analysis of PEX5 proteins in and of PEX14-(1–78) molecular of PEX5 proteins and their complexes with PEX14-(1–78) were by chromatography as shown in molecular from the amino acid molecular in a The molecular of PEX5 proteins and their complexes with PEX14-(1–78) were by chromatography as shown in molecular from the amino acid molecular In order to a structural analysis of the import for peroxisomal matrix we recombinant human PEX5 and its binding to human at the peroxisomal in vitro. In these studies we an N-terminal fragment of the amino acid the full-length protein as the binding site for the PTS1 receptor of S. been to the corresponding fragment of and W.-H. Kunau, manuscript in preparation. PEX5, as of the protein is of two different the conserved C-terminal half seven motifs and the N-terminal which possesses a amino that are conserved among the region was shown to the binding to proteins G. N. C. A. P. Valle D. Gould S.J. Nat. Genet. 1995; PubMed Scopus Google Scholar, C. T. Hartig A. Biochem. Biophys. 1994; PubMed Scopus Google Scholar, S.R. D. E. Subramani S. EMBO J. 1995; PubMed Scopus Google Scholar), a was to the N-terminal half. In this we that the N-terminal half of PEX5 binds PEX14. human PEX5 is a protein with a as by chromatography and by electron microscopy However, it also forms the C-terminal fragment in to the N-terminal fragment as a the to form to be a of the amino the suggest that be by the A C-terminal fragment which of the motifs is while the N-terminal fragment A of the recombinant PEX5 is the of multiple six or seven binding sites for the fragment which to bind with very high sequence analysis of PEX5L seven pentapeptide repeats the of amino These repeats are by the of have been reported to in the form of human PEX5 G. N. C. A. P. Valle D. Gould S.J. Nat. Genet. 1995; PubMed Scopus Google Scholar). An of the proteins that these repeats are found in each the number and the N-terminal half of the Pex5p of possesses of these motifs whereas Pex5p of S. analysis of these repeats in of the orthologues revealed that the position between the conserved amino acid be amino whereas is a for at the position and the position is in out of motifs by aspartic glutamic or structure B. 1996; PubMed Google Scholar) for proteins revealed that the motifs are of that the conserved of or are to the of the the number and of the repeats in human PEX5 the number of binding sites for the fragment in the PEX5 forms it is to that the repeats provide the structural for this It that the binding sites found for the PEX14-(1–78) fragment in in the of possible interacting protein are in by full-length It is very to a protein of this and with its into seven binding sites a of about amino acid the which the multiple binding sites of PEX5 for have in be that the number of binding sites the of binding between the two partners the that have to be In the that the binding sites out to have different binding it is that interacts with low affinity binding sites and is to the high affinity this additional fragments and forms of PEX5, as well as full-length PEX14, have to be the that recombinant PEX5 be in high and for in binding plasmon resonance to the structural of interactions between essential components of peroxisomal protein complexes are in for Stier for the of for and for the are most to of the for and throughout the of this