Supplementary Material

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1 Supplementary Material Rescuing the Rescuer: On the Protein Complex between the Human Mitochondrial Acyl Carrier Protein and ISD11 María Georgina Herrera, María Florencia Pignataro, Martín Ezequiel Noguera, Karen Magalí Cruz and Javier Santos* Institute of Biological Chemistry and Physical Chemistry, Dr Alejandro Paladini (UBA-CONICET), University of Buenos Aires, Junín 956, (C1113AAD), Buenos Aires, Argentina *Corresponding Author: Javier Santos. Institute of Biological Chemistry and Physicochemistry, Dr Alejandro Paladini (UBA-CONICET). Junín 956, 1113AAD, Buenos Aires, Argentina. Telephone: ext. 108, Fax: Running Title: The Human ACP-ISD11 Protein Complex 1

2 Figure S1. Conservation Among ISD11 Protein Sequences. LOGO for the ISD11 proteins was prepared using 125 sequences retrieved from CONSURF 1, Numbering was according to the 2 precursor form of the human ISD11. LOGO was prepared using WEBLOGO at 2

3 Cycle 1 Cycle 2 Cycle 3 Cycle 4 Absorbance (269 nm) Cycle 5 Cycle 6 Cycle 7 Cycle 8 Cycle 9 Markers Elution time (min) Figure S2. N-terminal Sequencing of Recombinant Human Mitochondrial ACP. C4-RP-HPLC purified protein (pool of peaks labelled as 2 in Figure 1B) was submitted to Edman degradation. The sequence obtained after nine degradation cycles was -SDMPPLTLE- indicating that the Met1 is not present. 3

4 B Figure S3. ESI-MS Analysis of the Fractions Obtained by Reversed Phase HPLC of the ACP-ISD11 Complex. ESI-MS of peaks eluted after a reversed phase HPLC using a C4 column are shown. (A) Deconvoluted spectra corresponding to peak 1 (assigned as ISD11) and (B) deconvoluted spectra corresponding to a fraction containing all peaks marked as 2 in Figure 1 (assigned as ACP). The most important corresponding ions are shown in Table S1. 4

5 Table S1: Mass Charge Relation of the Most Abundant Ions Obtained by ESI-MS. ISD11 and ACP were separated by Reversed Phase HPLC as described in Figure S1. ISD11 (m/z) ACP (m/z)

6 Figure S4. Temperature-induced Denaturation of ACP-ISD11 Monitored by the Change in Sypro Orange dye Fluorescence. Protein (10 µm) was prepared in buffer 75 mm sodium phosphate, 300 NaCl, ph 8.0. Samples without protein was also included as controls. The final concentration of the dye was 1 (Thermo Fisher Scientific). The temperature ramp (from 20 to 90 C) was carried out at 1 C min -1. The excitation and emission ranges were and nm, respectively. The fluorescence signal is quenched in the aqueous environment but becomes unquenched when the probe binds to the apolar residues upon unfolding. Experiments by triplicate were carried out in a Step One Real-Time-PCR instrument (Applied Biosystems). 6

7 Figure S5. Conservation Among Acyl Carrier Proteins. (A) Sequence alignment of ACPs from human mitochondria and E. coli is shown. Alignment was carried out using Seaview and muscle algorithm. pacp and macp indicate the human mitochondrial precursor of ACP and the recombinant mature form of the variant produced in this work. Numbering corresponds to that of the human ACP precursor. (B) LOGO for the ACP proteins was prepared using 135 sequences retrieved from CONSURF 1, Numbering was according to macp, where Met 1 was taken as the first residue. LOGO was prepared using WEBLOGO 2 at 7

8 Controlled Proteolysis of ACP-ISD11 Complex with Chymotrypsin Proteolysis assay was performed to evaluate the conformation of ACP-ISD11 complex and ISD11 refolded in the presence of 1mM DDM. The experiment was carried out using chymotrypsin that cuts at aromatic residues. High accessibility of proteolytic sites and/or high flexibility at specific regions of the protein chain may result in accentuated proteolysis. ACP contains 3 Phe and 4 Tyr, whereas recombinant ISD11 contains 3 Phe and 6 Tyr (neither ACP nor ISD11 have Trp residues). In the case of recombinant ISD11, one Tyr and one Phe are included in the designed proteolytic site between His tag and the protein. For this reason, these two residues are expected to exhibit full-accessibility to the solvent and be good substrate for proteolysis. In addition, Tyr26, Tyr28, Tyr31 and Tyr76 show high accessibility as judge by the analysis of the structure of the complex between E. coli ACP and human ISD11 (Figure S6). By contrast, the solvent accessibility profile of ACP (based on PDB: 5OOI.pdb) is characterized by the relatively high protection of aromatic residues (Figure S7, accessibility is lower than 50%), with the exception of Tyr88, which is not present in the model and most likely it is full-accessible because of its location, just before the last residue of the protein. Therefore, we expected a moderated resistance to proteolysis of ISD11, and in principle, we anticipated lower digestion of ACP compared to ISD11. The latter might change its flexibility upon ACP and/or NFS1 binding. In addition, the conformation of the heterodimer ACP-ISD11 may change in the absence of NFS1 and the existence of an equilibrium between dimer and tetramer (ACP-ISD11 and [ACP-ISD11] 2 ) may also alter the conformational stability and/or the accessibility of the aromatic residues. Protein samples were analyzed by HPLC to gain more resolution and we decided to use the RP C4- HPLC column that separates well ISD11 from ACP. The experimental condition of controlled proteolysis was 100:1 protein to protease mass ratio, incubation at 20 C. After 0, 10, min and 1h incubation (20 C) with or without protease (1:100, protease: protein, mass ratio), proteolysis was stopped by a sample dilution (1:10) in H 2 O 0.05% TFA µg of protein was loaded onto C4 equilibrated under acidic condition (H % TFA) and the elution was performed through a linear gradient from 0 to 100% ACN (0.05% TFA). In addition, we included an over-night incubation at 20 C, in the same experimental conditions. Interestingly, near 40% of the ISD11 (in the context of ACP-ISD11 complex, Figure S8, C and D) was resistant to proteolysis during the first hour of incubation. This firmly suggested that most of the heterodimer ACP-ISD11 is moderately resistant, and most likely, well-folded. The analysis of the profile corresponding to the over-night incubation showed that ISD11 was complete digested (Figure S8 A and C), whereas more than 80% of ACP remains intact (Figure S8 A and D), suggesting the existence of higher flexibility and high accessibility to proteolysis sites in the case of the former. On the other hand, when ISD11 was refolded in the presence of 1 mm DDM, the protein exhibited significantly lower resistance to proteolysis compared to the observed for ACP-ISD11 complex. In fact, near 70% of the refolded protein was digested during the first 10 min of the reaction and after 30 min full-length ISD11 was not detected by HPLC (Figure S9). Finally, ACP-ISD11 was not digested faster when 1 mm DDM was added to the reaction compared with the same reaction in the absence of detergent, showing that chymotrypsin was equally active in both conditions and revealing that the effect is not on the protease activity (Figure S10). 8

9 A B Figure S6. Solvent Accessibility Analysis of ISD11. Accessibility of the aromatic side-chains in (A) ISD11 in the context of ACP-ISD11 complex, assuming the conformation found in PDB ID: 5WGB for the complex between E. coli ACP and human mitochondria ISD11, or (B) in the isolated ISD11, assuming the ISD11 conformation found in PDB ID: 5WGB, in which E. coli ACP was deleted. ASAView 3 was used for calculation of the relative accessible surface area per residue. Tyr13 is part of the LYR motif; the N-terminal sequence of the recombinant ISD11 includes MHHHHHHENLYFQG and this sequence is not considered for this figure. Tyr and Phe included in this sequence are expected to be full accessible to the protease. Numbering corresponds to the natural sequence of full-length human ISD11. Figure S7. Accessibility of the Aromatic Side-chains in Human Mitochondrial ACP. The calculation was carried out assuming the protein conformation found in PDB ID: 5OOI, in which the rest of the protein chains were deleted. ASAView 3 was used for calculation of the relative accessible surface area per residue. Numbering corresponds to the sequence of the recombinant ACP that readers may find here as supplementary information. The last five residues (not present in the model) were not included in the calculation (KDVYE). 9

10 Figure S8. Resistance to Proteolysis of ACP-ISD11. (A) RP-HPLC profiles (C4 column) corresponding to 0, 10, 60 and 720 min of incubation with protease (1:100, protease: protein, mass ratio, at 20 C). (B) Zoom of the region Peptides corresponding to the peptides generated by digestion of the protein sample. (C) Zoom of the region 1 that corresponds to the ISD11 protein. (D) Zoom of the region 2 that corresponds to the recombinant mitochondrial ACP and the same protein with different acyl chains. (E) Peaks corresponding to ISD11 and ACP were integrated and the values obtained for each incubation time were plotted. Profiles for times 20 min and 30 min are not shown in panels A, B, C and D for clarity. In all cases, proteolysis was stopped by a sample dilution (1:10) in H 2 O 0.05% TFA. 20 µg of protein were loaded onto C4 equilibrated under acidic condition (H % TFA) and the elution was performed through a linear gradient from 0 to 100% ACN (0.05% TFA). Elution was followed by absorbance at 215 nm. 10

11 Figure S9. Resistance to Proteolysis of ISD11 refolded in the presence of 1 mm DDM detergent. (A) RP- HPLC profiles (C4 column) corresponding to 0, 10 and 20 min of incubation with protease (1:100, protease: protein, mass ratio, at 20 C). (B) Zoom of the region Peptides corresponding to the peptides generated by digestion of the protein sample. (C) Zoom of the region 1 that corresponds to the ISD11 protein. (E) Peaks corresponding to ISD11 were integrated and the values obtained for each incubation time were plotted. In all cases, proteolysis was stopped by a sample dilution (1:10) in H 2 O 0.05% TFA. 20 µg of protein were loaded onto C4 equilibrated under acidic condition (H % TFA) and the elution was performed through a linear gradient from 0 to 100% ACN (0.05% TFA). Elution was followed by absorbance at 215 nm. 11

12 Figure S10. Similar Resistance to Proteolysis of ACP-ISD11 in the Presence or Absence of DDM. RP-HPLC profiles (C4 column) corresponding to 10 min of incubation with protease (1:100, protease: protein, mass ratio, at 20 C), in the Presence (red) or Absence (blue) of 1 mm DDM. Proteolysis was stopped by a sample dilution (1:10) in H 2 O 0.05% TFA. 20 µg of protein were loaded onto C4 equilibrated under acidic condition (H % TFA) and the elution was performed through a linear gradient from 0 to 100% ACN (0.05% TFA). Elution was followed by absorbance at 215 nm. 12

13 ACP chain C ACP chain G Trp 97 Trp 440 ISD11 chain B ISD11 chain F NFS1 chain E NFS1 chain A Trp Trp 440 Trp 454 Figure S11. Representation of the Interaction Between NFS1 and ISD11 subunits. This representation was built with PDBID: 5WLW. NFS1 subunits forming a dimer are shown in ribbon representation, in orange and cyan (subunits A and E respectively), whereas ISD11 subunits are in green and violet (subunits B and F respectively). The VDW surface corresponding to Trp 97 (subunit A) is in gray, whereas VDW surface corresponding to the residues and 98 of NFS1 subunit A is in orange. VDW surface for residues and from NFS1 subunit E are in cyan (light and dark, respectively), and the surface corresponding to residues of ISD11 subunit F is in violet. E. coli ACP subunits are in brown and light green (subunits G and C, respectively, ribbon representation), and phosphopantetheine moieties linking ACP and ISD11 subunits are in sticks. Trp 97, Trp 440 and Trp 454 (the side-chain is truncated in the latter) from subunit E are in yellow (VDW representation). 13

14 Trp 97 from NFS1 seems to be the one that could report contact establishment between monomers of NFS1 and the interaction with ISD11. Even though this residue is not in direct contact with interface between subunits it could sense interactions throughout residues from NFS1, a stretch that is in contact with this Trp and directly interact with residues and from the other subunit of the NFS1 dimer. Moreover, stretch may sense interaction with ISD11 because it is in contact with residues from the latter. The other two tryptophan residues from NFS1 are located far from the interaction surface and show high solvent accessibility, as judged by the structure of the complex (PDBID 5WLW). Remarkably, amino acid sequences of human mitochondrial ACP and ISD11 do not contain tryptophan residues. 14

15 Protein Sequences Human mitochondrial Acyl carrier protein (ACP). This recombinant variant of the mature form starts at residue Ser69 of the precursor protein (highlighted in yellow, see below). In addition, Ser112 (highlighted in grey) is the serine residue that undergoes posttranslational modification, homolog to Ser37 in E. coli ACP (numbering from Met1): >recombinant mature mitochondrial ACP variant [ Homo sapiens] MSDMPPLTLEGIQDRVLYVLKLYDKIDPEKLSVNSHFMKDLGLDSLDQVEIIMAMEDEFGFEIPDIDAEKLMCPQEIVDYIADKKDV YE >NP_ acyl carrier protein, mitochondrial precursor [Homo sapiens] MASRVLSAYVSRLPAAFAPLPRVRMLAVARPLSTALCSAGTQTRLGTLQPALVLAQVPGRVTQLCRQYSDMPPLTLEGIQDRVLYVL KLYDKIDPEKLSVNSHFMKDLGLDSLDQVEIIMAMEDEFGFEIPDIDAEKLMCPQEIVDYIADKKDVYE >AAB acyl carrier protein [Escherichia coli] MSTIEERVKKIIGEQLGVKQEEVTNNASFVEDLGADSLDTVELVMALEEEFDTEIPDEEAEKITTVQAAIVYINGNQA Human mitochondrial ISD11 (isoform 1) was expressed with an his6 tag followed by a TEV protease site. The first residue corresponding to ISD11 sequence is Ala2 (highlighted in green). The Leu-Tyr-Arg motif is highlighted in magenta. >Human ISD11 (LYRM4) with a His6 tag in the N-terminal. MHHHHHHENLYFQGAASSRAQVLSLYRAMLRESKRFSAYNYRTYAVRRIRDAFRENKNVKDPVEIQTLVNKAKRDLGVIRRQVHIGQ LYSTDKLIIENRDMPRT >NP_ LYR motif-containing protein 4 isoform 1 [Homo sapiens] MAASSRAQVLSLYRAMLRESKRFSAYNYRTYAVRRIRDAFRENKNVKDPVEIQTLVNKAKRDLGVIRRQVHIGQLYSTDKLIIENRD MPRT The human variant of NFS1 D55 (it was prepared without the first 55 residues; with a RGS for antibody detection and a N-terminal His tag (yellow) for purification, followed by a thrombin cleavage site (Green); residue R57 is highlighted in grey NFS1: >human NFS1 MRGSHHHHHHLVPRGSRPLYMDVQATTPLDPRVLDAMLPYLINYYGNPHSRTHAYGWESEAAMERARQQVASLIGADPREIIFTSGA TESNNIAIKGVARFYRSRKKHLITTQTEHKCVLDSCRSLEAEGFQVTYLPVQKSGIIDLKELEAAIQPDTSLVSVMTVNNEIGVKQP IAEIGRICSSRKVYFHTDAAQAVGKIPLDVNDMKIDLMSISGHKIYGPKGVGAIYIRRRPRVRVEALQSGGGQERGMRSGTVPTPLV VGLGAACEVAQQEMEYDHKRISKLSERLIQNIMKSLPDVVMNGDPKHHYPGCINLSFAYVEGESLLMALKDVALSSGSACTSASLEP SYVLRAIGTDEDLAHSSIRFGIGRFTTEEEVDYTVEKCIQHVKRLREMSPLWEMVQDGIDLKSIKWTQH >NP_ cysteine desulfurase, mitochondrial isoform a precursor [Homo sapiens] MLLRAAWRRAAVAVTAAPGPKPAAPTRGLRLRVGDRAPQSAVPADTAAAPEVGPVLRPLYMDVQATTPLDPRVLDAMLPYLINYYGN PHSRTHAYGWESEAAMERARQQVASLIGADPREIIFTSGATESNNIAIKGVARFYRSRKKHLITTQTEHKCVLDSCRSLEAEGFQVT YLPVQKSGIIDLKELEAAIQPDTSLVSVMTVNNEIGVKQPIAEIGRICSSRKVYFHTDAAQAVGKIPLDVNDMKIDLMSISGHKIYG PKGVGAIYIRRRPRVRVEALQSGGGQERGMRSGTVPTPLVVGLGAACEVAQQEMEYDHKRISKLSERLIQNIMKSLPDVVMNGDPKH HYPGCINLSFAYVEGESLLMALKDVALSSGSACTSASLEPSYVLRAIGTDEDLAHSSIRFGIGRFTTEEEVDYTVEKCIQHVKRLRE MSPLWEMVQDGIDLKSIKWTQH 15

16 References [1] Ashkenazy, H., Erez, E., Martz, E., Pupko, T., and Ben-Tal, N. (2010) ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids, Nucleic Acids Res 38, W [2] Crooks, G. E., Hon, G., Chandonia, J. M., and Brenner, S. E. (2004) WebLogo: a sequence logo generator, Genome Res 14, [3] Ahmad, S., Gromiha, M., Fawareh, H., and Sarai, A. (2004) ASAView: database and tool for solvent accessibility representation in proteins, BMC Bioinformatics 5,