Peptide binding specificity of the chaperone calreticulin

Biochimica et Biophysica Acta 1774 (2007) 701 – 713 www.elsevier.com/locate/bbapap

Peptide binding specificity of the chaperone calreticulin Noreen Sandhu a , Karen Duus a , Charlotte S. Jørgensen b , Paul R. Hansen c , Susanne W. Bruun d , Lars Ø. Pedersen e , Peter Højrup f , Gunnar Houen a,⁎ a Department of Autoimmunology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen, Denmark Department of Bacteriology, Mycology and Parasitology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen, Denmark Department of Natural Sciences, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark d BioCentrum-DTU, Technical University of Denmark, Søltofts Plads, Building 224, 2800 Kgs. Lyngby, Denmark e Disease Biology, H Lundbeck, Ottiliavej 9, 2500 Valby, Copenhagen, Denmark f Institute of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark b

c

Received 13 October 2006; received in revised form 28 March 2007; accepted 29 March 2007 Available online 6 April 2007

Abstract Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composition. A large set of available synthetic peptides (n = 127) was tested for binding to calreticulin and the results analysed by multivariate data analysis. The parameter that correlated best with binding was hydrophobicity while β-turn potential disfavoured binding. Only hydrophobic peptides longer than 5 amino acids showed binding and a clear correlation with hydrophobicity was demonstrated for oligomers of different hydrophobic amino acids. Insertion of hydrophilic amino acids in a hydrophobic sequence diminished or abolished binding. In conclusion our results show that calreticulin has a peptide-binding specificity for hydrophobic sequences and delineate the fine specificity of calreticulin for hydrophobic amino acid residues. © 2007 Elsevier B.V. All rights reserved. Keywords: Calreticulin; Chaperone; Peptide specificity

1. Introduction Protein folding in vivo is a non-random co-operative process controlled by chaperones and folding catalyst such as disulphide isomerases and proline cis-trans isomerases [1–9]. In the endoplasmic reticulum (ER) protein folding occurs upon translocation of the nascent polypeptide chain into the lumen and involves several catalysts and chaperones including glucose-regulated protein (GRP) 78, GRP 94 (endoplasmin), calnexin, calreticulin, ERp57, protein disulfide isomerase (PDI) and others [2–9]. A large proportion of the proteins synthesized in the ER are N-glycosylated and folding of these has been found to be coordinated with glycan trimming [7–9]. Two abundant chaperones in the ER, calnexin and calreticulin, bind to folding glycoproteins by virtue of their lectin specificity and retain the glycoproteins until they are properly folded [7–9]. Interactions ⁎ Corresponding author. Tel.: +45 32683276; fax: +45 32683149. E-mail address: [email protected] (G. Houen). 1570-9639/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2007.03.019

of these chaperones with substrates appear to be determined by topological factors, and calnexin and calreticulin have also been reported to prevent thermal aggregation in vitro of nonglycosylated proteins, to promote folding of non-glycosylated proteins and to possess polypeptide binding properties [10–21]. A central question in this regard is the nature and specificity of the polypeptide binding site of calreticulin and its relation and possible co-operation with the lectin binding site. In order to characterize the polypeptide binding specificity of calreticulin, we have investigated the interaction of calreticulin with a large set of synthetic peptides. 2. Materials and methods 2.1. Chemicals Amino acids, 9-fluorenylmethyloxycarbonyl (FMOC)-protected amino acids, protease K, ovalbumin (OVA), para-nitrophenyl-phosphate (pNPP) substrate tablets, dimethylsulfoxide (DMSO), glycerol, dithiothreitol (DTT), sodium carbonate, Tris, Tris hydrochloride, Bis-Tris, urea, α-cyano-

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hydroxycinnamic acid, N-hydroxy-succinimidobiotin, 5-Iodacetamidofluorescein, and peptides (LL, LL-NH2, GGFL-NH2, APYA, YGGFL, YGGFL-NH2, YGGFM-NH2) were from Sigma (St. Louis, MO, USA). Acetonitrile, N,Ndimethyl formamide (DMF), diethanolamine, NaHCO3, NaH2PO4, Na2HPO4, MgCl2 and Tween 20 were from Merck (Darmstadt, Germany). The following peptides: RY, APYA, LPFFD, KLVFF, LDLLFL, LTRPRY, YGGFLR, RFKVVM, WKYMVM-NH2, LRRWSLG, RYLGYL, LRRASVA, ASQFETS, RFYVVMWK, APWLYGPA, WREMSVW-NH2, ASHLGLAR were from Bachem (Bubendorf, Switzerland). NaCl was from Unikem (Copenhagen, Denmark). Alkaline phosphatase (AP)-conjugated streptavidin was from DAKO (Glostrup, Denmark). MaxiSorp microtitre plates were from Nunc (Roskilde, Denmark). Diethanolamine was from Aldrich (Steinheim, Germany). Q Sepharose and Sephacryl S-100 HR were from Pharmacia (Uppsala, Sweden). Sodium dodecylsulfate (SDS) was from BDH (Poole, Dorset, England). Trifluoroacetic acid (TFA) was from Fluka (Buchs, Switzerland).

2.2. Purification of human placenta calreticulin Human placenta calreticulin was purified using a modification of a well established procedure [11,22,23]. A placenta was homogenized twice at 4 °C with 20 mM Bis-Tris, pH 7.2 and then twice with 20 mM Bis-Tris, pH 7.2, 1% Triton X-114. In between extractions, the supernatant and precipitate were separated by centrifuging at 16,300×g for 1 h (4 °C). The combined supernatants from extraction with 20 mM Bis-Tris, pH 7.2, 1% Triton X-114 were diluted with an equal volume 20 mM Bis-Tris, pH 7.2, 3% Triton X-114 and incubated at 37 °C over night in order to achieve temperature-induced separation of water and detergent phases. The upper water phase was separated from the lower detergent phase by aspiration, and ammonium sulfate (337 g/L) was added to the water phase. After stirring at 4 °C over night, the precipitate was removed by centrifuging at 16,300×g for 1 h (4 °C). The supernatant was subjected to ultradiafiltration against 6 volumes of 20 mM Tris, pH 7.5 using a 10-kDa cut off filter until a final volume of 500 mL, and then applied to a Q Sepharose ion exchange column (2.6 cm × 33 cm) equilibrated with 20 mM Tris, pH 7.5 (flow rate 3 mL/ min). The column was eluted stepwise with increasing concentrations of NaCl in the same buffer (0.3, 0.35, 0.4, 1.0 M NaCl). Fractions containing calreticulin were identified by SDS-polyacrylamide gel electrophoresis (PAGE), immunoblotting and enzyme-linked immunosorbent assay (ELISA) using antisera recognising the C-terminal end of calreticulin, pooled and concentrated by ultradiafiltration against 20 mM Tris, pH 7.5, followed by size exclusion chromatography on a Sephacryl S-100 HR column (1.6 cm × 100 cm) using a flow rate of 0.5 mL/min. The purified protein showed a single band of apparent molecular weight 60 kDa in SDS-PAGE and a single band of pI 4.6 in isoelectric focusing.

2.3. Biotinylation of human placenta calreticulin The purified calreticulin was dialysed against 0.1 M NaHCO3, pH 9.0 at 4 °C, followed by addition of N-hydroxysuccinimidobiotin in DMF (10 mg/mL) to a final concentration of 4 mg/mg calreticulin. The solution was incubated for 2 h at room temperature with end-over-end agitation, and then dialysed against phosphate-buffered saline (PBS) (10 mM NaH2PO4/Na2HPO4 pH 7.3, 0.15 M NaCl) at 4 °C. The biotinylated calreticulin was added an equal volume of glycerol and stored at − 20 °C until use.

2.4. Denaturation and proteolytic fragmentation of ovalbumin Heat-induced denaturation was carried out by autoclaving OVA (1 mg/mL PBS) at 105 °C for 1 h. Heat-denatured ovalbumin (1 mg/mL) was incubated with end-over-end mixing at room temperature with protease K using an enzyme:substrate ratio of 1:100. The digestion was stopped by addition of 1/100 volume of 100 mM PMSF in DMF to a final concentration of 1 mM. Digests were kept at − 20 °C until use.

Melanoma-associated peptides were synthesized at a GMP facility by Clinalfa (Läufelfingen, Switzerland) and had a purity of at least 98%. VMAPCTLLL was labelled with 5-Iodacetamidofluorescein as described by Rizvi et al. [21].

2.6. Solid phase binding assay Unless stated otherwise, incubations and washings were performed at room temperature on a shaking table using 100 μL/well. TTN buffer (0.025 M Tris, 0.5% Tween 20, 0.15 M NaCl, pH 7.5) was used for blocking, incubation and washing. Peptides (dissolved in DMSO, 10 mg/mL) were coated 1:50 (20 μg/ml) or 1:100 (10 μg/ml) respectively, onto the surface of the wells of microtitre plates using 0.05 M sodium carbonate, pH 9.6 as coating buffer. Proteolytic digests (1 mg/mL) were coated 1:100 or 1:1000. After coating overnight at 4 °C, plates were washed 3 times 1 min, followed by blocking for 1 h. Subsequently, incubation with or without biotinylated calreticulin diluted 1:500 was carried out for 1.5 h, followed by another 3 washes. Finally, the plates were incubated for 1 h with AP-conjugated streptavidin diluted 1:1000. Following another 3 washes, bound calreticulin was quantified using pNPP (1 mg/mL) in 1 M diethanolamine, 0.5 mM MgCl2, pH 9.8. The absorbance was read at 405 nm with background subtraction at 650 nm on a VERSAmax microplate reader (Molecular Devices, Sunnyvale, CA, USA). Results are expressed as percentages of absorbance values relative to the absorbance readings for calreticulin's binding to the protease K digest on the same plate.

2.7. Solution binding assay (solid phase inhibition assay) Inhibition of calreticulin binding by peptides was tested by pre-incubation of biotinylated calreticulin diluted 1:500 with or without the addition of peptide (1 mg/mL) or protease K digest diluted 1:10 in TTN for 1 h at room temperature. The pre-incubated mixture was added to plates coated overnight with a protease K digest of OVA known to bind calreticulin or selected peptides and was incubated for 2 h at room temperature. Subsequently, the plates were washed, incubated with AP-conjugated streptavidin and developed as described above. Results are expressed as percentage inhibition of absorbance values relative to the absorbance readings for calreticulin's binding to the protease K digest on the same plate.

2.8. Multivariate data analysis Multivariate data analysis was performed using The Unscrambler, version 7.6 (Camo Process, Oslo, Norway). In order to reveal the largest variation in the data set and relate the peptide properties to their binding abilities, the data were subjected to principal component analysis and partial least squares regression with X = variables for α-helix, β-sheet, β-turn [26] and hydrophobicity propensity [25] divided by the number of amino acids (NA) and Y = indicator variables for solid phase binding and solution binding. The indicator variable x has the possible values of +1 and −1 for binding and no binding, respectively, whereas the indicator variable y is just the opposite of x. Principal component analysis uses singular value decomposition of a single data table to extract the principal components (PCs), which summarises the systematic patterns of variation between samples. Unsystematic variation is left unmodelled in the residuals. Each PC reflects a dominant variation type and is built from a loading (p) and a score vector (t), where p expresses the relations between the original variables and the new PC variable, and t holds the contents of this PC for all samples. In partial least squares regression, both X and Y are modelled by PCs, and the Y-variance is used as a guide for decomposition of X to ensure that the first PCs are relavant to Y. The outcome of the analyses was visualized by graphical presentations of score plots and correlation plots of partial least squares regression analyses.

3. Results 2.5. Peptide synthesis Synthetic peptides (non-commercial) were synthesized by FMOC solid phase peptide synthesis as described by Atherton and Sheppard [24]. The identity and purity of the peptides were ascertained by high performance liquid chromatography (HPLC) and mass spectrometry (MS).

3.1. Binding assays Two assays were used to investigate the ability of peptides to bind calreticulin: a solid phase binding assay with peptides

N. Sandhu et al. / Biochimica et Biophysica Acta 1774 (2007) 701–713

coated on the surface of polystyrene microtitre plates and then incubated with biotin-labelled calreticulin followed by incubation with AP-conjugated streptavidin and enzyme-catalyzed colour development with pNPP. This assay requires that peptides are able to bind to the solid phase and in the immobilized state bind to calreticulin, which may be impossible for some calreticulin-binding peptides. For this reason, we also employed a solution binding assay (solid phase inhibition assay), in which the ability of peptides in solution to inhibit the binding of biotinlabelled calreticulin to an immobilized protease K digest of OVA was determined. The protease K digest is known to bind strongly to calreticulin and to contain peptides of a suitable length for binding to calreticulin [16]. Both assays were conducted in an incubation buffer assuring low non-specific binding (25 mM Tris, pH 7.5, 0.5% Tween 20, 0.15 M NaCl) and results of the two assays will be referred to as “solid phase” peptide binding and “solution” peptide binding. 3.2. Screening of peptides for binding to calreticulin Using the solution binding assay we tested a selection of commercially available amino acids, dipeptides, tripeptides and tetrapeptides for binding. None of these showed binding to calreticulin (results not shown). Screening of a hexapeptide library in the two assays indicated a preference for adjacent hydrophobic amino acids but did not reveal a distinct motif, presumably due to a large redundancy of the library (results not shown). Thus, with respect to peptide binding it appeared that calreticulin favoured longer peptides with hydrophobic amino acids. Testing of a larger collection of available peptides (n = 127) in both assays (Appendix A) revealed binding to a limited subset of these. In general, with only a few exceptions, peptides that showed binding in the solid phase also showed binding in the liquid phase, while the reverse was not necessarily true. Values for peptide hydrophobicity/hydrophilicity and structural potential were calculated using amino acid values for these parameters as defined by Kyte and Doolittle [25] and Chou and Fasman [26]. Statistical regression analysis of the data by multivariate methods, which determine the largest variation (principal components) in the data set, showed that peptides with high β-turn potential were disfavoured whereas a preference for hydrophobicity was evident (Fig. 1). The score plot (A) of PC1 vs. PC2 scores from partial least squares regression shows the grouping of binding peptides (x-points) and non-binding peptides (y-points) along the two axes (Principal components) describing the largest (PC1, 70%) and second largest (PC2, 24, 16%) variation in the X-data set. Together the two PCs explain 17% of the Y-variance. The correlation loading plot (B) for the two PCs graphically visualizes the relation between all X- and Y-variables, i.e. binding peptides (x), non-binding peptides (y), and the four calculated peptide propensities (hydrophobicity, β-turn, βsheet, α-helix) divided by the number of amino acids (NA) in the peptides. In this plot, correlations of the original variables to the new PC variables are plotted for PC1 vs. PC2. The positively correlated variables are placed close together,

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whereas negatively correlated variables are placed oppositely. No preference for β-strand or α-helix potential was revealed by the data, presumably because the hydrophobic amino acids have quite similar β-strand and α-helix propensities, i.e., they can be accommodated equally well in both structures. This, however, cannot be taken as evidence that calreticulin binds to both types of structure. 3.3. Analysis of major histocompatibility complex (MHC) I-binding peptides Calreticulin has been found to participate in the MHC I loading complex [7]. In order to test whether calreticulin would bind directly to peptides being loaded on MHC I molecules, we analysed a series of melanoma-related MHC I-binding peptides [27]. Both assays revealed that none of the peptides bound except for a peptide SLLQHLIGL, which showed a very weak solution binding (results not shown). Thus, there was no correlation between the peptide binding specificities of MHC I and calreticulin, but the results nevertheless contribute to the definition of calreticulin's peptide specificity. 3.4. Improving the binding of a peptide The melanoma-related peptide SLLQHLIGL had turned out as a very weak calreticulin-binding peptide during screening of the melanoma-associated HLA peptides despite a rather high content of hydrophobic residues. Several variants of this peptide were synthesized and tested in the assays (Table 1). None of the peptides exhibited binding to calreticulin, when they were immobilized on the polystyrene solid phase. However, note the strong solution binding by peptide SLLQLLIGL. The peptides SLLQHLIGL and QLLQHLIGL only had a very low activity in the solution binding assay, whereas the other 3 peptides showed no binding in solution. Thus, the substitution of a single histidine with leucine converted a non-binding peptide to a good solution binding peptide. Conversely, these results showed that the interruption of a hydrophobic sequence by the charged residue, histidine, abolished binding, while the neutral residue, glutamine, was tolerated. 3.5. Conversion of binding to non-binding peptides In order to further define the specificity of peptide binding we attempted to transform a binding peptide to a non-binding peptide. The prion protein (PrP) peptide CLVLFVAMWSD was previously found to be a calreticulin-binding peptide [16], and the following variants of this peptide were synthesized and tested for binding to calreticulin (Table 2); successively N- and C-terminally truncated versions, peptides with each residue substituted by alanine, a small residue of medium hydrophobicity (alanine scan), a peptide with inverted sequence, and peptides with internal deletion of one or more hydrophobic residues. In the solid phase assay, deletion of the 6 C-terminal residues did not diminish binding but further removal of the Phe at position 5, leaving the tetrapeptide CLVL, completely abolished binding. Removal of the N-terminal Cys had no

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Fig. 1. Multivariate data analysis of peptide binding to calreticulin. The score plot (A) and correlation loading plot (B) of a partial least squares regression with X = 4 variables (α-helix, β-sheet, β-turn [26] and hydrophobicity parameters [25] divided by the number of amino acids (NA) and Y = indicator variables for x, y is shown (x: solution binding peptides, y: non-binding peptides). PC1 and PC2: Arbitrary axes (Principal components) one and two, accounting for the largest (70%) and second largest (24%) variation in the X-data set, respectively. The inner circle of the correlation loading plot represents 50% explanation and the outer circle represents 100% explanation of the variables.

significant effect on binding, while further removal of the Leu abolished binding. For the alanine scan, we chose to use the peptide without Cys at position 1, and for the Ala at position 7, we chose to make instead an Ala to Leu substitution, thus increasing the hydrophobicity of the peptide. Substitution of the C-terminal hydrophilic Asp and Ser by the slightly hydrophobic Ala had no significant effect on solid phase binding, but substitution of the aromatic Trp or the hydrophobic Met by Ala reduced solid phase binding. Reduced solid phase binding was also seen for the peptides with alanine substituted for the very hydrophobic amino acid residues, and the largest effect was seen for the peptides with V/A or F/A substitutions at positions 6 and 5, where solid phase binding was completely abolished. For the peptides with L/A or V/A substitutions at positions 4, 3, and 2, respectively, diminished binding was observed, with the

smallest effect in the outermost position. Slightly increased solid phase binding was seen for the peptide with an A/L substitution at position 7 and for the peptide with inverted sequence. Reduced solid phase binding was observed for the peptides with internal deletions. Deletion of Val at position 6 strongly diminished binding, while further deletion of the Ala at position 7 compensated for the loss of Val resulting in only slightly diminished solid phase binding. Deletion of FVor VAM totally abolished solid phase binding. For the C-terminally deleted peptides a decrease in solution binding to 72% was not observed until 5 amino acids had been deleted (CLVLFV peptide) followed by a large decrease upon removal of one more amino acid (CLVLF peptide). The Nterminal Cys and Leu were dispensable for solution binding, but a sharp decrease in solution binding was seen upon removal of

N. Sandhu et al. / Biochimica et Biophysica Acta 1774 (2007) 701–713 Table 1 Variants of SLLQHLIGL tested for binding to calreticulin Peptide a

Number of Solid phase Solution Comments amino acids binding (%) b binding (%) c

SLLQHLIGL-NH2 QLLQHLIGL-NH2 SLLAHLIGL-NH2 SLLQLLIGL-NH2 SLLQHLPGL-NH2 SLLQHLISL-NH2

9 9 9 9 9 9

1 1 1 2 1 2

9 11 0 89 0 0

– S/Q substitution Q/A substitution H/L substitution I/P substitution G/S substitution

a

NH2: C-terminal amide. Expressed as a percentage of the observed binding of calreticulin to a protease K digest of heat-denatured ovalbumin. Maximum binding was set at 100%. c Expressed as a percentage of the binding to a protease K digest of heatdenatured ovalbumin seen without added peptide. b

the three N-terminal amino acid residues (CLV) resulting in peptide LFVAMWSD. The alanine-substituted variants (alanine scan) showed strong solution binding, although a slight reduction was seen for two peptides with F/A or V/A substitutions at positions 4 and 5 respectively. The peptide with inverted sequence likewise bound strongly (Table 2). However, an approximately 50% decrease in solution binding was observed for the peptide with internal deletion of FV, while the other internal deletions had no effect. Another calreticulin-binding peptide, GYVIIKPLVWV derived from serum amyloid P (SAP) was also studied in Nand C-terminally truncated versions (Table 3). Deletion of the C-terminal valine abolished both solid phase and solution binding. Deletion of the N-terminal glycine reduced both solid phase and solution binding, while deletion of GY abolished solid phase binding and reduced solution binding. Deletion of GYV reduced solution binding dramatically and deletion of GYVI abolished binding completely. Thus, for this peptide, hydrophobic residues at both ends were important for binding in both assays. 3.6. Binding to short hydrophobic peptides Analysis of the binding to oligomers of leucine showed that a minimum of 6 residues were required for optimal binding in the solid phase assay and that 5 residues were required for prominent binding in the solution assay (although tetraleucine did show some solution binding) (Table 4). In both assays, peptides with alternating leucine and glycine did not bind or showed a very low binding at the most. This indicated a preference for continuous stretches of hydrophobic amino acids and we therefore investigated the binding to pentamers and hexamers of different hydrophobic amino acids (Table 5). Pentaalanine showed no solid phase binding and only a very low level of solution binding. Hexaalanine exhibited significant solid phase binding but only a slight solution binding. Likewise, pentamethionine did not show solid phase binding and only some solution binding, whereas hexamethionine was both a good solid phase and solution binder. Hexatyrosine but not pentatyrosine bound to calreticulin in the solid phase assay, but

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none of them exhibited significant solution binding, presumably due to a low solubility. Pentaphenylalanine showed some solid phase but no solution binding, but hexaphenylalanine bound strongly in both assays. Finally, the pentamers of valine, leucine and isoleucine and hexamers of leucine and isoleucine (hexavaline had a low solubility) exhibited binding in both assays, and the strength of binding increased in the order mentioned. The hexamers of these amino acids, together with hexatyrosine and hexaphenylalanine were the best binders as judged from the solid phase binding assay, and the hexamers of isoleucine, leucine and phenylalanine were the best binders in the solution binding assay. 3.7. Influence of specific amino acid residues The contribution of specific amino acid residues to peptide binding was analysed by synthesizing pentamers and hexamers Table 2 Prion protein peptides analysed for calreticulin binding Peptide a

Number of amino acids

Solid phase binding (%) b

Solution binding (%) c

Comments

CLVLFVAMWSD-NH2

11

N100

93

CLVLFVAMWS-NH2 CLVLFVAMW-NH2 CLVLFVAM-NH2 CLVLFVA-NH2 CLVLFV-NH2 CLVLF-NH2 CLVL-NH2 LVLFVAMWSD-NH2

10 9 8 7 6 5 4 10

77 79 N100 N100 N100 76 1 72

94 92 96 96 72 21 14 71

VLFVAMWSD-NH2 LFVAMWSD-NH2 FVAMWSD-NH2 VAMWSD-NH2 AMWSD-NH2 MWSD-NH2 WSD-NH2 LVLFVAMWSA-NH2 LVLFVAMWAD-NH2 LVLFVAMASD-NH2 LVLFVAAWSD-NH2 LVLFVLMWSD-NH2 LVLFAAMWSD-NH2 LVLAVAMWSD-NH2 LVAFVAMWSD-NH2 LALFVAMWSD-NH2 AVLFVAMWSD-NH2 DSWMAVFLVL-NH2 LVLFAMWSD-NH2 LVLFMWSD-NH2 LVLFWSD-NH2 LVLAMWSD-NH2

9 8 7 6 5 4 3 10 10 10 10 10 10 10 10 10 10 10 9 8 7 8

5 1 1 1 1 1 1 70 57 13 30 85 2 2 18 36 47 84 17 64 2 2

97 2 6 0 0 0 0 96 98 98 97 98 75 76 95 98 98 98 93 98 83 53

C-terminal truncation – – – – – – – N-terminal truncation – – – – – – – Alanine scan – – – A/L substitution Alanine scan – – – – Inverted sequence Internal deletion – – –

a

NH2: C-terminal amide. Expressed as a percentage of the observed binding of calreticulin to a protease K digest of heat-denatured ovalbumin. Maximum binding was set at 100%. c Expressed as a percentage of the binding to a protease K digest of heatdenatured ovalbumin seen without added peptide. b

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Table 3 Serum amyloid P (SAP) peptides analysed for calreticulin binding

Table 5 Binding of calreticulin to pentamers and hexamers of hydrophobic residues

Peptide a

Number of amino acids

Solid phase binding (%) b

Solution binding (%) c

Comments

Peptide a

Number of amino acids

Solid phase binding(%) b

Solution binding (%) c

GYVIIKPLVWV-NH2

11

80

95

GYVIIKPLVW-NH2 GYVIIKPLV-NH2 GYVIIKPL-NH2 GYVIIKP-NH2 GYVIIK-NH2 GYVII-NH2 GYVI-NH2 YVIIKPLVWV-NH2

10 9 8 7 6 5 4 10

10 10 9 2 2 2 2 37

8 3 11 8 0 9 3 76

AAAAA-NH2 AAAAAA-NH2 VVVVV-NH2 VVVVVV-NH2

5 6 5 6

0 40 31 N100

6 8 76 nd a

9 8 7 6 5 4 3

4 2 1 1 3 1 1

67 11 0 0 18 0 6

C-terminal truncation – – – – – – – N-terminal truncation – – – – – – –

LLLLL-NH2 LLLLLL-NH2 IIIII-NH2 IIIIII-NH2 FFFFF-NH2 FFFFFF-NH2 YYYYY-NH2 YYYYYY-NH2

5 6 5 6 5 6 5 6

62 N100 N100 N100 32 N100 1 N100

80 78 78 70 1 92 5 nd a

MMMMM-NH2 MMMMMM-NH2

5 6

0 N100

10 91

VIIKPLVWV-NH2 IIKPLVWV-NH2 IKPLVWV-NH2 KPLVWV-NH2 PLVWV-NH2 LVWV-NH2 VWV-NH2 a

NH2: C-terminal amide. Expressed as a percentage of the observed binding of calreticulin to a protease K digest of heat-denatured ovalbumin. Maximum binding was set at 100%. c Expressed as a percentage of the binding to a protease K digest of heatdenatured ovalbumin seen without added peptide. b

of leucine with amino acid substitutions at positions 3 and 4 respectively (Tables 6, 7). As previously noticed, pentaleucine showed borderline behaviour in the solid phase assay with 31% binding, which is intermediate between the values seen in Tables 4 and 5. All other peptides with a substitution at position 3 showed no binding in the solid phase assay, irrespective of the nature of the amino acid. In the solution binding assay, peptides with hydrophobic amino acids substituted at position 3 exhibited prominent binding (I, V, F, Y), whereas, surprisingly, substitution with M and W

Table 4 Binding of calreticulin to leucine-containing peptides Peptide a

Number of amino acids

Solid phase binding (%) b

Solution binding (%) c

LLLL-NH2 LLLLL-NH2 LLLLLL-NH2 LLLLLLL-NH2 LGLGL-NH2 LGLGLG-NH2 LGLGLGL-NH2 LGGLGGL-NH2 LGLGGL-NH2 LGLGLL-NH2

4 5 6 7 5 6 7 7 6 6

0 4 N100 N100 2 0 0 0 2 0

13 91 91 89 0 17 2 5 2 8

a

NH2: C-terminal amide. Expressed as a percentage of the observed binding of calreticulin to a protease K digest of heat-denatured ovalbumin. Maximum binding was set at 100%. c Expressed as a percentage of the binding to a protease K digest of heatdenatured ovalbumin seen without added peptide. b

Comments

a Not determined due to precipitation

a

Not determined due to precipitation

a

NH2: C-terminal amide. Expressed as a percentage of the observed binding of calreticulin to a protease K digest of heat-denatured ovalbumin. Maximum binding was set at 100%. c Expressed as a percentage of the binding to a protease K digest of heatdenatured ovalbumin seen without added peptide. b

abolished binding. Peptides with small aliphatic amino acids (A, P) showed some binding and T and K were also tolerated, while substitution with Q, C and H led to a lower level of

Table 6 Binding of calreticulin to position 3-substituted variants of pentaleucine Peptide a

Number of amino acids

Solid phase binding (%) b

Solution binding (%) c

Comments

LLLLL-NH2 LLILL-NH2 LLVLL-NH2 LLFLL-NH2 LLMLL-NH2 LLYLL-NH2 LLALL-NH2 LLWLL-NH2 LLCLL-NH2 LLPLL-NH2 LLGLL-NH2 LLTLL-NH2 LLSLL-NH2 LLHLL-NH2 LLQLL-NH2 LLNLL-NH2 LLELL-NH2 LLDLL-NH2 LLRLL-NH2 LLKLL-NH2

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

31 0 1 1 1 1 1 1 1 1 1 2 0 1 1 1 0 0 0 1

54 96 53 36 0 51 53 0 25 37 0 61 4 29 13 8 0 0 0 42

Control L/I substitution L/V substitution L/F substitution L/M substitution L/Y substitution L/A substitution L/W substitution L/C substitution L/P substitution L/G substitution L/T substitution L/S substitution L/H substitution L/Q substitution L/N substitution L/E substitution L/D substitution L/R substitution L/K substitution

a

NH2: C-terminal amide. Expressed as a percentage of the observed binding of calreticulin to a protease K digest of heat-denatured ovalbumin. Maximum binding was set at 100%. c Expressed as a percentage of the binding to a protease K digest of heatdenatured ovalbumin seen without added peptide. b

N. Sandhu et al. / Biochimica et Biophysica Acta 1774 (2007) 701–713 Table 7 Binding of calreticulin to position 4-substituted variants of hexaleucine Peptide a

Number of Solid phase Solution Comments amino acids binding (%) b binding (%) c

LLLLLL-NH2 LLLILL-NH2 LLLVLL-NH2 LLLFLL-NH2 LLLMLL-NH2 LLLYLL-NH2 LLLALL-NH2 LLLWLL-NH2 LLLCLL-NH2 LLLPLL-NH2 LLLGLL-NH2 LLLTLL-NH2 LLLSLL-NH2 LLLHLL-NH2 LLLQLL-NH2 LLLNLL-NH2 LLLELL-NH2 LLLDLL-NH2 LLLRLL-NH2 LLLKLL-NH2

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

N100 N100 N100 N100 N100 3 6 3 28 3 3 10 4 2 12 7 34 6 0 1

69 43 83 49 76 69 8 33 86 12 26 15 10 21 50 0 21 11 21 4

Control L/I substitution L/V substitution L/F substitution L/M substitution L/Y substitution L/A substitution L/W substitution L/C substitution L/P substitution L/G substitution L/T substitution L/S substitution L/H substitution L/Q substitution L/N substitution L/E substitution L/D substitution L/R substitution L/K substitution

a

NH2: C-terminal amide. Expressed as a percentage of the observed binding of calreticulin to a protease K digest of heat-denatured ovalbumin. Maximum binding was set at 100%. c Expressed as a percentage of the binding to a protease K digest of heatdenatured ovalbumin seen without added peptide. b

binding. Substitution with acidic amino acids abolished solution binding as did substitution with G, R, N and S. Overall, the results show that calreticulin prefers hydrophobic residues and that the most hydrophilic residues disfavours binding, whereas residues of intermediate polarity are better tolerated. For the position 4-substituted variants of hexaleucine, all peptides with a very hydrophobic residue substituted for leucine (I, V, F) exhibited prominent binding in the solid phase assay, as did also the M-substituted peptide. All other peptides exhibited non-significant or low solid phase binding. In the solution binding assay prominent binding was seen for the hydrophobic residue-substituted peptides but also for M, Y, C, Q and Wsubstituted peptides, while non-significant or low levels of binding was seen for the rest of the peptides. These results reinforce the conclusion reached above: Hydrophobic residues are necessary for binding, whereas hydrophilic residues disfavour binding. Thus, it is evident that, in general, a peptide length of at least 6 amino acid residues is required for solid phase binding as well as a requirement for hydrophobic residues in order to show optimal binding. Pentaleucine is the exception to the rule and exhibits borderline behaviour. However, this actually illustrates the limitations of the solid phase assay and at the same time indicates a structural/conformational aspect of the interaction besides the requirement for hydrophobic sequences. With regard to solution binding, the same pattern is evident; a length of at least 5 amino acid residues is required, and hydrophobic

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sequences are the best binders, but there seems to be more room for substitutions. 3.8. Binding assay dependency on coating peptides and assay conditions In order to rule out a dependency on the OVA protease K digest used for coating in the solution binding assay, peptides were tested for solution binding using several different peptides (AMVLVNAIVFKGL-NH2, VYSFSLASRLYAE-NH2, PFLFCIKHI-NH2, GILKINSRWW-NH2, GNWVCAAKFE-NH2, TASVNCAKKIVS-NH2) coated on the solid phase. Identical results were obtained as in the experiments using the OVA protease K digest for coating (results not shown). Analysis of calreticulin binding to the protease K digest of ovalbumin in the presence of various compounds, known to bind calreticulin, revealed that addition of 1 mM Ca2+ or 1 mM EDTA did not affect binding (Fig. 2), indicating that Ca2+ is not essential for polypeptide binding. However, it cannot be ruled out that a tightly bound structural Ca2+ ion may remain bound in the presence of 1 mM EDTA. Similarly, 1 mM ATP had no influence on binding but 1 mM Zn2+ inhibited binding completely (Fig. 2). Increasing the Ca2+ concentration to 5 mM had no effect, whereas lowering the Zn2+ concentration abolished the inhibitory effect (results not shown). Similar results were obtained with heptaleucine coated on the solid phase (results not shown). Finally, a Fluorescein-labelled peptide (VMAPCFluoresceinTLLL), previously shown to bind calreticulin in a fluorescence-based solution binding assay [21], was tested for calreticulin binding in the two assays. This revealed very low binding in the solid phase assay but 65% binding in the solution binding assay. The two peptides KVFFKR and KLGFFKR showed no binding in the two assays (results not shown).

Fig. 2. Interaction of calreticulin with immobilized peptides in the presence of various additives. A protease K digest of ovalbumin was used for coating the wells of a polystyrene plate and then incubated with biotin-labelled calreticulin in the presence of 1 mM of the indicated compounds. Controls were included with incubation buffer alone (TTN), incubation buffer without Tween 20 (TN), and with another biotin-labelled protein (OVA) instead of calreticulin. Identical results were obtained, when heptaleucine was used for coating instead of the protease K digest of ovalbumin (not shown).

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4. Discussion Two assays were used to study the interaction of calreticulin with polypeptides. Both assays were conducted in a buffer assuring low non-specific binding. Calreticulin is a calciumbinding protein, but addition or chelation of Ca2+ had no effect on binding. This probably reflects, that the low affinity Ca2+ binding sites in the C-domain has no effect on polypeptide binding. In fact, results by Rizvi et al. have shown that a polypeptide binding conformation of calreticulin is induced by Ca2+ depletion [21]. ATP had no effect, whereas Zn2+ , previously shown to affect the structure of calreticulin, abolished binding. The functional significance of this is unclear at the present time. In the solid phase binding assay, peptides bind to the surface of polystyrene wells and calreticulin then interacts with the immobilized peptide side chains. This makes it likely that peptides bind to calreticulin in an extended β-strand conformation, with residues on both sides of the strand interacting with the binding site. In the solution binding assay, peptides compete with immobilized peptides for binding to calreticulin, and may require rather high concentrations to inhibit binding to the coated peptides, since peptides are more conformational flexible in solution. Despite these differences, a correlation was found between results of the two assays. With only a few exceptions, peptides showing solid phase binding also showed liquid phase binding, and peptides exhibiting more that 10% solid phase binding exhibited maximal binding in solution. The use of the solution binding assay for comparison with the solid phase binding assay assures that binding results do not merely reflect an artefact of the peptides coated on the solid phase. From the observation that amino acids, di-, tri-, tetra-, and most pentapeptides did not bind calreticulin, it may be concluded that in general, a length of at least five amino acid residues is required for significant binding to calreticulin. This is in agreement with previous results, showing that protease K or chymotrypsin digestion of OVA generates peptides binding to calreticulin and that prolonged digestion generates peptides with shorter length and decreased binding to calreticulin [16]. In the solid phase assay, pentaleucine was a special case and its binding ranged from low to high. This can be ascribed to the large hydrophobicity but suboptimal length of this peptide. All other peptides showed consistent binding patterns, and a hydrophobic sequence of 6 residues was required for binding to both the polystyrene surface and to calreticulin. In the solution assay, many pentapeptides failed to bind, but hydrophobic peptides showed significant binding and pentaleucine exhibited prominent binding. Together, these results confirmed the requirement for hydrophobic residues and showed that peptide binding required a minimum of 5 residues. From the various deletion, inversion and alanine substitution peptides tested, it can be concluded that calreticulin has no absolute sequence specificity. Rather, an affinity for hydrophobic sequences determines binding. The hydrophobic sequence must span at least 5 amino acids but may be longer (6–9 residues) and can be interrupted by some non-hydrophobic amino acids.

A correlation between hydrophobicity of a residue and binding to calreticulin was apparent with valine, leucine, isoleucine and phenylalanine as the preferred side chains. Alanine, tyrosine, methionine and tryptophan showed much less potential for binding, but were readily accommodated in a hydrophobic sequence. From the presented results, it is not possible to make an absolute ranking of other amino acids, and the ability of a hydrophobic sequence to accommodate hydrophilic residues may very well depend on the context as well as on the structure, size, hydrophobicity/hydrophilicity and charge of the amino acid side chains. However, it was evident that residues of medium hydrophilicity and/or basic nature were better accommodated than more hydrophilic or acidic residues. The results presented here are in agreement with previous results on binding of peptides to calreticulin. Calreticulin has been shown to suppress heat-induced aggregation of both glycosylated and non-glycosylated proteins [12] and to bind both glycosylated and non-glycosylated peptides [13,15,16,21]. Rizvi et al. [21], using the fluorescein-labelled peptide VMAPCFluorescein TLLL, showed that heat shock induced oligomerization and peptide binding of calreticulin, and similar results were reported by Jørgensen et al. [18]. Calreticulin has also been shown to bind denatured polypeptides in an extended conformation and to have a strong affinity for hydrophobic peptides [10,11,16]. Results on the interaction of calreticulin with nuclear hormone receptors and integrins have shown binding to the conserved motifs KVFFKR [28] and KLGFFKR [29] respectively, when these are embedded in a native protein sequence or immobilized on a carbohydrate matrix as peptides. Also, binding of calreticulin to leucinecontaining, neutrophil-activating peptides of the structure KLKLxKLK has been reported for x ≥ 5 [30] and binding to a neuronal survival peptide, Y-P30 (YDPEAASAPGSGNPCHEASAAQCENAGEDP) has been reported [31]. Apart from minor discrepancies, probably resulting from the use of different assays, these results are in agreement with the present ones, and also point to the existence of a hydrophobic binding site, which may be flanked by basic residues, in particular lysine. Despite the studies mentioned above, no peptide binding motif for calreticulin has been described before. Based on these previous studies and the results presented here, it can be concluded that calreticulin binds to hydrophobic sequences, which may be flanked by hydrophilic/basic residues and may be interrupted by some non-hydrophobic residues, possibly depending on the context. This agrees well with the aggregation suppressing activity of calreticulin, since hydrophobic sequences of proteins are exposed upon heat treatment. The peptide specificity of calreticulin dissected here is similar to that of other chaperones. The HSP 70/Dna K family has been reported to have a specificity for extended peptides of 6 or more amino acid residues with a preference for hydrophobic residues, while basic residues were tolerated to some extent [32–37]. The HSP 60/GRO EL family has a similar preference for hydrophobic residues, although some non-hydrophobic

N. Sandhu et al. / Biochimica et Biophysica Acta 1774 (2007) 701–713

residues are also tolerated, especially when GRO EL is in complex with GRO ES [38,39]. The specificities of other chaperones have been less studied but seem to involve hydrophobic interactions in general. However, functional and specificity differences are evident compared to the HSP 70 and HSP 60 families [40–43]. The geometry of the hydrophobic binding site on calreticulin is not directly deducible from the present results. However, in the solid phase assay it would appear that peptides must bind to the polystyrene surface in an extended conformation in order to both interact with the surface and the calreticulin binding site. This also implies that the hydrophobic binding site on calreticulin is accessible to the immobilized peptide. In agreement with this, it was found that peptides had to be one (hydrophobic) residue longer in order to show the same degree of binding in the solid phase assay as the solution assay. A preference for extended β-strand conformation over α-helix conformation was not revealed by the multivariate data analysis using α-helix and β-sheet propensities as defined by Chou and Fasman [26]. However, for the short (5–6 residues) hydrophobic peptides it is reasonable to assume that they bind in extended conformation rather than in an unstable helical conformation. Results by Leach et al. [44] have localised the lectin and peptide binding sites in calreticulin to neighbouring regions of the calreticulin globular domain (residues 152–191 and 282– 326), but also showed that the intermittent P-domain was necessary for full chaperone action. By comparison with the Xray crystal structure model of the luminal soluble calnexin fragment [45], the calreticulin globular domain is predicted to consist of a β-sandwich formed by the N-domain (residues 1– 180) and two short β-strands and an α-helix donated by the Cdomain (residues 293–320). Mutagenesis and modelling studies have located the lectin site to a cavity on the globular domain at the base of the P-domain and have implicated residues Tyr92, Lys94, Tyr111, Met114, Asp118 and Asp300 in the binding of

709

monoglucosylated N-linked glycans and His153 Trp244 and Trp302 in the chaperone action towards glycosylated and nonglycosylated substrates [46–49]. The lectin site has a specificity for monoglucosylated N-linked glycans, which are located at turns/loops [50,51]. Since the polypeptide binding site disfavours β-turns and has specificity for hydrophobic residues, it is tempting to speculate that the lectin and peptide sites may collaborate in the folding of glycoproteins. For this purpose, the lectin site in calreticulin may serve to anchor the folding substrate by a carbohydrate located at a flexible β-turn sequence, thus restricting the conformational space of the polypeptide chain and directing it to the hydrophobic binding site, which may be optimised for promoting formation of antiparallel β-hairpins. Thus, calreticulin appears as a chaperone with a preference for β-strands. Theoretically, it would be expected that β-sheets, which are formed by structural elements separated by longer distances, are less likely to fold spontaneously than α-helices, formed by a single continuous stretch of amino acids. Hence, the probability of malfolding is larger for β-sheets compared with αhelices, and the assistance of chaperones more needed. Another possibility could be that the polypeptide site is less accessible than the lectin site and mainly comes into play in cases of malfolding. In conclusion, calreticulin is a polypeptide- and carbohydrate-directed chaperone with a specificity for exposed hydrophobic sequences and monoglucosylated carbohydrate trees. Together, this may assure that polypeptides go through an ordered folding, glycosylation and sorting sequence of events. Acknowledgements Kirsten Beth Hansen, Dorthe Tange Olsen, Inger Christiansen and Jette Petersen are thanked for excellent technical work. The Novo Nordisk Foundation is thanked for grants to P. Højrup and K. Duus.

Appendix A Peptides tested for binding to calreticulin. The peptides were grouped in four groups (x–u) according to whether the solid phase binding and solution binding were ≥ 10% of the controls, respectively. Amino acid α-helix, β-sheet and β-turn values were taken from [26]. Amino acid hydrophobicity and hydrophilicity values were taken from [25]. x: solid phase binding ≥ 10%, solution binding ≥ 10%, y: solid phase binding b 10%, solution binding b 10%, z: solid phase binding ≥ 10%, solution binding b 10%, u: solid phase binding b 10%, solution binding ≥ 10%. No. Peptide sequence

Group Solid Solution Number Hydrophobicity Hydrophobicity/ Betanumber of aa sheet x–u phase binding of binding % amino value % acids

Beta- alpha- alpha- Beta- Betasheet helix helix turn turn value/ value value/ value value/ number number number of aa of aa of aa

1 2 3 4 5 6 7 8

u x u x x x x x

1.3 1.1 1.2 1.2 1.3 1.3 1.3 1.3

RFYVVMWK RVTEQESKPVQMMYQIGLF-NH2 KLTNYSVTL-NH2 CLVLFVAMWSD-NH2 CLVLFVAMWS-NH2 CLVLFVAMW-NH2 CLVLFVAM-NH2 CLVLFVA-NH2

7 79 4 100 77 79 100 100

79 94 24 93 94 92 96 96

8 19 9 11 10 9 8 7

2.5 − 7.4 0.9 20.5 23.3 24.1 25.0 23.1

0.3 − 0.4 0.1 1.9 2.3 2.7 3.1 3.3

10.3 20.2 10.5 13.4 12.6 11.8 10.5 9.4

8.6 20.4 8.4 12.0 11.1 10.3 9.2 7.8

1.1 1.1 0.9 1.1 1.1 1.1 1.2 1.1

6.3 16.9 8.7 9.0 7.6 6.2 5.2 4.6

0.8 0.9 1.0 0.8 0.8 0.7 0.7 0.7

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Appendix A (continued) No. Peptide sequence

Group Solid Solution Number Hydrophobicity Hydrophobicity/ Betax–u phase binding of number of aa sheet value binding % amino % acids

Beta- alpha- alpha- Beta- Betasheet helix helix turn turn value/ value value/ value value/ number number number of aa of aa of aa

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69

x x x u x x u x x x x x x x x x x x x x x y u u u u u u y y u u u u y y y u y u u y y u y y u u y y y y y y y y y y y y y

1.4 1.4 1.2 1.2 1.3 1.4 1.4 1.2 1.2 1.1 1.2 1.2 1.1 1.1 1.1 1.2 1.1 1.2 1.1 1.0 1.0 1.1 1.0 1.0 1.2 1.1 1.1 1.2 1.0 1.2 1.0 1.0 1.1 0.9 1.1 0.9 1.0 0.9 1.0 1.1 1.1 1.5 1.1 1.2 1.2 1.0 1.1 1.1 0.7 0.7 1.3 1.4 1.3 1.3 1.1 0.9 0.9 1.1 1.1 1.2 1.3

CLVLFV-NH2 CLVLF-NH2 LVLFVAMWSD-NH2 VLFVAMWSD-NH2 GYVIIKPLVWV-NH2 YVIIKPLVWV-NH2 VIIKPLVWV-NH2 LVLFVAMWSA-NH2 LVLFVAMWAD-NH2 LVLFVAMASD-NH2 LVLFVAAWSD-NH2 LVLFVLMWSD-NH2 LVAFVAMWSD-NH2 LALFVAMWSD-NH2 AVLFVAMWSD-NH2 DSWMAVFLVL-NH2 LVLFAMWSD-NH2 LVLFMWSD-NH2 SLYIGR GQPMYGQPMY GQMYGQPMYGQPMY SLLQHLISL-NH2 VTSIHSLLDEGKQSLTKLAAAWGG GKQSLTKLAAAWGGSGSEAYQGVQ SLLQLLIGL-NH2 LVLFAAMWSD-NH2 LVLAVAMWSD-NH2 LVLFWSD-NH2 APWLYGPA RYLGYL GQPMY GQPMYGQPMYGQPMYGQPMY LVLAMWSD-NH2 LVAMWSD-NH2 SLLAHLIGL-NH2 WREMSVW-NH2 LRRWSLG GSIGAASMEFC SSLRDILNQITKPNDVY-NH2 MSLQRQFLR LTLAKFSPY LVWV-NH2 YGGFM-NH2 LYIGR GYVIIKP-NH2 MTEQQWNFAGIEAAASAIQG YMDGTMSQV FLWGPRALV KKKK KKKKK LLL LLY LL-NH2 FL-NH2 YGGFL-NH2 ASHLGLAR ASQFETS YGGFLR LTRPRY LDLLFL KLVFF

100 76 72 5 80 37 4 70 57 13 30 85 18 36 47 84 17 64 22 34 13 2 3 3 2 2 2 2 1 1 2 2 2 2 1 1 1 1 1 3 3 1 2 7 2 3 3 3 1 1 2 9 2 2 2 1 1 1 1 1 1

72 21 71 97 95 76 67 96 98 98 97 98 95 98 98 98 93 98 11 11 11 0 35 91 89 75 76 83 0 0 20 10 53 51 0 0 0 17 0 29 13 0 0 12 8 8 10 28 0 0 0 0 0 0 0 0 0 0 0 0 0

6 5 10 9 11 10 9 10 10 10 10 10 10 10 10 10 9 8 6 10 14 9 24 24 9 10 10 7 8 6 5 20 8 7 9 8 7 11 17 9 9 3 5 5 7 20 9 9 4 5 3 3 2 2 5 8 7 6 6 6 5

21.3 17.1 17.3 13.5 17.3 17.7 19.0 22.6 19.9 20.0 17.2 19.3 15.3 14.9 15.3 17.3 13.1 11.3 1.3 − 9.8 − 13.7 11.4 1.9 − 10.1 18.8 14.9 19.3 9.4 1.6 0.1 − 4.9 − 19.6 10.3 6.5 17.1 − 5.1 − 3.5 9.4 − 10.3 − 4.5 3.9 8.5 2.6 2.1 6.0 − 1.5 − 2.2 9.0 − 15.6 − 19.5 11.4 6.3 7.6 6.6 4.5 2.3 − 4.7 0.0 − 8.8 14.5 9.7

3.6 3.4 1.7 1.5 1.6 1.8 2.1 2.3 2.0 2.0 1.7 1.9 1.5 1.5 1.5 1.7 1.5 1.4 0.2 − 1.0 − 1.0 1.3 0.1 − 0.4 2.1 1.5 1.9 1.3 0.2 0.0 − 1.0 − 1.0 1.3 0.9 1.9 − 0.6 − 0.5 0.9 − 0.6 − 0.5 0.4 2.8 0.5 0.4 0.9 − 0.1 − 0.2 1.0 − 3.9 − 3.9 3.8 2.1 3.8 3.3 0.9 0.3 − 0.7 0.0 − 1.5 2.4 1.9

8.6 6.9 11.9 10.6 14.5 13.7 12.3 12.2 12.0 11.4 11.7 12.4 11.15 11.1 11.5 11.9 10.2 94 6.8 9.8 14.2 10.3 23.6 22.8 10.7 111 11.4 8.3 7.7 7.2 4.9 19.7 8.8 6.2 10.0 7.5 7.3 10.3 17.8 9.8 9.5 4.4 5.4 6.1 8.4 19.5 9.6 10.1 3.0 3.7 3.9 4.1 2.6 2.7 5.7 7.6 6.4 6.6 6.4 7.1 6.5

6.4 5.3 11.4 10.2 10.6 10.1 9.4 11.8 12.1 11.7 11.4 11.2 11.6 11.8 11.6 11.4 10.3 8.9 5.3 8.8 12.6 9.6 25.0 24.1 9.6 11.8 11.7 7.5 7.5 5.4 4.4 17.6 9.2 8.0 9.7 7.9 6.8 11.4 15.9 10.0 9.0 3.4 4.4 4.5 6.2 22.7 8.9 9.2 4.6 5.8 3.6 3.1 2.4 2.3 4.2 8.6 7.5 5.2 5.3 7.0 5.7

1.1 1.1 1.1 1.1 1.0 1.0 1.0 1.2 1.2 1.2 1.1 1.1 1.2 1.2 1.2 1.1 1.1 1.1 0.9 0.9 0.9 1.1 1.0 1.0 1.1 1.2 1.2 1.1 0.9 0.9 0.9 0.9 1.2 1.1 1.1 1.0 1.0 1.0 0.9 1.1 1.0 1.1 0.9 0.9 0.9 1.1 1.0 1.0 1.2 1.2 1.2 1.0 1.2 1.2 0.8 1.1 1.1 0.9 0.9 1.2 1.1

4.0 3.5 7.9 7.3 9.2 7.7 6.5 7.1 7.1 7.6 8.0 7.8 8.0 8.1 8.0 7.9 7.4 6.7 6.1 11.6 15.9 7.6 23.3 25.2 7.4 8.1 8.0 6.1 8.6 6.0 5.8 23.2 6.8 6.2 7.4 6.1 7.0 10.9 18.1 7.7 8.5 2.1 5.5 4.7 6.7 17.9 9.2 7.9 4.0 5.1 1.8 2.3 1.2 1.2 5.5 7.4 6.8 6.4 6.1 4.4 3.3

0.7 0.7 0.8 0.8 0.8 0.8 0.7 0.7 0.7 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 1.0 1.2 1.1 0.8 1.0 1.0 0.8 0.8 0.8 0.9 1.1 1.0 1.2 1.2 0.8 0.9 0.8 0.8 1.0 1.0 1.1 0.9 0.9 0.7 1.1 0.9 1.0 0.9 1.0 0.9 1.0 1.0 0.6 0.8 0.6 0.6 1.1 0.9 1.0 1.1 1.0 0.7 0.7

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Appendix A (continued) No. Peptide sequence

Group Solid Solution Number Hydrophobicity Hydrophobicity/ Betax–u phase binding of number of aa sheet value binding % amino % acids

Beta- alpha- alpha- Beta- Betasheet helix helix turn turn value/ value value/ value value/ number number number of aa of aa of aa

70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127

y y y y u u x y u y u u u y u y y y y u u y y y u u u u y y u y u u u u u u u y y y y y y z z u y y y u y y u y u u

1.0 0.9 1.2 1.2 1.1 1.0 1.3 0.9 1.0 0.9 1.0 0.9 1.1 1.1 1.2 1.0 1.1 1.1 1.1 1.1 1.0 1.1 1.1 1.0 1.0 1.0 1.1 1.0 1.2 1.0 1.1 1.2 1.2 1.1 1.2 1.2 1.1 1.0 1.4 1.1 1.1 1.0 0.9 0.9 0.9 1.3 1.3 1.2 1.3 1.4 1.4 1.3 1.3 1.2 1.3 1.6 0.9 0.9

LPFFD APYA RY YSLYIGR EYSLYIGR GEYSLYIGR Ac-IVYWIRKGVPIL-NH2 AWGGSGSEAYQGVQQKWDATATEL QGVQQKWDATATELNNALQNLART ATELNNALQNLARTISEAGQAMAS LARTISEAGQAMASTEGNVTGMFA EAAASAIQGNVTSIHSLLDEGKQS AVLFFGRCVSP SLLQHLIGL-NH2 QLLQHLIGL-NH2 SLLQHLPGL-NH2 KLTNYSVTD-NH2 SLTNYSVTD-NH2 KLLNYSVTD-NH2 KLTQYSVTD-NH2 KLTNHSVTD-NH2 KLTNYLVTD-NH2 KLTNYSITD-NH2 KLTNYSVGD-NH2 EVDPIGHLY ILGDKKLL AFLPWHRLF SEIWRDIDF AAGIGILTV YLEPGPVTA LLDGTATLRL ITDQVPFSV VLYRYGSFSV QLLALLPSL NLTHVLYPV SLFRA VITK LIYRRRLMK MEVDPIGHLY CLVL-NH2 LFVAMWSD-NH2 FVAMWSD-NH2 VAMWSD-NH2 AMWSD-NH2 MWSD-NH2 WSD-NH2 GYVIIKPLVW-NH2 GYVIIKPLV-NH2 GYVIIKPL-NH2 GYVIIK-NH2 GYVII-NH2 GYVI-NH2 IIKPLVWV-NH2 IKPLVWV-NH2 KPLVWV-NH2 PLVWV-NH2 VWV-NH2 CEPAVYFKEQFLDGD CEDVPGQALDEL

3 1 1 1 1 1 12 3 3 3 3 3 1 1 1 1 1 1 3 1 1 1 1 1 4 3 3 3 3 3 4 3 3 3 4 3 4 6 1 1 1 1 1 1 1 10 10 9 2 2 1 2 1 1 3 1 3 3

0 0 0 5 10 13 92 7 29 6 11 27 11 9 11 0 0 0 0 11 10 0 5 0 39 18 20 16 1 0 23 9 17 29 32 21 13 21 14 3 6 0 0 0 0 8 3 11 0 9 3 11 0 0 18 6 24 15

5 4 2 7 8 9 12 24 24 24 24 24 11 9 9 9 9 9 9 9 9 9 9 9 9 8 9 9 9 9 10 9 10 9 9 9 9 10 4 8 7 6 5 4 3 10 9 8 6 5 4 8 7 6 5 3 15 12

4.3 0.7 − 5.8 0.0 − 0.4 − 3.9 13.1 − 17.4 − 20.5 − 3.8 4.7 − 4.8 14.8 11.8 9.1 5.7 − 6.4 − 3.3 − 1.9 − 6.4 − 8.3 − 1.8 − 6.1 − 6.1 − 1.0 4.2 4.8 − 4.9 19.1 0.7 7.2 5.6 5.9 14.9 5.7 7.2 − 4.7 0.9 14.3 9.3 5.5 2.7 − 1.5 − 3.3 − 5.2 13.1 14.0 9.8 7.6 11.5 7.0 14.8 10.3 5.8 9.7 7.5 − 6.8 − 3.4

0.9 0.2 − 2.9 0.0 0.0 − 0.4 1.1 − 0.7 − 0.9 − 0.2 0.2 − 0.2 1.3 1.3 1.0 0.6 − 0.7 − 0.4 − 0.2 − 0.7 − 0.9 − 0.2 − 0.7 − 0.7 − 0.1 0.5 0.5 − 0.5 2.1 0.1 0.7 0.6 0.6 1.7 0.6 0.8 − 0.5 0.1 3.6 1.2 0.8 0.5 − 0.3 − 0.8 − 1.7 1.3 1.6 1.2 1.3 2.3 1.8 1.9 1.5 1.0 1.9 2.5 − 0.5 − 0.3

5.2 3.7 2.4 8.3 8.6 9.4 15.3 22.7 24.3 22.7 23.2 22.5 12.5 10.3 10.6 9.2 9.8 9.8 9.9 10.0 9.2 10.3 9.7 9.3 9.2 8.3 9.9 9.1 10.6 8.7 10.6 10.5 12.2 9.7 11.0 10.4 10.3 10.2 5.5 8.9 7.6 6.2 4.5 37 2.7 12.8 11.4 9.7 7.9 7.1 5.5 10.6 9.0 7.4 6.6 4.8 14.2 10.5

5.1 4.1 1.7 6.0 7.5 8.1 11.6 24.8 25.6 26.7 26.0 25.5 10.6 9.4 9.7 8.9 8.2 7.8 8.6 8.7 8.5 8.7 8.3 8.0 8.7 8.6 9.7 9.7 9.2 8.4 10.5 8.6 8.9 9.9 8.3 9.6 9.7 10.2 4.2 9.1 7.9 6.8 5.7 4.3 2.9 9.6 8.5 7.4 5.6 4.5 3.4 8.3 7.2 6.1 5.0 3.2 15.8 12.9

1.0 1.0 0.8 0.9 0.9 0.9 1.0 1.0 1.1 1.1 1.1 1.1 1.0 1.0 1.1 1.0 0.9 0.9 1.0 1.0 0.9 1.0 0.9 0.9 1.0 1.1 1.1 1.1 1.0 0.9 1.0 1.0 0.9 1.1 0.9 1.1 1.1 1.0 1.0 1.1 1.1 1.1 1.1 1.1 1.0 1.0 0.9 0.9 0.9 0.9 0.9 1.0 1.0 1.0 1.0 1.1 1.1 1.1

4.8 4.0 2.1 7.3 8.0 9.6 10.1 24.7 23.1 22.2 22.1 23.5 10.1 7.8 7.3 8.8 9.6 10.0 9.2 9.0 9.4 8.8 9.6 10.2 8.9 7.3 7.4 8.5 7.4 9.2 8.9 8.4 9.8 7.5 8.3 7.2 7.3 9.5 2.9 6.8 6.2 5.6 5.1 4.5 3.9 8.7 7.8 7.3 5.2 4.1 3.7 6.0 5.6 5.1 4.1 2.0 14.8 12.0

1.0 1.0 1.0 1.0 1.0 1.1 0.8 1.0 1.0 0.9 0.9 1.0 0.9 0.9 0.8 1.0 1.1 1.1 1.0 1.0 1.0 1.0 1.1 1.1 1.0 0.9 0.8 0.9 0.8 1.0 0.9 0.9 1.0 0.8 0.9 0.8 0.8 1.0 0.7 0.9 0.9 0.9 1.0 1.1 1.3 0.9 0.9 0.9 0.9 0.8 0.9 0.8 0.8 0.8 0.8 0.7 1.0 1.0

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