Investigating the oxidative refolding mechanism of Cripto-1 CFC domain

BIOMAC-12792; No of Pages 11 International Journal of Biological Macromolecules 137 (2019) xxx

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Investigating the oxidative refolding mechanism of Cripto-1 CFC domain Emanuela Iaccarino a,b,1, Annamaria Sandomenico b,1, Giusy Corvino a,1, Giuseppina Focà a, Valeria Severino a, Rosita Russo a, Andrea Caporale b, Domenico Raimondo c, Marco D'Abramo d, Josephine Alba d, Angela Chambery a,⁎, Menotti Ruvo b,e,⁎⁎ a

Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche (DISTABIF), via Vivaldi, 43, 80100 Caserta, Italy Istituto di Biostrutture e Bioimmagini, CNR and Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPeB), via Mezzocannone 16, 80134 Napoli, Italy Dipartimento di Medicina Molecolare, Sapienza Università di Roma, 00161, Italy d Dipartimento di Chimica, Sapienza Università di Roma, 00161, Italy e Anbition srl, via A. Manzoni, 1, 80123 Napoli, Italy b c

a r t i c l e

i n f o

Article history: Received 18 March 2019 Received in revised form 5 July 2019 Accepted 6 July 2019 Available online 08 July 2019 Keywords: Cripto CFC domain Oxidative folding Disulfide bridges

a b s t r a c t Using a combined approach based on MS, enzyme digestion and advanced MD studies we have determined the sequential order of formation of the three disulfide bridges of the Cripto-1 CFC domain. The domain has a rare pattern of bridges and is involved in the recognition of several receptors. The bridge formation order is C1-C4, C3-C5, C2-C6, however formation of C1-C4 plays no roles for the formation of the others. Folding is driven by formation of the C3-C5 bridge and is supported by residues lying within the segment delimited by these cysteines. We indeed observe that variants CFC-W123A and CFC-ΔC1/C4, where C1 and C4 are replaced by serines, are able to refold in the same time window as the wild type, while CFC-K132A and CFC-W134A are not. A variant where cysteines of the second and third bridge are mutated to serine, convert slowly to the monocyclic molecule. Data altogether support a mechanism whereby the Cripto-1 CFC domain refolds by virtue of long-range intramolecular interactions that involve residues close to cysteines of the second and third bridge. These findings are supported by the in silico study that shows how distant parts of the molecules come into contact on a long time scale. © 2019 Published by Elsevier B.V.

1. Introduction Disulfide bonds play a crucial role in the folding and assembly of secretory proteins. The mechanism underlying the acquisition of the native state by unfolded proteins is one of the most stimulating issues in structural biology because understanding the progression of protein folding events may provide valuable insights in studying protein's misfolding mechanisms associated with pathological diseases or in assisting protein structure prediction and design of protein modules with predefined functions. Elucidating the mechanism of folding of a given protein has also broader implications for the comprehension of Abbreviations: RP-HPLC, reverse-phase high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization- time of flight; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; LC/MS, liquid chromatography mass spectrometry; TFA, trifluoroacetic acid; TRIS, tris(hydroxymethyl)aminomethane; MD, molecular dynamics. ⁎ Correspondence to: A. Chambery, Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche (DISTABIF), Università della Campania L. Vanvitelli, via Vivaldi, 43, 80100 Caserta, Italy. ⁎⁎ Correspondence to: M. Ruvo, Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone, 16, 80134 Napoli, Italy. E-mail addresses: [email protected] (A. Chambery), [email protected] (M. Ruvo). 1 EI, AS and GC share the first authorship.

the general principles regulating the self-recognition of protein internal elements and how they contribute to adopt the structures more advantageous for protein function, stability and versatility [1]. The folding of a protein or of a protein domain is indeed a very complex process resulting from the interplay between the conformational space accessible to side chains of adjacent residues, the non-covalent interactions between different protein regions and the formation of covalent bonds, mostly between cysteine thiols to form disulfide bridges. Disulfide bridges strongly contribute to stabilize protein architectures during both the refolding process or in the course of their life cycle, favoring interactions, preventing potential misfoldings and forcing in some cases the adoption of kinetically accessible though thermodynamically unfavored conformations [2]. We have previously reported that the synthetic CFC domain (Cripto-1/Frl-1/Cryptic domain) of both human and mouse Cripto-1 is able to spontaneously refold under mild oxidative conditions and to form the right pattern of disulfide bridges [3–6]. Cripto-1 is a growth factor expressed in the early weeks of embryogenesis and remerges in adult tumor tissues, mostly breast, colon and stomach [7]. The protein contains two independent domains of about 40 residues, the EGF-like and the CFC domain, both with three disulfide bridges. It has been reported that the two domains are able to independently interact with relevant protein partners [8] and to fulfil distinct biological functions. Specifically, the Cripto-1 CFC domain is involved in the

https://doi.org/10.1016/j.ijbiomac.2019.07.040 0141-8130/© 2019 Published by Elsevier B.V.

Please cite this article as: E. Iaccarino, A. Sandomenico, G. Corvino, et al., Investigating the oxidative refolding mechanism of Cripto-1 CFC domain, , https://doi.org/10.1016/j.ijbiomac.2019.07.040

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interaction with at least the ALK4 receptor, and with GRP78 [8]. The ability of the isolated CFC domain to refold suggests that it behaves like an independent structural and functional module of the protein and, as such, could work as a soluble receptor interactor thus as a Cripto-1 antagonist or agonist. The domain contains six cysteinyl residues engaged in three disulfide bonds arranged in an unusual C1-C4, C3-C5, C2-C6 pattern [3,5,6,9,10]. The domain contributes to the protein tumorigenic activity by directly engaging the ALK4 and GRP78 receptors and mediating their downstream antiapoptotic signals [8,11–13]. The interaction with ALK4 is mostly mediated by histidine 120 (H120) and tryptophan 123 (W123), localized in the domain largest loop comprised between cysteine 115 (C1) and cysteine 128 (C2) [5]. We have previously investigated the structure and the refolding kinetics of the CFC human and mouse variants [4,5], however the role of some specific residues outside this region and of cysteines in the refolding mechanism has never been studied. We have now examined in further details the process of refolding of the CFC domain of human Cripto-1, elucidating the sequential order of formation of the three bridges and the role played by cysteines in the mechanism of assembly of the final structure. These studies might pave the way to the comprehension of the mechanism regulating the assembly of this as well as other similar domains and to the design of new molecules with an improved ability to bind and regulate the activity of relevant receptors.

A

B

2. Material and methods All protected amino acids and resins for peptide synthesis were from either Novabiochem (Laufelfingen, Switzerland) or from GL Biochem (Shanghai, PRC). Solvents for peptide synthesis and purification were from LabScan (Dublin, Ireland). Other reagents for peptide synthesis were from Sigma-Aldrich (Milan, Italy). Preparative RP-HPLC were performed on a Waters Prep 150 LC preparative system, equipped with a 2489 UV/Visible detector and with a ONYX monolithic C18 column (100x10mm ID). Matrices and consumables for MALDI-TOF mass analysis were also from Waters SpA, Milano, Italy. LC-MS analyses were performed utilizing an Agilent ESI-TOF LC-MS system comprising an Agilent 1290 Infinity LC coupled to an Agilent 6230 TOF mass spectrometer. A Xbridge C18 150 × 2 mm ID column from Waters was used for all analyses applying a gradient from 20% solvent B to 60% solvent B in 40 min. Solvent A was H2O, 0.08% TFA; solvent B was CH3CN, 0.05% TFA. Flow rate was 0.2 mL/min. Detection in positive ion mode from m/z 300–3000. Other experimental conditions for mass analyses are reported elsewhere [14–17]. 2.1. Wild type and mutated Cripto-1 CFC domains preparation The wild type CFC domain of human Cripto-1, residues 112–150, whose primary structure and disulfide arrangement are reported in Fig. 1A, was chemically synthesized as reported elsewhere [18,19] and purified by an improved method involving a three-step strategy. The crude polypeptide was in the first step extensively reduced in 100 mM DTT/100 mM TRIS, pH 7.0 at room temperature for 30 min. It was then purified by reversed-phase HPLC using a C18 NUCLEODUR 15 × 2.1 cm ID (Macherey Nagel) column, flow rate 20 mL/min, applying a gradient of CH3CN, 0.1% TFA from 20 to 45% over 30 min. Purified fractions were immediately frozen and lyophilized. In the second step the polypeptide was refolded as reported in Calvanese et al. [9] and purified again by RP-HPLC using the same method, collecting only the main peak. In the third step the lyophilized material was reduced again using DTT and the reducing agent removed by a rapid desalting step in HPLC. The purified material was stored frozen at −80 °C as working aliquots and used for the experiments of disulfide bridge determinations and for determining the refolding kinetics. Refolding experiments were performed in 100 mM Tris-HCl buffer, 0.5 mM EDTA, 20% DMSO, pH 8.5 at the concentration of 56 μM (0.25 mg/mL, 500 μL), maintaining the solution under stirring, exposed to air oxygen. 70 μL aliquots

Fig. 1. (A) Single letter amino acid sequence and disulfide pattern of the CFC domain of human Cripto-1. Cysteine residues are reported in red. The three disulfide bridges are indicated by connecting lines. (B) NMR Structure of the CFC domain [4]. Disulfide bridges and residues mutated to alanine are labelled. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(containing about 4 nmoles of CFC) were taken from the reaction mixture at time 0, 10, 20, 30, 40, 50, 60, 90, 120,180 and 270 min. T0 was the time just after addition of the refolding buffer. The mutated domains CFC(112–150)-W123A (CFC-W123A) and CFC(112–150)-C115S/C133S (CFC-ΔC1/C4), were similarly prepared by solid phase chemical synthesis. CFC(112–150)-W134A (CFCW134A), CFC(112–150)-K132A (CFC-K132A) and CFC(112–150)C128S/C149S-C131S/C140S (CFC-ΔC2/C6-C3/C5) were instead prepared by solid phase chemical synthesis and repeatedly purified to homogeneity by RP-HPLC in presence of the reducing agent under the conditions reported above. The fully reduced polypeptides were characterized by LC-MS using an Agilent ESI-TOF LC-MS system to assess molecular weight and final purity. All refolding experiments were performed in the same buffer. Aliquots were taken at the indicated time points and analyzed by LC-MS as described above. 2.2. CFC domain alkylation Cysteines in the free state during the refolding reaction were alkylated with IAM. 50 nmoles of IAM in 5 μL water were added to each aliquot (containing about 4 nmoles of polypeptide, about 24 nmoles thiols) and the sample left for 15 min in the dark. 2.3. Trypsin and chymotrypsin digestion Aliquots taken at the indicated time points were treated with either trypsin or chymotrypsin. 25 μL aliquots of all alkylated samples, were digested with trypsin by adding the enzyme at 1:100 and incubating at 37 °C for 16 h [20,21]. Samples were then centrifuged at 15800g for

Please cite this article as: E. Iaccarino, A. Sandomenico, G. Corvino, et al., Investigating the oxidative refolding mechanism of Cripto-1 CFC domain, , https://doi.org/10.1016/j.ijbiomac.2019.07.040

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10 min before MALDI-TOF analyses. Digestions with chymotrypsin were similarly carried out using the same volume of sample and the same enzyme:substrate ratio. Resulting fragments were analyzed by MALDI-TOF mass spectrometry using a Waters MICRO MX spectrometer in Reflectron mode after accurate calibration with a tryptic mixture of Alcohol Dehydrogenase. α-cyano-4-hydroxycinnamic acid at 10 mg/mL in 50% acetonitrile was used as ionizing matrix. Data were processed using the MassLynx software, v. 4.1 [22]. 2.4. H/D exchange experiments 10 nmoles of the synthetic polypeptide dissolved in 1 mL of NH4HCO3 1 mM pH 8.0 (concentration 10 μM) were allowed to refold under stirring upon exposure to air oxygen. 25 μL aliquots were removed after 0, 10, 30, 60, 90, 180, and 270 min and diluted 1:1 with D2O or the same volume of water. The D2O-treated and non-treated aliquots were then alkylated as described before, acidified at pH ≈ 1.5 by adding formic acid and analyzed by MALDI-TOF mass spectrometry. 2.5. Analysis of the folding kinetics A preliminary comparative evaluation of the folding kinetics of the various domains was attempted by fitting the data points referred to disappearance of the reduced polypeptides (decay) and appearance of the final oxidized species with a non-linear algorithm. The analysis was carried out on the wild type domain (disappearance of the reduced polypeptide and appearance of the 3-bridged species), on CFC-W123A (disappearance of the reduced polypeptide and appearance of the 3bridged species), on CFC-K132A (disappearance of the reduced polypeptide and appearance of the 3-bridged species), on CFC-W134A (disappearance of the reduced polypeptide), on CFC-ΔC1/C4 (disappearance of the reduced polypeptide and appearance of the 2bridged species) and on CFC-ΔC2/C6-C3/C5 (disappearance of the reduced polypeptide and appearance of the 1-bridged species). The fitting was performed on the isolated curves using the One-Phase decay (disappearance of reduced species) and One-Phase Association algorithms (appearance of most oxidized species) implemented in Prism GraphPad v. 5.0 obtaining the corresponding K (min−1) and tau (min) values. 2.6. Molecular dynamics simulations To obtain a molecular description of the pathways connecting the selected unfolded structures to the folded ones of the wild type human CFC domain, we applied the Essential Dynamics Sampling (EDS) procedure previously described [23]. Briefly, in EDS, a “free” MD step is accepted if the system goes towards the target structure, i.e. if the distance between the structure obtained after an MD step and the target structure is decreased compared to the same distance obtained using the previous MD structure. In case such a distance increases, the new MD structure is projected onto the hypersphere defined by a set of eigenvectors obtained from the principal component analysis of the free MD trajectories of the two forms. These new coordinates are then used for the next MD step. In this way, the distance between the MD sampled structures can decrease but not increase along the trajectory, thus resulting in a very efficient sampling of the pathway connecting the starting structure to the target one. The all-atoms molecular dynamics simulations of the folded and unfolded forms of the CFC domain were performed by Gromacs software package [24]. The CFC solution structure obtained by NMR [9] was used as starting model of the folded form. The unfolded conformations were obtained by 200 ns long hightemperature (350 K) MD simulation of the folded form. Nine folded and nine unfolded structures were selected by a cluster analysis of the 300 K and 350 K MD simulations, respectively. By sensitivity analysis, the first 193 eigenvectors, as obtained by the essential dynamics analysis of the (backbone+Cβ) concatenated trajectory of the folded and the unfolded forms, were selected to apply the EDS algorithm.

3

This number of eigenvectors (equal to the number of the backbone and Cβ atoms of the CFC domain) represents the best compromise between an accurate description of the target (folded) structures and a limited restraint on the degrees of freedom of the system. In this way, 81 paths describing the folding process of the CFC domain were obtained by means of EDS simulations, each lasting 500 K steps. Concerning the disulfide bond analysis, disulfide bonds were considered formed when the S\\S distance was b5 Angstrom. All the selected unfolded structures used to start the EDS simulations have an RMSD with respect to the NMR structure N4 Angstrom and the three SS bonds not formed. 3. Results We have investigated the mechanism of oxidative refolding of the CFC domain of human Cripto-1, a small protein module (Fig. 1A, B) able to refold forming the correct pattern of disulfide bonds under a variety of conditions [5,6,9]. We have here determined the temporal sequence of bridges formation and studied the impact of some selected mutations on the progress of the polypeptide refolding over time, including W123A, located in the segment comprised between C1 and C2, K132A, located between C3 and C4, and W134A, located next to C4. W123 was selected because, together with H120, is involved in the recognition of the ALK4 receptor. We have previously shown that when H120 and W123 are mutated to alanine the resulting polypeptides are still able to refold [3,5], suggesting that the stretch comprised between C1 and C2 can only poorly influence the structural behaviour of the domain. Also, preliminary experiments with the mutant lacking the first bridge (CFC-ΔC1/C4, see below) showed that bridge C3-C5 was readily and invariably formed, suggesting that these and adjacent residues may play a crucial role in the overall assembly process of the domain. We therefore focused our attention on the region around C3 (and the adjacent C4) whose ability to form disulfide bridges might be strongly impaired by mutations of residues in its immediate vicinity, such as K132, which is in-between C3 (C131) and C4 (C133), and W134, which is next to C4 and also bears the bulkiest side chain (see Fig. 1B). We have also investigated the influence of replacing selected cysteines with the isosteric serine on the ability of the domain to refold within a defined time interval (270 min). The domain mutants studied were thus CFC-W123A, CFC-W134A, CFC-K132A, CFC-ΔC1/C4 and CFCΔC2/C6-C3/C5. 3.1. Chemical synthesis of the polypeptides All linear peptides were obtained with good yields and in sufficient amounts to perform all refolding studies. The experimental MWs determined by LC-MS analysis were in very good agreement with those expected (see Table 1). 3.2. Refolding kinetics of Cripto-1 CFC domain studied by MALDI-TOF mass spectrometry The refolding kinetic of the wild type domain was studied by MALDITOF analysis at various time points monitoring the alkylation of the Table 1 Calculated and experimental molecular mass values of the human CFC variants prepared and investigated in this study. Molecular weights have been determined as described in the section of Methods. CFC variant

MWcalcd. (monoisotopic) (amu)

MWexpm. (monoisotopic) (amu)

hCFC hCFC-W123A hCFC-K132A hCFC-W134A hCFC-ΔC1/C4 hCFC-ΔC3/C5-C2/C6

4472.07 4357.03 4415.01 4357.03 4440.12 4408.16

4472.08 4357.03 4415.04 4357.34 4440.13 4408.34

Please cite this article as: E. Iaccarino, A. Sandomenico, G. Corvino, et al., Investigating the oxidative refolding mechanism of Cripto-1 CFC domain, , https://doi.org/10.1016/j.ijbiomac.2019.07.040

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residual free cysteines. This analysis preceded subsequent experiments performed to establish the order of disulfide bridge formation. Mass analyses were performed after cysteine alkylation with IAM (Iodoacetamide, Δmass: +57.02 amu) to facilitate the detection of residual free thiols. In Fig. 2A the MALDI-TOF MS spectra of the alkylated samples collected during the refolding process at the indicated time points are reported. As shown, at T0 the polypeptide was mostly in the fully reduced form (fully alkylated product, six IAMs incorporation, m/z 4818.8), but a substantial fraction of polypeptide (approximately 33%, as calculated by peak height) contained already a single disulfide bridge (four IAMs incorporation, m/z 4702.5). At 10 min the occurrence of species containing a second disulfide bridge (about 30%) was recorded, with two IAMs incorporated (m/z at 4586.4), together with single disulfide species (about 50%) and a fraction of about 20% of the fully reduced polypeptide (six IAM incorporation). The fully oxidized species appeared firstly at T30 (about 40%) and its formation progressed steadily to completion over the next 60 min (T90, N95% refolding achieved) (Fig. 2B). At T30, species with one disulfide were about 10%

and those with two bridges were about 50%. At T60, species with one disulfide were absent and those with two were only about 20%. At 180 min only the fully oxidized polypeptide was present and persisted up to 270 min, suggesting that no reshuffling reactions were occurring (Fig. 2B). These findings were in agreement with our previous study [4] reporting an almost complete domain assembly after the same time span. This observation was also in very good agreement with the refolding kinetic monitored by means of LC-MS analyses (Fig. S1) showing that the first disulfide was already formed at 10 min and disappeared at 60 min, the second disulfide appeared at 10 min and disappeared at 60–90 min and the third bridge formed and persisted starting from 60 min onward. Refolding mechanism of the wild type Cripto-1 CFC domain characterized by MALDI-TOF MS. We next determined the sequential order of bridge formation in the wild type CFC domain by treating samples taken at the various time points with either trypsin or chymotrypsin and analyzing the resulting fragments by MALDI-TOF MS. As shown in Fig. 3, analysis of fragments obtained by trypsin at T0 and up to T30, provided a mass peak at m/z 2649.2 (Fig. 3A; monoisotopic value), that accounts for the fragment 112–126 + 133–139 interconnected by the disulfide bridge between C1 and C4, Fig. 3B. This fragment originated by the cleavage of trypsin on K126, K132 and R139 (See Table 2 for calculated and experimental MWs). In the sample treated with chymotrypsin, at the same time points, we also detected a mass peak at m/z 1901.8 (Fig. 3C; monoisotopic value), that accounted for the peptide 135–150, containing the alkylated C5 and C6 and originated by the cleavage on W134 (Fig. 3D). The presence of this isolated fragment excluded the concomitant occurrence of the other two single bridges (C3-C5 and C2-C6) and initially suggested that domain refolding was triggered by the formation of the C1-C4 pair. Within the spectra at time points T10-T90 collected on the trypsintreated samples, we also detected a mass peak at m/z 1988.84 (Fig. 4A; monoisotopic value), corresponding to peptides 127–132 + 140– 150 joined by one disulfide and alkylated on the other two cysteines (theoretical [M + H] + = 1988.89 Da). This peak was diagnostic for the formation of a second bridge (Fig. 4B), but did not enable its unambiguous assignment, since the two peptide fragments could be held together or by bridge C3-C5, species (*) either C2-C6, species (**). The identity of this bridge was assessed analyzing the mass peaks obtained on samples treated with chymotrypsin. Here, we detected a mass peak at m/z 4061.63 at time points T60 and T90 (Fig. 4C; average value), corresponding to fragment 112–123 + 124–145 connected by the bridges C1-C4 and C3-C5 and bearing the alkylated C2, see Fig. 4D. This fragment originated by cleavage on W123 and F145 and was diagnostic for the formation of the second disulfide bridge between C3 and C5, beyond the first assigned to C1 and C4. The occurrence of the ion peak at m/z = 4044.44 (average value), corresponding to the peptide 112–145 with a miscleavage on W123, further confirmed this assignment (Fig. 4C, D). Both ion peaks were no longer detected at T180, when the third bridge was fully formed and signals disappeared due to engagement of C2 in the bond with C6, in full agreement with the data shown in Fig. 4A, B obtained on the undigested sample. Altogether, the data demonstrate that the sequence of bridges formation of human Cripto-1 CFC domain is: 1) C1-C4; 2) C3-C5; 3) C2-C6.

3.3. Refolding kinetics of wild type CFC, CFC-W123A, CFC-K132A, and CFCW134A studied by LC-MS

Fig. 2. A. MALDI-TOF mass spectra of the undigested CFC domain at different time points and after alkylation with iodoacetamide (IAM). The mass difference of 114 Da accounts for the incorporation of two IAM groups on pairs of reduced cysteines. Fig. 2B. Time course progression of the formation of the different disulfide bridges during CFC refolding. Relative amounts of the different species have been roughly estimated by MALDI-TOF peak heights as they appear in Fig. 2A.

The refolding kinetics of the Cripto-1 CFC domain was further investigated analyzing the behaviour of the three mutants CFC-W123A, CFCK132A, and CFC-W134A, by characterizing the formation of disulfide bonds within a time span of 270 min. The time interval we considered was sufficient for complete renaturation of the wild type and some of the mutated domains [4].

Please cite this article as: E. Iaccarino, A. Sandomenico, G. Corvino, et al., Investigating the oxidative refolding mechanism of Cripto-1 CFC domain, , https://doi.org/10.1016/j.ijbiomac.2019.07.040

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Fig. 3. A: MALDI-TOF mass spectra with the tryptic peak at m/z 2649.2 accounting for the polypeptide fragment 112–126 + 133–139. Fig. 3B: tryptic peptides originated by cleavage on K126, K132 and R139. Fig. 3C: time progression of mass peak at m/z 1901.8 which accounts for the chymotryptic peptide 135–150, alkylated on C140 and C149 and originated by cleavage on W134 (Fig. 3D). Upwards arrows indicate the sites of trypsin or chymotrypsin cleavage.

The chromatograms and the plot of % products formation over time obtained for the refolding of the different domains are reported in Fig. S1–S6. Data obtained with the wild type domain, reported in Fig. S1, show that it was fully folded at 90 min and remained stably folded at least up to 270 min as also observed with the MALDI-TOF experiments. The formation of intermediates monitored by the different mass

spectrometry approaches had very similar kinetic courses with some differences likely resulting from the different sensitivities of the mass spectrometric approaches (MALDI-MS and ESI-MS). The mutant CFCW123A (Fig. S2) spontaneously refolded to a quasi-homogeneous species with the expected formation of 3 disulfide bridges. However, the process was much slower compared to the wild type polypeptide. We

Please cite this article as: E. Iaccarino, A. Sandomenico, G. Corvino, et al., Investigating the oxidative refolding mechanism of Cripto-1 CFC domain, , https://doi.org/10.1016/j.ijbiomac.2019.07.040

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Table 2 Calculated and experimental molecular mass values for the most relevant CFC tryptic and chymotryptic fragments observed in the MALDI-TOF spectra used to assign the polypeptide disulfide arrangement. CFC peptide fragment

M + H+ calcd.

112–150

4702.52

112–150

# ##

##

(average of values at T10 and T30 min)

4586.30 (average of values at T10, T30, T60 and T90 min) 4470.21## (average of values at T30, T60, T90, 180 and T270 min) 2649.20###

Notes Fully reduced, 6 IAM. Average molecular mass. See Fig. 2A. One disulfide bridge, 4 IAMs. Average molecular mass. See Fig. 2A. Two disulfide bridges, 2 IAMs. Average molecular mass. See Fig. 2A. Three disulfide bridges. No IAMs. See Fig. 2A.

##

112–150

127–132 + 140–150 112–123 + 124–145

Δmass (Da)

4818.70## (average of values at T0 and T10 min)

112–150

112–126 + 133–139 135–150

M + H+ expm.

−0.05

One disulfide bridge C1-C4. Trypsin cleavage. See Fig. 3A, B.

1901.88

1901.81

###

−0.07

1988.89

1988.84###

−0.05

4044.74#; 4062.76#

4044.44#; 4061.43#,###

−0.30; −1.33#

IAM alkylated C5 and C6, originated by chymotrypsin cleavage on Trp134. See Fig. 3C, D. The bridge was either C3-C5, species (*) or C2-C6, species (**) with 2 IAMs. See Fig. 4A, B. Bridges are between C1-C4 and C3-C5 and bearing the alkylated C2. See Fig. 4C, D.

2649.25

Values were taken from the most intense peak and were not the real average experimental molecular mass. Average molecular mass.

indeed observed that while the first and second disulfide formed rapidly, appearing yet at 10 min, the third one started to form only at 50 min, reaching an almost complete conversion at 270 min. At variance with the wild type domain, the conversion of the reduced polypeptide to the fully oxidized form passed through the formation of many intermediates which resolved completely only after several hours (not shown). The refolding kinetic of the CFC-W134A variant, reported in Fig. S3, showed that while a single bridged species still appeared at 10 min, a high number of intermediates did not convert to a single homogeneous molecule within the time window of 270 min. The rapid appearance of the first bridge (b10 min) reinforces the hypothesis that its formation is driven by intramolecular interactions involving residues within the first loop, which are indeed unaltered in this polypeptide. Data also suggests that W134, instead, strongly contribute to drive the formation of the other bridges, since its absence prevented their formation, at least in the explored time window. In agreement with this hypothesis, the CFC-K132A variant, containing a K to A mutation close to W134 and in-between two cysteines (see Fig. S4), commenced to convert to a single disulfide bridge after 10 min, but its amount did not change between 40 and 270 min. Data show that it was only able to form a minor amount of a 2-bridged molecule (peak at 16.0 min), while no species with 3 disulfide bridges appeared in the LC-MS chromatograms in the investigated time window. This behaviour suggested that K132 and other nearby residues strongly influence the formation of 2- and 3-bridged species that is likely triggered by the single bridged form. 3.4. Refolding kinetics of CFC-ΔC1/C4 and CFC-ΔC2/C6-C3/C5 In order to investigate into a greater detail the impact of the C1-C4 disulfide bridge on the Cripto-1 refolding kinetics, we analyzed the kinetics of two domain mutants: CFC-ΔC1/C4 and CFC-ΔC2/C6-C3/C5 where cysteines C1 and C4 and C3-C5 and C2-C6 were replaced by isosteric serines. Despite the absence of the pair of cysteines forming the first bridge, the refolding kinetic of CFC-ΔC1/C4, reported in Fig. S5, was surprisingly similar to that of the wild type domain. In fact, a species with one disulfide appeared yet at 10 min and increased over the next 20 min (up to 30 min; see inset of Fig. S5). At 20 min we observed the appearance of the 2-bridged molecule whose amount progressively increased over the subsequent 70 min (up to 90 min; see inset of Fig. S5) and persisted for the next 180 min, suggesting that it was very stable over time. Remarkably, also a small percentage (about 10%) of the starting material remained unaltered, likely in equilibrium with the main product. We next analyzed by LC-MS the CFC-ΔC2/C6-

C3/C5 polypeptide that contains only the two cysteines forming the bridge C1-C4. The overlaid chromatograms at the various time points are reported in Fig. S6 and show that the fully reduced polypeptide formed the internal C1-C4 disulfide very slowly compared to the wild type and the other mutants. At 270 min, only about 70% of the starting material was converted to the cyclic form, although this species was observed yet at t0, just after dissolution. Data obtained with the CFC-ΔC1/ C4 and CFC-ΔC2/C6-C3/C5 mutants thus strongly support the hypothesis that formation of the first bridge is not necessary to trigger the formation of the others that indeed seem to drive the entire folding process. 3.5. H/D exchange studies on the wild type CFC domain We next performed a H/D exchange experiment on the wild type CFC domain to determine the number of hydrogens not exchanged during the refolding progression and thus potentially involved in strong intra-chain H-bonds. Mass spectra were collected on the undigested alkylated polypeptide at T0 and on samples taken at the subsequent time points. As shown in Fig. 5, the fully alkylated polypeptide at T0 incorporated on average 51–54 deuterium atoms (mass peak at about m/z 4867/4870 compared to the expected value of about m/z 4818). The fully refolded polypeptide (T270) incorporated on average only 39 deuterium atoms (mass peak at about m/z 4509 compared to m/z 4470) suggesting that upon refolding some 10–15 deuterium atoms were protected from exchange. Considering the extent of deuterium incorporation for the same fully refolded species at T30 (see mass peak at about m/z 4517) and the back-exchange occurring over time during the experiments, the number of protected hydrogens was likely higher than about 20. This observation suggested that an average number of hydrogens comprised between 11 and 20 was protected by the exchange with deuterium during refolding progression. 3.6. Fitting of kinetics The fitting curves and parameters (K and tau) obtained with the different domains are reported in Fig. S7A–F and Table S1. K and tau can be used interchangeably for data interpretation, since tau is simply defined as 1/K. Data in Table S1 are a rough indication of the reaction speed for the conversion of reduced polypeptides into intermediates (decay) and of intermediates to the final oxidized species. The higher is the K value, the faster is the initial speed of reaction for a given species. Also, the more similar are the speed constant values for the disappearance of the starting molecule and for the formation of the final oxidized species,

Please cite this article as: E. Iaccarino, A. Sandomenico, G. Corvino, et al., Investigating the oxidative refolding mechanism of Cripto-1 CFC domain, , https://doi.org/10.1016/j.ijbiomac.2019.07.040

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Fig. 4. A: MALDI-TOF mass spectra showing mass peak at m/z 1988.84 (theoretical [M + H]+ = 1988.89 Da) accounting for the peptide pair 127–132 + 140–150 containing the four cysteines C128, C131, C140, C149. Fig. 4B: schematic view of peptides originated by chymotrypsin cleavage. On the basis of the molecular weight, cysteines C131 and C140 are connected by a disulfide bridge, the others are alkylated with IAM. Mass spectra do not elucidate whether species (*) or (**) are detected. C: MALDI-TOF mass spectra showing peaks referred to chymotryptic peptides at m/z 4061.43 and m/z 4044.44 diagnostic for the second disulfide bridge between C131 (C3) and C140 (C5). D: Sequences and disulfide pattern of fragments detected. “I” indicates the fragment 112–145 with a cleavage on W123, while “II” indicates the same fragment without the cleavage on W123. Mass peak values are reported as average values. Upwards arrows indicate the sites of trypsin or chymotrypsin cleavage.

Please cite this article as: E. Iaccarino, A. Sandomenico, G. Corvino, et al., Investigating the oxidative refolding mechanism of Cripto-1 CFC domain, , https://doi.org/10.1016/j.ijbiomac.2019.07.040

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Fig. 5. MALDI-TOF mass spectra at the indicated time points of the undigested CFC domain incubated in a buffer with D2O, after alkylation with IAM. Mass increase (Δm, Daltons, Da) at the different time points is reported for all the expected molecular species. Vertical lines are placed at the MW values expected without deuterium incorporation. Δm decreases over time indicating that several hydrogen atoms exchange very slowly with deuterium because - of their engagement in H-bonds.

the more concerted is expected to be the folding process. As shown in Table S1, the fastest decay, beyond that of CFC-W134A which did only convert to non-homogeneous species, was observed for CFC-ΔC1/C4, while the slowest was observed for CFC-ΔC2/C6-C3/C5 which leads to the formation of the single bridged species C1-C4. Also the decay speeds of wt CFC and of CFC-K132A are slow, while that of CFC-W123A is high, likely due to the rapid formation of many intermediates which are resolved towards homogeneous species only after about 120 min (see Fig. S2). This observation suggests that the formation of the first bridge C1-C4 is actually braking the decay and subsequent reactions. In agreement with this interpretation, reaction speeds of formation of final species recorded for CFC-W123A, CFC-K132A and CFC-ΔC2/C6-C3/C5 are all particularly slow, with that of CFC-K132A that could not be even estimated. That of CFC-ΔC1/C4 was instead the fastest, confirming that the absence of the first bridge can improve the reaction progression. The K determined for the wt domain for the decay and formation speeds

are quite similar, suggesting that the conversion is highly concerted. A similar behaviour is qualitatively observed for CFC-ΔC1/C4 although the formation of the second bridge C2-C6 is slower than that of the decay due to the rapid formation of the first bridge C3-C5. 3.7. Molecular dynamics simulations The concatenated trajectory, as obtained by joining the 300 K and the 350 K molecular dynamics simulations, was analyzed by means of principal component analysis (essential dynamics). As expected, the essential subspace defined by the two first eigenvectors was able to discriminate between the folded and the unfolded forms (Fig. 6). The EDS procedure was then used to describe the folding pathways connecting the unfolded structures to the folded ones (Fig. 6, green and red lines and SM File1). By such an approach, it is possible to describe conformational transitions occurring on a time scale unreachable

Please cite this article as: E. Iaccarino, A. Sandomenico, G. Corvino, et al., Investigating the oxidative refolding mechanism of Cripto-1 CFC domain, , https://doi.org/10.1016/j.ijbiomac.2019.07.040

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by standard molecular dynamics simulations at a reasonable computational cost [23,25,26]. As expected, all the selected unfolded structures folded towards the corresponding folded forms very efficiently, reaching a value of the RMSDs between the structures of the last EDS frame and the corresponding target structures below 1 Angstrom (data not shown). Very interestingly, the EDS trajectories show that the C3-C5 disulfide bridge was formed in all the 81 simulations. Similarly, the distance between the C2 sulphur and that of C6 reaches a value of 5 Angstrom in 62 of the 81 trajectories (77%). Most of the trajectories show a mechanism of formation of the 3 disulfide bridges in agreement with our experimental data, leading to a properly folded CFC folding. In particular, according to our simulations, the most common mechanism (82% of the EDS trajectories) is represented by the formation of the C3-C5 bond, followed by that connecting C2 and C6. A representative picture of the folding behaviour with respect to disulfide bridges formation is reported in Fig. 7. Remarkably, the bridge C1-C4, which links the Cripto-1 N-terminal segment to the central strand of the domain and the first to be experimentally detected, is formed before, after and between the formation of the C3-C5/C2-C6 bonds in 28%, 50% and 22% of the trajectories, respectively. This multiplicity of mechanisms, in apparent divergence with the experimental data, might instead reflect the substantial dispensability of the C1-C4 bridge for the assembly of the final refolded domain and the fact that its formation is complete only after the closure of the second and third bridge too (see Figs. S5 and S6). The overall rearrangement of the CFC chain described by the EDS approach shows a global concerted motion involving almost all the CFC regions. The EDS trajectories describe a plausible three-dimensional pathways leading to the typical CFC ellipsoidal compact shape with three antiparallel strands connected by disulfide bridges [3]. The orientation of the C-terminal tail correctly points to the first strand and the N-terminal portion to the turn formed by residues 136–138. As expected, all the selected unfolded structures evolved towards the corresponding folded forms very efficiently, reaching a value of the RMSDs between the structures of the last EDS frame and the corresponding target structures below 1 Angstrom (data not shown). It is worth noting that the system suddenly reaches the folded conformational region in the very first frames of the EDS trajectories. After, and until the end of the simulations, there is an improvement of the local contacts which also includes the formation of the disulfide bridges. To further substantiate the results obtained by this analysis, we compared the intramolecular interactions occurring within the CFC structure solved by NMR and those recorded in the final structures

Fig. 6. The projections of the folded (black region on the left) and unfolded (black region on the right) MD trajectories on the essential subspace. The pathways connecting the two conformational states as provided by the EDS are depicted as red and green lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 7. Disulfide bridge evolution along a representative EDS trajectory. The black, red and green lines indicate the C1-C4, C3-C5 and the C2-C6 distance variation along the EDS simulation, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

obtained by means of EDS. The disposition of the β-strands occurring in the CFC experimental structure, represented as narrow contact stretches orthogonal to the diagonal of the matrix, and that of the average contact map obtained with the 81 final EDS structures are reported in Fig. S8A and S8B, respectively. This figure reveals that the stability of almost all intramolecular contacts responsible and characteristic of the CFC domain three-dimensional folding is very similar in the experimental and post-EDS structures. 4. Discussion Elucidating the refolding mechanisms of proteins is a very fascinating field of investigation. Such studies also have thoughtful applicative implications for the insightful help they provide in the de novo design of protein modules with predetermined structures and functions [27]. General rules to accurately predict how polypeptides adopt organized structures in absence of adequate sequence similarity or experimental structural information, are however still far to be identified. Several approaches based on primary structure [28], extrapolated short and longrange contacts [29] or amino acids composition [30] have been proposed and used. However, all approaches need experimental data to support and substantiate predictions. On the other hand, oxidative folding pathways of well-known 3-disulfide small proteins have been experimentally investigated and, on these bases, some simplified models have been proposed [31,32]. The two extreme models emerged by these studies are identified as the BPTI-like and the Hirudin-like mechanisms [31]. In between some mixed mechanisms have also been identified [31–34], delineating a complex scenario of many alternative folding pathways, that can take place also in dependence of the oxidation conditions [31]. Along these lines we have undertaken a study of the oxidative folding mechanism of the CFC Cripto-1 domain, a relatively short polypeptide characterized by an uncommon pattern of disulfide bridges that exhibits a C1-C4, C3-C5, C2-C6 array [3–6,35]. The domain encompasses residues 112–150 of the human protein or residues 96–134 of the mouse variant. We previously investigated the structural properties of both domains, observing that they have a high propensity to spontaneously refold under mild oxidizing conditions, also in presence of several mutations. Notably, both adopt rather flexible conformations, featuring elongated modules [3,5,9] devoid of canonical secondary structures. Only short-living β-strand conformations are observed across some

Please cite this article as: E. Iaccarino, A. Sandomenico, G. Corvino, et al., Investigating the oxidative refolding mechanism of Cripto-1 CFC domain, , https://doi.org/10.1016/j.ijbiomac.2019.07.040

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residues, suggesting that the polypeptide structures are essentially pulled together by the 3 disulfides, without strong intramolecular interactions. In this view, the domains behave like a highly malleable, “stapled” thread [3] whereby bridges stabilize the structure, avoiding large fluctuations and promoting internal interactions. This feature has been also previously evidenced by CD studies showing the absence of canonical secondary structures [4] and has been here confirmed for the human domain by a preliminary H/D exchange study that indeed shows a limited retention of hydrogen bonds after formation of the folded structure. However, in spite of its flexibility and of the lack of stable intermolecular interactions, the domain efficiently refolds to form the expected pattern of disulfide linkages, clearly suggesting that a network of yet weak interactions must exist before the formation of the disulfide network and in some way triggers the oxidative self-assembly. Experimental data collected during the refolding of the wild type domain indicate that the bridge between C1-C4 forms very rapidly, suggesting that a portion of the polypeptide, likely positioned around L124, P125, K126 and K127, at the corner of a large turn, is highly prone to bending and might trigger the entire process. However, kinetics observed with the CFC-ΔC1/C4 and CFC-ΔC2/C6-C3/C5 mutants indicate that formation of the first bridge could only be a collateral event of the formation of bridge C3-C5 and, subsequently, of bridge C2-C6. In fact the rapid and quantitative folding of the CFC-ΔC1/C4 variant and the quite slow formation of bridge C1-C4 in CFC-ΔC2/C6-C3/C5 support together the view that is the formation of C3-C5 to drive the entire folding, also pushing the complete formation of bridge C1-C4 which is, alone, less stable and unable to form completely in the time interval explored. This hypothesis was also supported by data from simulations obtained applying the EDS procedure where the formation of bridges C3-C5 and C2-C6 was clearly the most represented mechanism (observed in 82% of the EDS trajectories). Looking at the structure evolution appearing in SM File1, we can also speculate that the region included between C4 and C5, between W134 and R139, rapidly bends leading to the formation of bridge C3-C5. This evolution, that also involves the neighbouring K132 and C133 (C1), might well explain why CFCK132A and CFC-W134A do not properly fold. Looking at the domain structure obtained in solution by NMR [36] (see Fig. S9A\\B), we deduce that formation of the second bridge C3-C5 could be driven by compaction of residues H135, G136, Q137, L138 and R139, that shape the corner of the relatively small bulge connecting the first and the second bridge. Also, formation of the third disulfide bond could be likely guided by interactions between residues F141, P142, Q143, A144, F145, L146 and S129-L130 on the adjacent strand. On this basis, we hypothesize that the mechanism of folding is driven by the formation of the C3-C5 bond that, in turn, promotes the formation of the C2-C6 and stabilizes that between C1 and C4. In this view, the consecutive formation of the three bridges appears highly concerted and dynamic, as suggested by the concomitant appearance and disappearance of the different species with 1, 2 and 3 bridges (Fig. 2B). This interpretation is also corroborated by a preliminary analysis of the kinetics suggesting that formation of bridges C3-C5 and C2-C6 is highly favoured, while that of C1-C4 may even reduce the overall speed of folding reaction. This mechanism is in agreement with the concept of foldon units introduced by Maity et al. [36] that predicts protein folding as a process promoted by the sequential stabilization and assembly of discrete structural units rather than one residue at a time, and that the folding pathway originates from a stepwise stabilization process. However, at variance with the concept of pre-organized foldons, which entails the formation of stable secondary structures, we hypothesize the occurrence of weakly interacting substructures that drive and stabilize increasingly ordered structures, progressively shaping the native protein. The sequence of the CFC domain is highly conserved among the mouse and human variants of both Cripto-1 and CFC1 proteins, while it is only poorly retained in evolutionary more distant species like zebrafish, Xenopus and chicken CFC1 (Fig. S10). Remarkably, H120

and W123 (human numbering), reportedly involved in receptor(s) binding, are conserved across different proteins and species. Also disulfide-engaged cysteines (together with L138) are fully preserved, denoting that, despite huge sequence differences, domains are likely capable of forming the expected disulfide bond network and tridimensional assembly. Of note, only few residues are mutated passing from the human to the mouse variant, and two of them, D121 mutated to G and A144 mutated to T, are completely divergent. In a previous study we analyzed the refolding kinetic of the mouse CFC domain and of the related mutants H104A and W107A (mouse numbering) [3,4,9,37]. As reported in these studies, refolding of the wild type mouse domain was complete only after N180 min. Also, refolding of the two mutants took much longer times to go to completion, thus suggesting that slight modifications of the primary structure largely affected the kinetics and possibly also the mechanisms of self-assembly. Although further studies are needed, the mechanistic details we have here unveiled for this domain may provide a rationale for understanding the refolding dynamics of other proteins or related domains, sharing or not similar structure or disulfide pattern, and may provide insights for predicting and elucidating relevant interactions that drive oxidative protein refolding. Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2019.07.040. Acknowledgments Authors acknowledge the support from Regione Campania for the projects: i) “Fighting Cancer resistance: Multidisciplinary integrated Platform for a technological Innovative Approach to Oncotherapies (Campania Oncotherapies)”; ii) “Development of novel therapeutic approaches for treatment-resistant neoplastic diseases (SATIN)”; iii) NANOCAN, NANOfotonica per la lotta al CANcro. Also, support from MIUR for project PRIN n°20155ACHBN to AS and 2015783N45 to MR is gratefully acknowledged. Authors also acknowledge the technical advice and support for mass spectrometry by Maurizio Amendola. The authors declare no conflicts of interests. References [1] V. Munoz, M. Cerminara, When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches, Biochem. J. 473 (17) (2016) 2545–2559. [2] C.B. Anfinsen, Principles that govern the folding of protein chains, Science 181 (4096) (1973) 223–230. [3] L. Calvanese, A. Saporito, D. Marasco, G. D'Auria, G. Minchiotti, C. Pedone, L. Paolillo, L. Falcigno, M. Ruvo, Solution structure of mouse Cripto CFC domain and its inactive variant Trp107Ala, J. Med. Chem. 49 (24) (2006) 7054–7062. [4] L. Calvanese, A. Saporito, R. Oliva, D.A. G, C. Pedone, L. Paolillo, M. Ruvo, D. Marasco, L. Falcigno, Structural insights into the interaction between the Cripto CFC domain and the ALK4 receptor, J. Pept. Sci. 15 (3) (2009) 175–183. [5] D. Marasco, A. Saporito, S. Ponticelli, A. Chambery, S. De Falco, C. Pedone, G. Minchiotti, M. Ruvo, Chemical synthesis of mouse cripto CFC variants, Proteins 64 (3) (2006) 779–788. [6] S.F. Foley, H.W. van Vlijmen, R.E. Boynton, H.B. Adkins, A.E. Cheung, J. Singh, M. Sanicola, C.N. Young, D. Wen, The CRIPTO/FRL-1/CRYPTIC (CFC) domain of human Cripto. Functional and structural insights through disulfide structure analysis, Eur. J. Biochem. 270 (17) (2003) 3610–3618. [7] X.F. Hu, P.X. Xing, Cripto as a target for cancer immunotherapy, Expert Opin. Ther. Targets 9 (2) (2005) 383–394. [8] A. Sandomenico, M. Ruvo, Targeting Nodal and Cripto-1: perspectives inside dual potential theranostic cancer biomarkers, Curr. Med. Chem. (2018). [9] L. Calvanese, A. Foca, A. Sandomenico, G. Foca, A. Caporale, N. Doti, E. Iaccarino, A. Leonardi, G. D'Auria, M. Ruvo, L. Falcigno, Structural insights into the interaction of a monoclonal antibody and Nodal peptides by STD-NMR spectroscopy, Bioorg. Med. Chem. 25 (24) (2017) 6589–6596. [10] L. Calvanese, A. Sandomenico, A. Caporale, A. Foca, G. Foca, G. D'Auria, L. Falcigno, M. Ruvo, Conformational features and binding affinities to Cripto, ALK7 and ALK4 of Nodal synthetic fragments, J. Pept. Sci. 21 (4) (2015) 283–293. [11] J.A. Kelber, A.D. Panopoulos, G. Shani, E.C. Booker, J.C. Belmonte, W.W. Vale, P.C. Gray, Blockade of Cripto binding to cell surface GRP78 inhibits oncogenic Cripto signaling via MAPK/PI3K and Smad2/3 pathways, Oncogene 28 (24) (2009) 2324–2336. [12] M. Klauzinska, D. Bertolette, S. Tippireddy, L. Strizzi, P.C. Gray, M. Gonzales, M. Duroux, M. Ruvo, C. Wechselberger, N.P. Castro, M.C. Rangel, A. Foca, A. Sandomenico, M.J. Hendrix, D. Salomon, F. Cuttitta, Cripto-1: an extracellular

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Please cite this article as: E. Iaccarino, A. Sandomenico, G. Corvino, et al., Investigating the oxidative refolding mechanism of Cripto-1 CFC domain, , https://doi.org/10.1016/j.ijbiomac.2019.07.040