Structural Model for the Spider Silk Protein Spidroin-1 - Journal of

Article pubs.acs.org/jpr

Structural Model for the Spider Silk Protein Spidroin‑1 José Roberto Aparecido dos Santos-Pinto,†,‡ Helen Andrade Arcuri,† Helga Priewalder,§ Heliana Clara Salles,† Mario Sergio Palma,*,† and Gert Lubec*,‡ †

Center of the Study of Social Insects, Department of Biology, Institute of Biosciences of Rio Claro, São Paulo State University, Rio Claro, SP 13500, Brazil ‡ Department of Pediatrics, Medical University of Vienna, Vienna 1090, Austria § Department of Paleontology, Geological Survey of Austria, Vienna 1230, Austria S Supporting Information *

ABSTRACT: Most reports about the 3-D structure of spidroin-1 have been proposed for the protein in solid state or for individual domains of these proteins. A gel-based mass spectrometry strategy using collision-induced dissociation (CID) and electron-transfer dissociation (ETD) fragmentation methods was used to completely sequence spidroins-1A and -1B and to assign a series of post-translational modifications (PTMs) on to the spidroin sequences. A total of 15 and 16 phosphorylation sites were detected on spidroin-1A and -1B, respectively. In this work, we present the nearly complete amino acid sequence of spidroin-1A and -1B, including the nonrepetitive N- and C-terminal domains and a highly repetitive central core. We also described a fatty acid layer surrounding the protein fibers and PTMs in the sequences of spidroin-1A and -1B, including phosphorylation. Thus, molecular models for phosphorylated spidroins were proposed in the presence of a mixture fatty acids/water (1:1) and submitted to molecular dynamics simulation. The resulting models presented high content of coils, a higher percentage of α-helix, and an almost neglected content of 310-helix than the previous models. Knowledge of the complete structure of spidroins-1A and -1B would help to explain the mechanical features of silk fibers. The results of the current investigation provide a foundation for biophysical studies of the mechanoelastic properties of web-silk proteins. KEYWORDS: silk proteins, Nephila clavipes, mass spectrometry, post-translational modification, molecular dynamics

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INTRODUCTION Spider web silk is among the most interesting known biomaterials and is produced by the assembly of web-silk proteins known as spidroins.1,2 Spidroin-1 is one of the silk proteins produced by the major ampullate gland from spiders, which is used in the construction of the frame and radii of orb webs and as a dragline to escape from predators; the another protein is spidroin-2.3 The structure of these proteins results in fibers exhibiting an exceptional combination of tensile strength and extensibility;4 therefore, the biophysical properties of these materials have attracted great interest in areas such as chemistry, biochemistry, biotechnology, and biology. Spidroin-1 is present in large quantities in silk fibers and is uniformly found throughout the fiber core;5,6 it is known to encode repetitive short amino acid sequence motifs that predominantly contain Ala and Gly residues.7 The mechanical properties of silk fibers depend on its highly repetitive backbone region, which has a structure reminiscent of a block copolymer and is composed of alternating blocks of a Gly-rich block followed by an Ala-rich block.3 Spidroin-1 contains GGX, (GA)n, and (A)n motifs, and the tensile strength is apparently provided by the (A)n or (GA)n repeats, which form crystalline intra- and intermolecular β-sheet structures in the fiber, © 2015 American Chemical Society

whereas the elasticity is dependent on intervening Gly-rich repeats (GGX and GPGXX motifs).8 The Gly-rich segments are postulated to form different structures such as β-spirals and coil structures.9,10 The mechanical and physicochemical features of spider silk proteins are perfectly suited for many biotechnological applications;11−13 significant academic interest has been devoted to understanding the properties of these biomaterials at the molecular level and their potential uses for biomedical and industrial applications. However, up to now, the mechanical properties of spidroins have been characterized without any consideration about the existence of posttranslational modifications (PTMs) to the sequence; both the natural protein and the recombinant spidroins have been biochemically and physically characterized as they would have the same physicochemical properties.7,9,14 The most sequences of spidroin-1 were obtained from gene sequencing;15,16 only a few complete sequences of spidroins were obtained directly from protein sequencing, and 3-D structures have been Received: March 20, 2015 Published: July 27, 2015 3859

DOI: 10.1021/acs.jproteome.5b00243 J. Proteome Res. 2015, 14, 3859−3870

Article

Journal of Proteome Research

extraction was performed using 0.5% (v/v) formic acid and 0.5% (v/v) formic acid in 30% (v/v) acetonitrile. The extracted peptides were then pooled for nanoLC−ESI−CID/ETD−MSn analysis.

proposed for spidroin in solid state and for individual domains of these proteins.6 In the present study a gel-based mass spectrometry strategy involving collision-induced dissociation (CID) and electrontransfer dissociation (ETD) fragmentation methods was used to sequence and determine the presence/location of any PTMs on spidroins-1A and -1B. The present study presents structural models for the spider silk proteins spidroin-1A and -1B, reporting phosphorylations as PTMs into the sequence spidroin-1, and the contribution of these PTMs to the 3-D models of this protein. On the basis of the results, dynamic structural 3-D models for the entire spidroins-1A and -1B sequences are proposed. Taken together, the findings reported here might be valuable for understanding the physicochemical properties of the silk proteins and in the design of recombinant spider silk proteins.

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NanoLC−ESI−CID/ETD−MSn

The HPLC instrument used in the analysis was an Ultimate 3000 system (Dionex, Sunnyvale, CA, USA) equipped with a PepMap100 C-18 trap column (300 mm × 5 mm) and a PepMap100 C-18 analytic column (75 mm × 150 mm). The gradient (A, 0.1% formic acid in water; B, 0.08% formic acid in acetonitrile) was 4−30% B from 0 to 105 min, 80% B from 105 to 110 min, and 4% B from 110 to 125 min. An HCT ultra ETD II instrument (Bruker Daltonics, Bremen, Germany) was used to record the peptide spectra over a mass range of m/z 350−3500, and MS/MS spectra were acquired in informationdependent data acquisition mode over a mass range of m/z 100−3500. MS spectra were recorded repeatedly, and then three data-dependent CID MS/MS spectra and three ETD MS/MS spectra were generated from the three precursor ions of highest intensity. An active exclusion of 0.4 min was used after two spectra to detect low abundance peptides. The voltage between the ion spray tip and the spray shield was set to 1500 V. The nitrogen gas used for drying was heated to 150 °C, and the flow rate used was 10 L/min. Multiple charged peptides were selected for analysis in the MS/MS experiments, and the collision energy was set automatically according to the mass and charge state of the peptides that chosen for fragmentation (due to their good fragmentation characteristics). The MS/MS spectra were interpreted and peak lists were generated using DataAnalysis 4.0 (Bruker Daltonics) as described previously by Bae et al.19 The combined use of CID MS/MS and ETD MS/ MS generated spectral data, which were analyzed using a MASCOT protein engine search and Modiro.

EXPERIMENTAL SECTION

Web-Silk Samples

Web silk from N. clavipes was collected at the campus of the University of São Paulo State at Rio Claro, SP in southeast Brazil. The web-silk samples were processed as described previously by Santos-Pinto et al.17 Approximately 20 mg (dry weight) of spider silk was dissolved in 2 mL of saturated lithium thiocyanate (38 M LiSCN hydrate, Sigma, Deisenhofen, Germany) at 25 °C for 2 h with shaking. Subsequently, LiSCN was removed by exchange in urea buffer prior to 2-DE analysis. Protein concentration was determined using the Bradford assay.18 Two-Dimensional Gel Electrophoresis

Samples (200 μg) of spider silk protein were subjected to rehydration on 18 cm IPG strips and pH 7−10 nonlinear gradient strips. Isoelectric focusing (IEF) began at 200 V and was gradually increased to 8000 V (approximately 150 000 Vh). The IPG strips were incubated in equilibration buffer [50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS] containing 1% (w/v) DTT for 15 min and then in equilibration buffer containing 4% (w/v) iodoacetamide for 15 min. The second dimension was run in precast SDS-PAGE gels (7−10% gradient) at 50 V (which was held constant overnight) and then at a constant 200 V for a further 4 h at 10 °C. The gels were stained with Coomassie Brilliant Blue R-250 (CBB) and were scanned and digitized for documentation.

Protein Identification and PTMs

Searches were carried out using MASCOT 2.2.06 (Matrix Science, London, UK) against the latest NCBI database (http://blast.ncbi.nlm.nih.gov, on August 18, 2014) for protein identification; for this purpose, we selected all 57 321 entries contained in the taxa Araneae (spider). The databanks mentioned earlier were appended with common external contaminants from cRAP, a maintained list of contaminants, laboratory proteins, and protein standards provided through the Global Proteome Machine Organization (http://www.thegpm. org/crap/index.html). The search parameters were set as follows: enzyme selected as trypsin, or alternatively chymotrypsin, pepsin, Glu-C/V8 protease, proteinase 10, Asp-N protease, proteinase K, and subtilisin, according to the type of proteinase used; two maximum missing cleavage sites allowed, 0.2 Da peptide mass tolerance for MS and 0.2 Da tolerance for MS/MS spectra. Carbamidomethyl (C) was specified as a fixed modification, while methionine oxidation and phosphorylation (of Y, T, and S) were specified in MASCOT as variable modifications. After protein identification, an error-tolerant search was performed to detect nonspecific cleavage and unassigned modifications.20 Proteins identified after database search were subjected to additional filtering using Scaffold 4.3.2 (Proteome Software Inc., Portland, OR) to validate peptide identification and to obtain a false discovery rate (FDR) of less than 1%; FDR was calculated from the forward and decoy matches by requiring significant matches to at least two distinct sequences. According to a Local FDR algorithm implemented into Scaffold, the peptide probability was set to a minimum of

In-Gel Digestion

Gel pieces were destained twice for 30 min at 25 °C with 10 mM ammonium bicarbonate/50% (v/v) acetonitrile, dehydrated in acetonitrile, dried, and subsequently treated with the following eight proteolytic enzymes: 40 ng/mL of trypsin (Promega, Madison, USA) in 5 mM octyl β-D-glucopyranoside (OGP) and 10 mM ammonium bicarbonate pH 7.9 at 37 °C for 18 h; 50 ng/mL of chymotrypsin (Roche Diagnostics) in 5 mM OGP and 25 mM ammonium bicarbonate pH 7.9 at 30 °C for 2 h; pepsin (Sigma, Deisenhofen, Germany) in 0.1 M HCl pH 1.0 at 37 °C for 4 h; proteinase 10 (syn.: Thermolysin) in 25 mM Tris-HCl pH 7.5 at 50 °C for 2 h; 40 ng/μL of Glu-C/ V8 protease (Sigma) in 50 mM ammonium bicarbonate pH 7.8 at 37 °C for 16 h; 40 ng/μL of proteinase K (Sigma) in 50 mM ammonium bicarbonate pH 7.8 at 37 °C for 2 h; 40 ng/μL of subtilisin (Sigma) in 6 M urea and 1 M Tris-HCl pH 8.8 at 37 °C for 2 h; and 40 ng/μL of Asp-N protease (Promega) in 25 mM ammonium bicarbonate pH 7.8 at 37 °C for 16 h. Peptide 3860


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studied in a Vega II scanning electron microscope (Tascan, Brno, Czech Republic) at 7 kV with a working distance of approximately 8 mm, as described previously by Santos-Pinto et al.17

90%, whereas the protein probability was set at 95%. PTM searches were performed using Modiro PTM Explorer 1.1 software (Protagen AG, Dortmund, Germany) with the following parameters: enzyme selected as trypsin, or alternatively chymotrypsin, pepsin, Glu-C/V8 protease, proteinase 10, Asp-N protease, proteinase K, and subtilisin, according to the type of proteinase used; two maximum missing cleavage sites allowed, 0.2 Da peptide mass tolerance for MS, 0.2 Da tolerance for MS/MS spectra, and carbamidomethyl (C) and methionine oxidation were specified as modifications 1 and 2, respectively. Searches for unknown mass shifts, amino acid substitution, and calculation of significance were selected in advanced PTM-explorer search strategies. The Modiro software is complementary to the MASCOT software and uses already identified sequences and has the advantage that also unknown mass shifts can be handled.21 A list of 172 common modifications including phosphorylation was selected and applied to virtually cleaved and fragmented peptides that were compared with experimentally obtained MS/MS spectra. The potential sites of PTMs indicated by Modiro were identified and ranked based on internal scoring list for each sequence presenting ions score >200 and significance score >80; the fragmentation pattern of each PTM was then assigned in the corresponding spectrum. The gel-based proteomic approach used was based on that described by Kang et al.22

Lipid Detection

To determine the fatty acids coating the silk fibers, web silks were exposed to 4% (v/w) OSO4 vapor in water for 1 h and then postfixed in 37% (v/v) formaldehyde. Subsequently, the fibers were incubated in 0.1 M imidazole buffer at pH 7.5 at room temperature for 30 min in the dark and then washed in PBS buffer. The fibers were embedded in Epon Araldite; thin sections were stained with 1H-imidazole and photographed using a Jeol scanning electron microscope (model JSM-P15). Secondary Structure Analysis and Molecular Modeling

The secondary structures of spidroins-1A and -1B were predicted using the JNET Secondary Structure Prediction program23 and edited using JALVIEW.24 The secondary structure was estimated using the 2Struc server.25 Spidroin was subjected to molecular modeling using the restrained-based modeling approach as implemented in the program MODELLER 9v11. Templates were searched using THREADER v3.526 and BLASTp.27 Template searching by sequential identity using the program BLASTp found only templates that exhibited 50% identity to the N-terminal (24−155) and Cterminal (934−1008) regions. Thus, we decided to search for templates using the program THREADER, which searches according to structural similarity: two proteins are considered homologous if they usually share similar structures. The output of these tools was formatted and used as input into the MODELLER program, which implements an automated approach for comparative modeling based on the fulfilment of spatial restraints. Spidroins-1A and -1B were modeled using PDB IDs: 3LR2,6 2KHM,28 and 3SYJ29 as template proteins. One-thousand models were generated, and the final model was selected based on stereochemical quality and a MODELLER objective function. Images of the 3-D structures of the models were generated using PyMOL.30 PROCHECK31 was used to check bond lengths, bond angles, peptide bonds and side-chain ring planarities, chirality, and main-chain and side-chain torsion angles. The results of this analysis are shown in Ramachandran diagrams and as values of the complete G-factor.

Western Blotting

To verify the phosphotyrosine and phosphoserine modifications of the spidroin sequences, 20−50 μg of web silk protein extracts was loaded onto 1D-SDS-PAGE gels; after electrophoresis, the proteins were transferred onto PVDF membranes (Millipore) at 21 °C using a semidry Bio-Rad transfer system. The membranes were blocked by incubation for 1 h in T-TBS containing 0.1% (v/v) Tween 20 and 5% BSA (bovine serine albumin). After washing, the membranes were incubated with diluted primary antiphosphotyrosine or antiphosphoserine antibodies (1:2000, Abcam) at 4 °C overnight. The membranes were then washed three times by gentle agitation in T-TBS (containing 0.1% (v/v) Tween 20) and incubated with HRPcoupled antimouse IgG secondary antibodies (1:10 000, Abcam). As an experimental control, the samples were treated with phosphatase (calf intestine alkaline phosphatase, New England Biolabs) prior to resolving on 1D-SDS-PAGE gels. The same procedure was performed to verifiy nitrotyrosine modifications; in this case, membranes were blocked by incubation for 1 h in T-TBS containing 0.1% (v/v) Twen 20 and 5% nonfat dried milk powder (Bio-Rad) and then were incubated with diluted primary antibodies antinitrotyrosine (1:1000, Millipore) and incubated with HRP-coupled secondary antibodies antirabbit IgG (1:3000, Abcam). Nitro-BSA (nitrated bovine serum albumin MW 68 kDa, Sigma) was used as positive control. The membranes were developed with the Amersham ECL-plus Western blotting detection system (GE Healthcare).

Multiple Primary Sequence Alignment

Spidroin primary sequences were aligned using CLUSTAL W program32 and edited using JALVIEW program.24 Molecular Dynamics Simulations

Molecular dynamics (MD) simulations were performed using the GROMACS 4.5.5 software package;33 the force field 43a334 and the flexible Simple Point Charge (SPC) water mode35 were implemented. Spidroin-1A and -1B proteins were subjected to MD simulation in a cubic box containing water and in a cubic box containing water with tridecanoic and octadecanoic fatty acids; a minimum distance to the box face of 1.0 nm was used in all directions. These are the most abundant fatty acids on N. clavipes silk fibers according to Salles et al.36 Information regarding the topology of these fatty acids was not available in the force fields used in this study. Therefore, to build this topology, we used the PRODRG server (http://davapc1.bioch. dundee.ac.uk/programs/prodrg/), which generates parameters for bonds, angles, and charges. The protein overall charge was neutralized, and the physiological salt concentration was set using Na+ and Cl− counterions. During simulations, the lengths

Scanning Electron Microscopy

To examine the structural characteristics of the spider silk fiber surface and the solubilization of those fibers, a short length (approximately 5 mm) was placed into a saturated solution of lithium thiocyanate. Next, aliquots were taken from the solution, placed on scanning electron microscopy stubs, and dried on a heating plate for several hours at 70 °C. Subsequently, the stubs were coated with gold in a Cressington sputter coater using a planetary drive. The stubs were then 3861


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Figure 1. SEM analysis of N. clavipes web silk. (A) Fibers that form the radii and spiral of the web. (B−C) Ultrastructure of the silk fibers at different points of the web highlighting silk fiber repair (white arrow) carried out by the spider. (D) Solubilization of silk in lithium thiocyanate. (E) A fatty acid layer on the fibers is indicated by black arrows.

Figure 2. Proteomic analysis of N. clavipes web silk. (A) A representative 2-DE profile of web silk stained with CBB. (B) Western blotting showing nitrotyrosine immunoreactivity (lanes 2 and 3). Lane 1 shows control sample Nitro-BSA. (C) Western blotting showing phosphotyrosine immunoreactivity (black arrows, lanes 1 and 3). Lanes 2 and 4 show no immunoreactivity after phosphatase treatment. (D) Western blotting showing phosphoserine immunoreactivity (black arrows, lanes 1 and 3). Lanes 2 and 4 show no immunoreactivity after phosphatase treatment.

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Figure 3. Representative amino acid sequence and PTM assignment of spidroin-1A and spidroin-1B identified in web silk. Multiple alignments between the primary sequences of the spidroins. Regions of identity are marked in dark blue, highlighting the highly conserved residues in two sequences. The residues marked with an asterisk and in red show the phosphorylation sites that were identified in each of the sequences. The highly conserved cysteine residue is marked with a black arrow.

of bonds within the protein were constrained under the conditions set by LINCS37 and SETTLE38 for water geometry. In the initial MD simulations, all hydrogen atoms, ions, and water molecules were subjected to 500 steps of energy minimization to remove close van der Waals contacts. Both systems (i.e., water and water with fatty acids) were subjected to a short MD simulation with position restraints for 1000 ps. The final MD simulations were performed under the same conditions, except that the position restraints were removed over an interval of 20 000 ps. Energy minimization and MD were carried out under periodic boundary conditions. Simulation was accomplished in the isothermal−isobaric ensemble at 300 K using temperature coupling and a constant pressure of 1 atm with isotropic molecule-based scaling.39,40 Temperature and pressure were modulated using coupling techniques39 with coupling and isothermal compressibility constants of 0.01 ps (solvent and protein) and 6.5 × 10−5 bar−1, respectively. Electrostatic interactions among nonligand atoms were evaluated using the particle-mesh Ewald method.41 Cutoff distances for the calculation of Coulomb and van der Waals interactions were 1.0 and 1.4 nm, respectively. The convergences of the simulations were analyzed in terms of the radius of gyration (Rg), root−mean−square deviation (RMSD) from the initial model structures, potential energy (E), and the intermolecular hydrogen bond types present. All analyses were performed on an ensemble of system configurations that were extracted at 0.5 ps time intervals from the simulation, and the MD trajectory collection was initiated after 1 ns of dynamic simulation to ensure that the evolution was completely

equilibrated. Molecular visualization was performed in the graphical environments VMD (Visual MD)42 and PyMOL.30 The STRIDE43 and the DSSP44 programs were used by GROMACS to calculate the percentage and number of secondary structures that were present.

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RESULTS AND DISCUSSION To obtain the chemical basis for the interpretation of mechanoelastic properties of spider silk proteins, the web silk produced by Nephila clavipes spider was submitted to proteomic analysis. The purpose of the present study was the structural proteomic characterization of the spidroin-1 from the Table 1. Sequence Coverage of N. clavipes Web Silk Spidroins by in-Gel Protein Digestion Using Various Proteolytic Enzymesa enzyme

spidroin-1A

spidroin-1B

chymotrypsin (%) trypsin (%) pepsin (%) Glu-C/V8 protease (%) proteinase 10 (%) Asp-N protease (%) proteinase K (%) subtilisin (%) total (%)

87.32 35.26 not identified 18.76 10.55 8.46 7.12 not identified 96.57

89.37 46.39 31.91 23.96 16.48 13.87 12.03 9.61 98.94

a

The enzyme efficiency was evaluated by combined sequence coverage obtained from all spots individually. 3863


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Journal of Proteome Research Table 2. PTMs of N. clavipes Web Silk Spidroins As Revealed by MASCOT and Modiro® (See Tables S1 and S2)a spot

access code

3

B5SYS6

PTMs by MASCOT

PTMs by Modiro

spidroin1B

protein

phosphorylation (T49, S95, S144); sulphone (M60); oxidation (M90, M96, M145) phosphorylation (Y31, S64, S156, S260, S526, Y575, S580)

spidroin1B

oxidation (M71, M96, M145); phosphorylation (S95, S144, S195); deamidation (Q191) phosphorylation (S64, S156, Y184, S260, Y575, S580); methylation (Q37, Q41) phosphorylation (T49, S119, S195); oxidation (M74, M96, M145); methylation (Q215) phosphorylation (S64, S156, S260, Y387, Y575, S580) methylation + deamidation (Q129, Q136) sulphone (M60); oxidation (M43, M71, M90, M96, M145); methylation (T113); phosphorylation (S164, S170, S211, S244) phosphorylation (Y31, Y59, Y151, S156, S260, Y353, Y387, Y478)

oxidation (M1, M71, M74, M90, M96, M145); hexose (N44); hydroxylation (D58); methylation + deamidation (Q86) nitrotyrosine (Y18); phosphorylation (S243); deamidation (R261, R308); methylation (S742) sulphone (M60); oxidation (M74, M90, M96, M145); methylation (E103); phosphorylation (T127); HexNAc2DHex2 (N237) nitrotyrosine (Y18); phosphorylation (Y151, S243); deamidation (R652) hexose (N44); sulphone (M60), oxidation (M1, M74, M90, M96, M145); deamidation (R140) deamidation (R615)

P19837 4

B5SYS6 P19837

5

B5SYS6

spidroin1B

P19837 6

B5SYS5 P19837

a

spidroin1A

phosphorylation (T53, T62, S164, S170, S211, S244); sulphone (M60); oxidation (M1, M43, M90, M96); methylation (E103, S108, Q241) deamidation (Q326, Q433); methylation + deamidation (Q572, Q588)

The position of amino acid residues was assigned according to the numbering of the original sequences observed in UNIPROT.

dragline silk of N. clavipes under natural conditions, including the assignments of the most abundant PTMs of this protein. The use of forced silking would have been an interesting system to obtain the major ampullate silk protein in high purity, if compared to the use of the whole web, as done in the present investigation; however, the effect of forced silking is unknown in the maturation of spidroin and PTMs occurrence. Even considering that the use of the whole webs resulted in heterogeneous silk samples, with the presence of different spider silk proteins (major ampullate spidroin, minor ampullate spidroin, and flagelliform silk protein), this decision permitted to characterize the spidroin-1 under a biologically natural condition. Scanning electron microscopy (SEM) analysis of the web silk protein revealed the ultrastructure of fibers at different web locations (Figure 1A−C) and showed that these materials comprise compact arrays of several layers of silk. The solubilization of spider web silk in lithium thiocyanate (Figure 1D) was a key step in dissolving the silk proteins prior to separation on 2-D gel electrophoresis (2-DE); Figure 1, panel E shows an SEM image of a fatty acid layer on the fibers. The electrophoretic profile of the solubilized web silk (Figure 2A) reveals six spots of spidroins (labeled 1−6) with MWs greater than 250 kDa. The proteins in these spots were identified using mass spectrometry as follows: flagelliform silk protein (spots 1 and 2) (GenBank ID access codes O44358 and O44359); spidroin-1A (spot 6) (GenBank ID access codes B5SYS5 and P19837); and spidroin-1B (spots 3, 4, and 5) (GenBank ID access codes B5SYS6 and P19837). Spidroin-1 is present in large quantities in silk fibers and is uniformly found throughout the fiber core.5 Therefore, we focused on the data obtained from spots 3−6 to characterize the major ampullate spidroin (MaSpi). By accessing UNIPROT (http://www. uniprot.org/uniprot/) and searching for “spidroin”, it was observed the existence of two different forms of this protein: MaSp1A and MaSp1B, which are differentiated from each other by a some polymorphisms in the N-terminal domain among these forms; the alignment of both sequences reveals that MaSp1A has 82.7% identity in relation to the sequence of MaSp1B. Both polymorphic spidroins were identified in the present research. The nearly complete sequences of spidroin1A and -1B obtained in the current study reveal that both forms

differ from each other by 89 polymorphic positions that are distributed among the three domains (3Figure 3). A gel-based mass spectrometry strategy using CID and ETD fragmentation methods was applied to completely sequence spidroins-1A and -1B (including the N- and C-terminal nonrepetitive domains and the central core) and to assign the presence/location of PTMs within the spidroin sequences. Table 1 shows the efficiency of protease action against each type of spidroin, with a total sequence coverage of 96.57% for spidroin-1A and 98.94% for spidroin-1B. The complete sequences of both forms of spidroin-1 are shown in Figure 3, and the theoretical MWs are 85 313 Da and 86 770 Da for spidroin-1A and 1B, respectively. Recently, the full-length sequence of Araneus ventricosus minor ampullate spidroin was published; however, no structural model was proposed for this protein.45 The observation of MW values apparently greater than 250 kDa for spidroins in the 2-DE gel profile (Figure 2A) might be due to the oligomerization of spidroins-1A/1B, cross-links with spidroin-2, or the occurrence of certain PTMs that are known to reduce protein mobility in gels.46,47 Spidroin forms presenting MW values (∼87 kDa) lower than that mentioned earlier were observed in Figure 2, panels C and D, indicating that incomplete or processed forms of spidroin-1 also may exist in the web, making the structure of silk fibers heterogeneous and complex; it is also necessary consider the possibility of some artifact of sample manipulation during silk solubilization caused cleavage of spidroin molecule. High-sequences coverage of spidroins-1A and -1B are required to reveal a series of PTMs; the combined use of CID and ETD generated spectral data that were analyzed both with MASCOT protein engine search and Modiro, permitting assignment of a series of PTMs in the sequences: phosphorylations, nitrotyrosinations, glycosylation, deamidations, hydroxylations, sulphonations, methylations, and oxidations (Table 2). Besides scoring the quality of a match between the interpreted sequence of a peptide, Modiro also calculates a significance value for this score (based on the quality of spectrum and the length of the peptide) with values changing from 0−100, whereas 100 means the maximum significance, and values higher than 80 are significantly meaningful; this strategy is very useful for the assignment of PTMs (Modiro PTM Explorer User Manual V1.1, 2008). Table S1 shows the 3864


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functional relevance of nitrotyrosinations observed on spidroin-1 (Figure S1) in the present study remains unclear, but previous work revealed the effects of tyrosine nitration on mechanoelastic properties and on protein−protein interactions.50,19 Evidence for glycosylation of spidroins was observed through mass spectral interpretation (Tables S1 and S2), which was also reported for other web silk proteins.47,51−53 Table 3 shows the mapping of the phosphorylation sites observed on spidroins, highlighting the positions of these modifications along the sequences; Figure 3 shows the sequences together with the assignment of phosphorylation sites. In total, 15 phosphorylation sites at T53, T62, S164, S170, S211, S244, Y296, Y324, Y416, S421, S525, Y618, Y652, Y743, and Y840 were detected on spidroin-1A, and 16 phosphorylation sites at T49, S95, S119, T127, S144, S195, Y296, S329, Y416, S421, Y449, S508, S525, S788, Y840, and S845 were detected on spidroin-1B. In general, the introduction of one or more phosphate groups in the backbone of proteins is known to induce significant conformational changes that affect protein activity or interactions with other proteins.54 Proteomic studies have revealed the presence of such PTMs in most known silk proteins (i.e., heavy-chain fibroin, light-chain fibroins, and the P25 protein),55,56 including the major ampullate silk spidroin-1 that is produced by Nephila spiders.17 Eight phosphorylation sites in spidroin-1 from N. clavipes, four in that from N. madagascariensis, and two in that from N. edulis have been reported.17 It has been postulated that phosphate residues might play important roles in the storage of silk proteins at high concentrations within the silk gland and during the selfassembly of the proteins during fiber spinning.2,8,57−59 Winkler et al.60 demonstrated that it is possible to control β-sheet assembly to enhance protein solubility via the enzymatic phosphorylation/dephosphorylation of spidroin-inspired recombinant short (ca. 25 kDa) proteins. Because phosphorylation was the major PTM assigned to the spidroin sequences, a few mass spectra that are representative of those used to assign the position of each phosphorylation site are presented (Figure 4); Figure 4, panel A shows the CID spectrum of chymotryptic peptide S*GQGAAAAAGGAGQGGY (244− 260) used to assign the phosphorylation site at *S244 observed on the spidroin-1A protein, while Figure 4, panel B shows the CID spectrum of the tryptic peptide AAAAGGAGQGGYGGLGS*QGAGR (313−334) used to assign the phosphorylation site at *S329 observed on the spidroin-1B protein. Lists containing detailed proteomics data for all peptide sequences and PTM assignments for spidroin-1A and -1B are shown in Tables S1 and S2. The phosphorylation sites that were detected on spidroins-1A and -1B are described in Figures S2−S23. The position in which phosphorylation sites occur in the sequence (Figure 3) was determined according to the primary sequence of spidroins-1A and -1B obtained in this study, after alignment and overlapping of all the peptide fragments identified. Thus, the numbering sequence obtained in this location does not match the position of these sequences in the access codes by which the proteins have been identified in databases (Tables S1 and S2). PTMs occur in endoplasmic reticulum (ER) and in Golgi apparatus and seem to be common features in many secreted proteins; phosphorylation generally occurs in the lumen of Golgi.61 It will be necessary future investigations to determine if the PTMs of spidroins occur in ER, Golgi, or in the secretory granules. The biological significance of the many phosphor-

Table 3. Mapping of the Phosphorylation Sites Observed on N. clavipes Web Silk Spidroins Showing the Position of These Modifications in the Sequences As Represented in Figure 3 protein

PTMs

enzyme

fragmentation method

comments

phosphorylation sites spidroin-1B

T49

spidroin-1A spidroin-1A spidroin-1B

T53 T62 S95

spidroin-1B

S119

spidroin-1B spidroin-1B

T127 S144

spidroin-1A

trypsin/ chymotrypsin trypsin trypsin trypsin

CID/ETD CID CID CID CID

Table S1 (spots 3 and 5) Table S2 (spot 6) Table S2 (spot 6) Table S1 (spots 3 and 4) Table S1 (spot 5)

CID/ETD CID

S164

Glu-C/V8 protease chymotrypsin Glu-C/V8 protease chymotrypsin

spidroin-1A spidroin-1B

S170 S195

chymotrypsin chymotrypsin

CID CID

spidroin-1A spidroin-1A spidroin-1A and -1B spidroin-1A

S211 S244 Y296

chymotrypsin chymotrypsin chymotrypsin

CID CID CID

Y324

CID/ETD

spidroin-1B

S329

CID

Table S1 (spots 3−5)

spidroin-1A and -1B spidroin-1A and -1B spidroin-1B

Y416

trypsin/ chymotrypsin trypsin/ chymotrypsin trypsin

Table S2 (spot 4) Table S1 (spots 3 and 4) Tables S1 (spot 6) and S2 (spot 6) Table S2 (spot 6) Table S1 (spot 4 and 5) Table S1 (spot 6) Table S1 (spot 6) Table S1 (spots 3 and 6) Table S1 (spot 6)

CID/ETD

S421

chymotrypsin

CID

Tables S1 (spot 6) and S2 (spot 4) Table S1 (spots 3−6)

Y449

CID

Table S1 (spot 4)

spidroin-1B

S508

trypsin/ chymotrypsin chymotrypsin

CID/ETD

spidroin-1A and -1B spidroin-1A spidroin-1A spidroin-1A spidroin-1B spidroin-1A and -1B spidroin-1B

S525

chymotrypsin

CID/ETD

Table S2 (spots 3 and 4) Table S1 (spots 4−6)

Y618 Y652 Y743 S788 Y840

trypsin trypsin trypsin trypsin trypsin

CID/ETD CID/ETD CID/ETD CID CID

S845

trypsin/ chymotrypsin

CID

CID

Table S1 (spot 6) Table S1 (spot 6) Table S1 (spot 6) Table S1 (spot 4) Table S1 (spots 4 and 6) Table S1 (spots 3−5)

values of MASCOT ions score, while Table S2 shows the values of Modiro ions score and significance scores. The use of eight proteolytic enzymes to digest the spidroins resulted in the occurrence of some sequences in different peptides, in which the sequence and PTM assignments identified from poor mass spectral data (presenting reduced values of MASCOT ions score) obtained for some peptides were reliably sequenced (or PTM assigned) using rich mass spectral data from another peptides with an overlapped their sequences with the previous peptide (Tables S1 and S2). PTMs such as oxidation, deamidation, and methylation might have occurred due to various artifacts arising from sample preparation or analytical procedures.48 However, a large series of phosphorylation sites was detected and confirmed by immunoblotting as well as nitrotyrosinations (Figure 2B−D). The tyrosine oxidations were reported to form oxidationmediated cross-links from two tyrosine radicals.49 The 3865


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Figure 4. Representative mass spectra of N. clavipes web silk spidroins PTMs. (A) CID spectrum of chymotryptic peptide S*GQGAAAAAGGAGQGGY (244−260) selecting the m/z 715.710 [M + 2H]2+ as precursor ion and showing the *S244 phosphorylation site observed on the spidroin-1A protein. (B) CID spectrum of the tryptic peptide AAAAGGAGQGGYGGLGS*QGAGR (313−334) selecting the m/z 738.059 [M + 3H]3+ as a precursor ion and showing the *S329 phosphorylation site observed on the spidroin-1B protein.

This may suggest that spidroins are stored is a suspension of fatty acids to preserve the structural features of these proteins. It was reported that the N- and C-terminal domains of spidroins form dimers when these domains are individually cloned and expressed;62,63 however, the oligomeric behavior of the entire spidroin molecules is not known. Anyway, the individual molecular models for both domains can be used as structural templates for modeling the entire spidroin-1A and 1B. The nearly complete sequence of spidroins-1A and -1B, the presence of multiple phosphorylations, the coating by fatty acids, and the known 3-D molecular structures of the isolated N- and C-terminal domains of the spidroins-1A and -1B enabled us to perform molecular modeling and MD simulations under natural conditions. The Ramachandran plots for the templates and models indicate that over 98% of the residues are in the most favorable stereochemical regions (Figure S24). The analysis of the structural quality of the homology model was performed using PROCHECK (for Ramachandran plots and G-factor [torsion angles = −0.18 to −0.43; covalent geometry = 0.22−0.51; global G-factor = −0.08 to −0.16]), which strongly indicated that the model was adequate for structural studies.

ylation sites observed on spidroins produced by orb-weaving spiders remains to be elucidated. Data from the literature suggest that these PTMs might determine the conformation of GGX and (A)n domains,4,8 which are responsible for the mechanical properties of spider silk. In fact, a careful observation of the localization of the phosphorylation sites on spidroins-1A and -1B reveals that most of these PTMs are localized within the GGX and (A)n structural motifs. PTMs often occur in reversible manner, but in the present investigation, the phosphorylation sites were identified in the web silk fiber, suggesting that the PTMs must be permanent, and probably are related to the stabilization of spidroins-1A and -1B in ideal conformations under environmental conditions in the nature, to permit the proper mechanical work of the web. By using GC−MS analysis, it was previously reported that an oily material was extracted from web silk fibers of N. clavipes, being mostly composed of a suspension of tridecanoic and octadecanoic acids in water, which was coating the silk proteins with a layer of fatty acids;36 the presence of a lipidic layer was confirmed by SEM analysis in the current study (Figure 1E). 3866


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Figure 5. Representative 3-D molecular models of the spidroins after 20 ns of MD simulation. (A) Phosphorylated spidroin-1A after simulation in water. (B) Phosphorylated spidroin-1A after simulation in water containing fatty acids. (C) Phosphorylated spidroin-1B after simulation in water. (D) Phosphorylated spidroin-1B after simulation in water containing fatty acids. (E) Secondary structure analysis of both proteins under different experimental conditions. The percentages and numbers of secondary structures were calculated using STRIDE and DSSP as implemented in GROMACS.

in the presence of water, whereas spidroin-1B is more stable in the presence of the fatty acid/water mixture than in the presence of water alone. The characterization of the secondary structure of the spider silk proteins was always challenging, requiring the use sophisticated spectroscopy instrumentation; the success with solid-state NMR to study the structure/property correlations of synthetic polymers stimulated the applications of this experimental strategy to characterize the structure of the major ampullate silk protein from N. clavipes; thus, by using solid state 13C NMR analysis and measurements of relaxation time constants in dragline silk of N. clavipes, it was demonstrated unambiguously the contribution of alanine residues to form the β-sheet conformations in these proteins.64 The use of fluorescence-free Raman spectra of major ampullate dragline silks from different spiders species permitted to correlate differences in the mechanical properties of native and supercontracted silk fibers to variations in β-sheet contents of these proteins, as well to permitted to attribute the contraction of the spidroin-1 to conformational changes in the supermolecular structure of the silk fibers.65 The major and minor ampullate silks from live N. senegalensis were investigated in situ by XRD, using wide- (WAXS) and small-angle (SAXS) scattering obtained at the same time, for characterizing the secondary structures of both proteins; the use WAXS permitted

The simulations were performed on the phosphorylated form of the spidroins (other PTMs were not considered). MD simulations were performed in a virtual box in the presence of water molecules (Figure S25A) and in the presence of a mixture of tridecanoic and octadecanoic acids (1:1) and water (Figure S25B). The MD simulations were run for 20 ns; after this time, all model validation parameters (protein backbone RMSD, radius of gyration, potential energy analysis, and number of hydrogen bonds) (Figure S26A−D) revealed that the molecular models were reliable. The data shown in Figure S26A indicate that the spidroin conformations were stabilized after 6 ns and remained stable throughout the 20 ns simulation. The phosphorylated spidroins exhibited higher molecular radii in the presence of the fatty acid/water mixture than in the presence of water alone (Figure S26B), and spidroin-1A exhibited a higher potential energy in the presence of the fatty acid/water mixture than in the presence of water alone (Figure S26C). The number of hydrogen bonds in a molecular structure can be considered as an indicator of its stability. Spidroin-1A contains 640 intramolecular hydrogen bonds in the presence of water and 560 in the presence of the fatty acid/ water mixture, whereas spidroin-1B contains 590 hydrogen bonds in the presence of water and 640 in the presence of the fatty acid/water mixture (Figure S26D). These results indicate that phosphorylated spidroin-1A is more structured and stable 3867


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Figure 6. Electrostatic potential for the 3-D molecular models highlighting the interaction of fatty acid with spidroins. (A) Spidroin-1A. (B) Spidroin-1B. The image at the top is positioned at 0° (front), and the image at the bottom is rotated by 180° (back). Fatty acids are represented using gray sticks.

The N- and C-terminal regions of all models include αhelical and loop elements, and the central repetitive domain is apparently more organized in the presence of the fatty acids/ water mixture, based on the simulations (Figure 5A−D). The percentage of each secondary structure type present is shown in Figure 5, panel E; spidroins-1A and -1B exhibited a higher content of β-strands and α-helices and a greater molecular volume in the presence of the fatty acid/water mixture than in the presence of water alone. Considering that the mechanical properties of spidroins directly depend on the extension of β-sheet elements in the highly repetitive central core region (which is rich in GGX, (GA)n, and (A)n motifs),4,26,27 it appears reasonable to suggest that the presence of fatty acids and water (which interact with specific sites on spidroin-1A and -1B, Figure 6A,B) provide different microenvironments on the spidroin surface, thus maintaining the silk proteins in ideal conformations for supporting the known mechanoelastic properties of the silk fibers.

to observe that the thread exiting from the spigots already contained β-sheet poly(alanine) crystallites, while the use of SAXS data obtained evidence about the presence of microfibrils with an axial repeating periods.66 Inspired in strategies successfully applied to characterize the detection of betaamyloid proteins in situ preparations, the combined use of differential interference and polarizing microscopes in the presence of Congo Red staining permitted to identify betaconformations in spider silk proteins formed by elongational flow in the draw-down taper within the extrusion dye.67 The studies described earlier paved the way for the use of molecular spectroscopy for characterizing the structural models for the major ampullate spidroins,68−71 in which the content of β-sheet seems to be much higher (up to 47%) than that observed in the present investigation (15.3% and 23.1% for spidroin-1A and -1B, respectively); meanwhile, the content of α-helix determined in the present study (15.3% and 15.9% for spidroin-1A and -1B, respectively) seems to be higher than those values previously observed for the same elements of secondary structure (which are almost neglected in the previous models. However, the 310-helices that occurred in very reduced content in the present models (Figure 5) are relatively abundant in the molecular models previously published (∼18%).72,73 The resulting models reported in the present study present high content of coils (61.0−74.1%) when compared to the previous models reported in the literature (∼19%). These differences seem to be related to the fact that the previous models were obtained for the spidroins in solid state, while in the present study, it was considered a virtual environment constituted of fatty acids and water; certainly there is also a contribution of the phosphorylations reported as PTMs in the present models, which were not considered in the previous models. When the secondary structure of spidroin-1 was studied in aqueous medium, the percentages of elements of secondary structure were more similar to the results of present investigation (38.2% α-helix and 11% β-sheets).74

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CONCLUSION The present proteomic study reveals novel insights into the sequence and 3-D structure of web silk proteins produced by spiders, which may be valuable for understanding the physicochemical properties of silk fiber proteins and may be relevant for scientists in material science, biology, biochemistry, and environmental scientists. Our results indicate that the presence of fatty acids and water, which interact with specific sites on the spidroins, provide microenvironments on spidroin surface suitable for maintaining protein conformations that are ideal for supporting the observed mechanical properties. The complete sequence of spidroin-1 can contribute to the future design of recombinantly produced spider silk proteins for biotechnological and biomedical applications. The proposed molecular models and their interaction with fatty acids and 3868


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water will aid in better understanding the extraordinary mechanical properties of silk fiber proteins and in creating novel applications for this biomaterial. Thus, these findings for spidroin-1A and -1B from Nephila clavipes certainly will provide the basis for understanding mechanical-elastic properties of spider silk.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.5b00243. Representative CID/ETD spectra; Ramachandran plot; schematic representations of MD simulations; structural analysis of 3-D molecular models of phosphorylated spidroins (PDF) Amino acid sequences of spidroin-1A and -1B by MASCOT and Modiro (XLS)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: 55-(19)-35348523. Fax: 55-(19)-35348523. *E-mail: [email protected]. Phone: 143-1404006065. Fax: 143-1-404006065. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from FAPESP (Proc. 2010/ 19051-6, Proc. 2011/51684-1, and Proc. 2013/26451-9), CNPq (Proc. 301656/2013-4), and Gert Lubec Proteomics Laboratory at the University of Vienna. M.S.P. is a researcher from the National Research Council of Brazil-CNPq; G.L. is a researcher from the Medical University of Vienna, Vienna, Á ustria; and J.R.A.S.P. is a postdoctoral research fellow from FAPESP at São Paulo State University-UNESP, Rio Claro, Brazil.

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Structural Model for the Spider Silk Protein Spidroin-1 - Journal of

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