doi:10.1016/j.jmb.2012.02.040 J. Mol. Biol. (2012) 418, 197–207

Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb

N-Terminal Domain of Bombyx mori Fibroin Mediates the Assembly of in Response to pH Decrease

Yong-Xing He†, Nan-Nan Zhang†, Wei-Fang Li, Ning Jia, Bao-Yu Chen, Kang Zhou, Jiahai Zhang, Yuxing Chen and Cong-Zhao Zhou⁎

Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, People's Republic of China

Received 20 October 2011; Fibroins serve as the major building blocks of silk fiber. As the major received in revised form component of fibroin, the fibroin heavy chain is a considerably large 18 February 2012; comprising N-terminal and C-terminal hydrophilic domains and 12 highly accepted 23 February 2012 repetitive Gly-Ala-rich regions flanked by internal hydrophilic blocks. Here, Available online we show the crystal structure of the fibroin N-terminal domain (FibNT) at 1 March 2012 pH 4.7, revealing a remarkable double-layered anti-parallel β-sheet with each layer comprising two FibNT molecules entangled together. We also Edited by M. Moody show that FibNT undergoes a pH-responsive conformational transition from random coil to β-sheets at around pH 6.0. Dynamic light scattering Keywords: demonstrates that FibNT tends to oligomerize as pH decreases to 6.0, and fibroin; electron microscopy reveals micelle-like oligomers. Our results are X-ray crystallography; consistent with the micelle assembly model of silk fibroin and, more N-terminal domain; importantly, show that the N-terminal domain in itself has the capacity to structural transition; form micelle-like structures in response to pH decrease. Structural and micelle mutagenesis analyses further reveal the important role of conserved acidic residues clustered in FibNT, such as Glu56 and Asp100, in preventing premature β-sheet formation at neutral pH. Collectively, we suggest that FibNT functions as a pH-responsive self-assembly module that could prevent premature β-sheet formation at neutral pH yet could initiate fibroin assembly as pH decreases along the lumen of the posterior silk gland to the anterior silk gland. © 2012 Elsevier Ltd. All rights reserved.

Introduction enveloping the filaments. The core filament is assembled from fibroins, which consist of three fi Silk produced by the silkworm Bombyx mori is a components: a 390-kDa heavy chain [ broin heavy fi chain (FibH)],3,4 a 26-kDa light chain,5 and a 25-kDa high-strength natural protein ber that has been 6 used to make textiles since 3000 BC.1 Each silk fiber glycoprotein. After having been synthesized in fi is about 10–25 μm wide2 and composed of two core posterior silk glands, broins are secreted into the – filaments bundled together through a coat lumen as an aqueous solution (12 15% by weight) and extruded by peristaltic motion to the ducts of middle silk glands, where the concentration in- creases to 20–30%.1 In the lumen of anterior silk *Corresponding author. E-mail address: [email protected]. glands, the pH of the processing solution falls to † Y.-X.H. and N.-N.Z. contributed equally to this work. around 4.9, salt concentration increases, and fibroins Abbreviations used: FibNT, fibroin N-terminal domain; are finally spun into water-insoluble fibers by FibH, fibroin heavy chain; SAD, single-wavelength mechanical shear and the stretching action of the anomalous diffraction; SeMet, selenomethionine; DLS, spinneret.7 Artificially reeling silk from immobi- dynamic light scattering. lized silkworms at faster and steady speeds greatly

0022-2836/$ - see front matter © 2012 Elsevier Ltd. All rights reserved. 198 Structure of N-terminal Domain of Fibroin improves silk strength and toughness, which can spinning process is initiated by the self-assembly of even be compared to those of spider dragline silk, micelle-like particles (100–200 nm), propagates suggesting that spinning conditions are crucial to through the aggregation of particles into larger the mechanical properties of B. mori silk.8 globules (0.8–15 μm) and gel-like states, and finally Silk fibroin has a crystalline portion of about 95%, ends with elongation and alignment under physical featuring abundant highly repetitive structural shear, leading to the formation of silk fiber.17 motifs GAGAGS, and the rest is amorphous, mostly We crystallized the fibroin N-terminal domain composed of hydrophilic residues. As the major (FibNT) at pH 4.7, and the crystal structure reveals component of fibroin complex, FibH is composed of an entangled β-sheet dimer. We also show that 5263 residues and comprises a highly conserved N- FibNT undergoes a pH-responsive conformational terminal hydrophilic domain with a signal peptide, transition from random coil to β-sheets at around a C-terminal hydrophilic domain, and 12 hydro- pH 6.0 and demonstrate the capability of FibNT to philic linkers interspersed among 12 GA-rich repet- form oligomers with micelle-like substructures in a itive regions that account for its crystalline nature3,4 pH-responsive fashion. Structural and mutagenesis (Fig. S1). Interestingly, the 12 linkers share highly analyses further reveal the important role of conserved sequences, yet their roles remain elusive. conserved acidic residues clustered in FibNT, such As early as the 1950s, it had been proposed, based as Glu56 and Asp100, in preventing premature β- on X-ray diffraction analysis, that polypeptides in sheet formation at neutral pH. Our results provide the crystalline portion of fibroin were arranged as the first structural insights into the assembly anti-parallel pleated β-sheets.9 The adjacent sheets mechanism of B. mori silk at the atomic level, pack together at distances of about 3.5 and 5.7 Å, of which could possibly apply to all hymenopteran which the longer intersheet distance is explained by because of the high conservation of their N- the presence of residues with bulky side chains, such terminal domains. as tyrosines. The planes of the β-sheets lie parallel with the axis of the fiber, which consists of bundles of nanofibrils with lateral dimensions of approxi- Results mately 20 Å×60 Å.10 These nanofibrillar structures, which represent well-ordered crystallites in silk, are suggested to be embedded in a matrix of less Overall structure of FibNT ordered noncrystalline regions.11 The hydrogen- bonding network of β-structured crystallites plays a The N-terminal hydrophilic domain devoid of the major role in determining the strength and rigidity signal peptide (Ile23-Ser126, referred to as FibNT) of the material,12 while the noncrystalline regions was overexpressed in Escherichia coli.Through adopting much more flexible conformations are exhaustive crystal screening trials, we eventually 13 likely to contribute to elasticity. crystallized FibNT in space group P6522 at pH 4.7. The folding and assembly mechanism of silk To determine the structure, we collected a single- fibroins has long been investigated. We previously wavelength anomalous diffraction (SAD) data set at fitted the FibH sequence to the anti-parallel β-sheet 50.0–3.0 Å resolution from a single crystal of model and proposed that ∼10 repeats of GAGAGS selenomethionine (SeMet)-substituted protein. Ini- in the crystalline region of FibH should form a long tial electron density map calculated from the β-strand of ∼200 Å3, which is around the upper experimental phases lacked the features of side limit of the crystallite size.14 It was found that chains. After B-factor sharpening had been applied, reconstituted fibroins, when incubated in water the quality of electron density improved, and under mechanical shear, can form a native-like unambiguous assignments of the side chains were fiber dominated by a so-called ‘parallel β’ structure, allowed (Fig. S2). The protein sequence registry was where the β-strand is arranged parallel with the confirmed by a difference Fourier analysis of SeMet. fiber axis. However, when alcohol is used to trigger The asymmetric unit of FibNT crystals contains a assembly (without shear), there emerge entangled homodimer of a twisted eight-stranded β-sheet with amyloid-like nanofibers with a ‘cross-β’ structural a 2-fold symmetric axis perpendicular to the β-sheet feature.15 This structural variation implies that (Fig. 1a). This is completely distinct from the α- fibroin has an incredibly flexible and moldable helical structures of thespidroinN-terminal structure, which senses the exterior conditions and domain,18,19 although the mechanisms of fibroin undergoes conformational transitions accordingly. and assemblies were thought to be similar.20 One essential step in silk spinning is the reduction of The two chains A and B are structurally similar, with a root-mean-square deviation (RMSD) of 2.1 Å over 64 pH from the posterior part of the silk gland to the α anterior part of the silk gland, which triggers the C atoms, but they differ profoundly in the N- gelation of the condensed fibroin.16 By mimicking terminal segments Phe26-Val35, which form a short the spinning conditions in vitro, Jin and Kaplan helix packing at the β-sheet in chain A but with a loop proposed an intriguing model of silk assembly: the protruding to the solvent in chain B. Each chain is Structure of N-terminal Domain of Fibroin 199

Fig. 1. Overall structure of FibNT. (a) Entangled dimer structure, subunit A (cyan), and subunit B (pink) are shown in cartoon representation. (b) Front view of the tetramer. (c) Side view of the tetramer. composed of two β-hairpins and part of the loop other chain (Fig. 2). The hydrogen bond registries in connecting β2andβ3, and the last 18 residues both chains are well ordered and strictly the same, (Gly109-Ser126) at the C-terminus are disordered. suggesting that the β-sheet alignment is well The dimer has a maximum length of ∼60 Å along the stabilized and represents the native registry of the β-strands and a width of ∼35 Å across the strands. hydrogen bonds. Two FibNT dimers further assemble into a tetramer (dimer of dimers) through a crystallographic 2-fold Characterization of pH-responsive conformational rotation axis (Fig. 1b and c). This crystallographic transition and size distribution of FibNT dimer–dimer interface involves a broad range of hydrophobic and hydrogen bonding interactions. The Due to the essential role of pH in fibroin assembly, distance between the centers of the two sheets is an we probed the secondary structures of FibNT in the average of 10 Å. pH interval 7.0–3.0 using circular dichroism (CD) spectroscopy (Fig. 3a).AtpH7.0,CDspectra Dimer entanglement demonstrate a strong negative band centered around 197 nm and a positive band around 212 nm, During model building, we found that disruptions of electron density between strands β2 and β3 result in ambiguous chain tracing, resulting in β-sheet β –β –β –β –β –β –β –β topologies of 1A 2A 4A 3A 3B 4B 2B 1B β –β –β –β –β –β –β –β and 1A 2A 4B 3B 3A 4A 2B 1B within the dimer (Fig. 2). However, given that the distances β β between the disrupted ends of 2A and 3A and β β ∼ between the disrupted ends of 2B and 3B are 38 and 30 Å, respectively, the missing residues cannot fill in the loop connecting the two β-hairpins in the β –β –β –β –β –β –β –β topology 1A 2A 4A 3A 3B 4B 2B 1B, even when assuming that all these residues adopt fully extended conformations. Therefore, we established that the topology of the dimer is β –β –β –β –β –β –β –β 1A 2A 4B 3B 3A 4A 2B 1B.This entangled topology increases (approximately twice) the number of FibNT interchain hydrogen bonds by exchanging the β-hairpins of each chain within the dimer, compared to the presumable dimer without entanglement. Residues Thr36-Asn65 form the first β-hairpin structure with a type 1 β-turn in residues Asp49-Gly52, and residues Glu78-Ser107 form the second β-hairpin with a type 1 β-turn in residues β Asp89-Gly92. These two -hairpins have four Fig. 2. Topology of FibNT dimer. The residue numbering available β-edges: strands β2 and β4 reciprocally β fi β of the disrupted ends of -hairpinsismarkedinthe gure. form intermolecular hydrogen bonds, and strand 3 Structural analysis unambiguously established the topology β –β –β –β –β –β –β –β forms hydrogen bonds with its counterpart from the of FibNT as 1A 2A 4B 3B 3A 4A 2B 1B. 200 Structure of N-terminal Domain of Fibroin

(a) (b) 25 4 20 15 pH7.0 0 10

Intensity(%) 5 0 -4 25 20 -8 15 pH6.0 pH3.0 10 pH4.6 -12 Intensity(%) 5 pH6.0 0 pH7.0 25 -16 20 15 pH5.5 -20 10 190 200 210 220 230 240 250 260

Intensity(%) 5 0 (c) 30 20 pH5.0

10

0 15

10 pH4.5 5 Intensity(%) Intensity(%)

0 20 15 10 pH4.0

Intensity(%) 5 0 1 10 100 1000 10000 size (d nm)

Fig. 3. (a) CD spectra of FibNT at pH 7.0 (down-pointing triangles), pH 6.0 (up-pointing triangles), pH 4.6 (circles), and pH 3.0 (squares). (b) Analysis of the oligomer size distributions of FibNT. Representative DLS spectra are shown at pH 7.0, 6.0, 5.5, 5.0, 4.5, and 4.0. (c) SEM image of FibNT oligomers formed at pH 5.0.

μ indicating the dominance of random-coil structure. molecules have Rh values at around 1 m. It is However, when the pH decreases to 6.0, the two important to note that a pH-responsive pattern of bands drastically invert to a positive band at ∼195 nm oligomerization coincides with that of conforma- and to a negative band at ∼215 nm, showing the tional transition, with turning points between emergence of β-sheet conformation. As the pH drops pH 7.0 and pH 6.0. The close association of these to 3.0, the fraction of the β-sheet structure increases to two events suggests that oligomerization of FibNT ∼40%. Titration from pH 3.0 to pH 7.0 turns FibNT is likely to rely on the emergence of a well-ordered back to the random-coil conformation (data not β-structure. shown). We also conducted dynamic light scattering (DLS) Morphology of FibNT oligomers studies to probe the size distribution of FibNT in the pH interval 7.0–4.0 (Fig. 3b). At pH 7.0, the size To determine morphological manifestations of distribution observed for FibNT reveals mainly FibNT oligomers, we examined the preparations of particles with a hydrodynamic radius (Rh)of FibNT with a scanning electron microscope (Fig. 3c). ∼9 nm, representing low-molecular-weight species. At pH 5.0 and a protein concentration of 1 mg/ml, The measurement also reveals particles with large FibNT oligomers adopt clustered micelle-like struc- ∼ μ Rh values ranging from 10 nm to 1 m. At pH 6.0, tures, with diameters of the micelles in the range of however, the peak of the size distribution curve 50–100 nm. A previously postulated model of silk shifts to ∼200 nm, indicating that most of the assembly suggests that internal hydrophobic blocks undergo higher oligomerization. With a in fibroin are likely to be the driving force of micelle further decrease in pH values, the size of the formation via extensive hydrophobic interactions, oligomers increases; at pH 4.0, most of the FibNT while the N-terminal and C-terminal hydrophilic Structure of N-terminal Domain of Fibroin 201 blocks undergo either homo-oligomerization or be self-assembled in response to pH decrease. It was hetero-oligomerization to seal the hydrophobic demonstrated that the α-helical spidroin N-terminal core and to form the outer edges of the micelles.17 domain readily self-assembles at pH 6.3 and The micelle-like morphology of FibNT oligomers displays delayed assembly when pH is above 7.0, that we show here not only is consistent with the and this pH-responsive assembly involves con- micelle-forming silk assembly model but also served acidic residues such as Asp40 and Glu8418. identifies FibNT as a self-assembly unit that could In FibNT, there are 19 acidic residues distributing on possibly initiate fibroin assembly in response to pH both faces of the dimeric β-sheet. Some of these decrease. acidic residues may have their pKa values up-shifted near the transition point and therefore ionize at Structural basis for pH-responsive conformational neutral pH to impede protein folding and assembly transition through charge repulsion (Fig. 4a). As clustering of acidic residues can increase side-chain pKa values, Despite the very distinct structures of FibNT and we looked into the FibNT structure to identify the spidroin N-terminal domain, both proteins can clusters of conserved acidic residues. Notably, the

Fig. 4. (a) Acidic residue distribution in the structure of FibNT. The hydrogen bonds formed between two pairs of acidic residues (Asp44-Glu56 and Glu98-Asp100) are highlighted on the right. (b and c) CD spectra of FibNT mutants E56A and E99A/D100A in the pH interval 7.0–3.0. The broad negative peaks between 200 and 220 nm for both proteins at pH 7.0 indicate that they have a fraction of β-sheet conformation and are more folded than the wild-type FibNT at neutral pH. 202 Structure of N-terminal Domain of Fibroin strictly conserved Glu56 and Asp100 cluster with the strands is analogous to the amyloidal cross-β Asp44 and Glu98, respectively, by forming hydro- structure, which is associated with many neurode- gen bonds (Fig. 4a). Ionization of these residues can generative diseases. A typical amyloid fiber is lead to charge repulsion of the side chains and can composed of repeating substructures that are possibly reduce structure stability or even impair made up of β-strands running perpendicular to the well-ordered registry of the β-sheets. To test this the fiber axis. An X-ray fiber diffraction pattern of hypothesis, we disrupted charge repulsion by amyloids features a meridional reflection at ∼4.7 Å constructing the Glu56Ala mutant and the (corresponding to inter-β strand spacing) and an Glu99Ala/Asp100Ala (Glu99 is also strictly con- equatorial reflection at ∼6–11 Å (corresponding to served) double mutant and characterized the sec- the distance between stacked β-sheets).21,22 The ondary structures of the mutant proteins by CD as a geometric parameters of FibNT β-sheets coincide function of pH (Fig. 4b and c). Both mutants still well with those of amyloid, with an inter-β strand show pH-responsive conformational transition but spacing of ∼4.8 Å and an intersheet distance of have β-sheet conformation emerging already at ∼10 Å. More importantly, we demonstrate that pH 7.0. It indicates that mutation of these residues FibNT undergoes higher oligomerization when pH can promote β-sheet formation at neutral pH rather decreases to 6.0 and can be self-assembled into than impair it, suggesting that charge repulsion micelle-like structures with diameters around 50– between closely located acidic residues plays an 100 nm. The dimer entanglement that we observed important role in preventing premature β-sheet in the crystal structure of FibNT could be essential formation at neutral pH. It seems that the same for FibNT oligomerization because, in solution, mechanism is also used in the spidroin N-terminal each FibNT monomer can donate one β-hairpin to domain, since Asp40 and Glu84 form a hydrogen reconstitute the eight-stranded β-sheet, resulting in bond and since the single mutant Asp40 or the oligomerization in a head-to-tail fashion (Fig. 5). double mutant Asp40-Glu84 was reported to pro- Note that the loops connecting the β-hairpins are mote self-assembly even at pH 8.0. The fibroin and very flexible and do not restrain the reconstituted spidroin N-terminal domains give us a nice example β-sheets in the same plane, so the oligomers of convergent evolution at the molecular level—two display the morphology of micelle-like particles structurally unrelated proteins evolving indepen- other than nanofibrils, which are usually associated dently to attain similar functions by using similar with two-dimensional molecular assembly and chemical mechanisms. propagation. Another possible mechanism for the oligomeriza- tion of FibNT could be the abundantly exposed Discussion hydrophobic residues on both sides of the dimeric β- sheet, which serve as alternative assembly inter- The FibNT structure presented here reveals a faces. One face of the dimeric β-sheet forms the remarkable two-layered entangled β-sheet struc- dimer–dimer interface through extensive hydropho- ture. To our surprise, the spatial arrangement of bic and polar interactions. This interface is very

Fig. 5. Oligomerization model of FibNT via β-hairpin entanglements. In this model, each FibNT monomer donates one β-hairpin to reconstitute the eight-stranded β-sheet and subsequently forms oligomers in a head-to-tail fashion. Structure of N-terminal Domain of Fibroin 203 similar to the so-called ‘steric zipper,’ which refers to which could possibly bind to the hydrophobic patch a compact dehydrated interface that makes two of another molecule in solution. layers of β-sheet interdigitated during amyloid Our crystal structure and mutagenesis analyses polymerization. A number of hydrophobic residues, also highlight the important role of clustered acidic such as Ile59, Ile81, Val85, Val97, and Val101, lie at residues in preventing premature β-sheet formation the center of the dimer–dimer interface (Fig. 6a), and at neutral pH. Note that the hydrophilic linkers also multiple sequence alignment (Fig. S3) indicates that contain a number of acidic residues with a these residues are well conserved in the FibNT of predicted pI of around 4.5; these interspersed hymenopteran insects, underscoring the functional regions may also play a role in preventing the importance of these residues. A well-conserved premature assembly of fibroin at neutral pH. In hydrophobic patch composed of residues Leu102, light of our results and the micelle assembly model, Phe84, Ile86, and Ile96 is also found on the other face we propose a model for how FibNT mediates silk of the dimeric β-sheet (Fig. 6b). This hydrophobic assembly: In the lumen of posterior silk glands patch is buried by the N-terminal short helix of one where the pH value is around 6.9, FibNT exists in FibNT monomer, with the side chain of hydropho- random-coil conformation and has net negative bic Phe26 pointing inwards and with hydrophilic charges due to the ionization of acidic residues (Fig. residues Asp25 and Glu28 pointing outwards. 7). Due to charge repulsion, the premature β-sheet However, in the other monomer, this N-terminal formation and the self-assembly of fibroin are segment flips away from the hydrophobic patch, prevented at this point. With the procession of

Fig. 6. Surface hydrophobic patches of FibNT. Hydrophobic residues on the surface are shown in yellow and marked in the figure, basic residues are shown in blue, acidic residues are shown in red, and the rest is shown in white. (a) Front view of FibNT (facing the dimer–dimer interface). (b) Back view of FibNT. 204 Structure of N-terminal Domain of Fibroin

Fig. 7. Assembly model of B. mori fibroin. At a pH of around 6.9, FibNT exists in random-coil conformation and has net negative charges due to the ionization of acidic residues, thus preventing premature β-sheet formation. When local pH decreases, the acidic side chains of FibNT begin to protonate, resulting in less negative charges and weaker electrostatic repulsion, and drives FibNT to fold into β-sheets and to oligomerize via β-hairpin entanglements. Increasing ionic strength in silk glands could also contribute to the oligomerization of FibNT by shielding electrostatic repulsion. The hydrophobic patches could possibly serve as micelle–micelle docking sites.

fibroin solution along the silk gland lumen, local Materials and Methods pH decreases, and the acidic side chains of FibNT (and perhaps the linker regions) begin to protonate; this results in less negative charges and weaker Construction, expression, and purification of FibNT charge repulsion and drives FibNT to fold into β- β The cDNA encoding the N-terminal domain of FibH sheets and to oligomerize via -hairpin entangle- – fi ments. Subsequently, micelle formation is promoted, (FibNT, residues 20 126) was cloned into a modi ed pET28a expression vector (Novgan) with an additional six- with FibNT sequestering the hydrophobic regions ′ ‘ ’ histidine coding sequence at the 5 end of the genes. The and forming water-tight micelle outer edges. In- recombinant plasmid was transformed into competent E. creasing ionic strength in silk glands could also coil Rosetta (DE3) cells, which were cultured in 400 ml of promote the oligomerization of FibNT by shielding 2×YT containing 0.01 mg/ml kanamycin. The culture was charge repulsion. The solvent-exposed hydrophobic grown at 310 K to an A600 of 0.6 and induced with 0.2 mM patches at one face of the FibNT β-sheet could interact IPTG for 4 h. After being harvested by centrifugation, the with hydrophobic repetitive regions, while those at cells were resuspended in 20 mM cold Tris–HCl (pH 8.0) the dimer–dimer interface could possibly serve as and 100 mM NaCl. After three steps of freeze thawing and micelle–micelle docking sites, leading to much larger subsequent sonication, the lysed cells were centrifuged at fi 16,000g for 20 min. The His-tagged proteins were purified globular structures that can be nally spun into silk fi fi with an Ni-NTA af nity chromatography column (Amer- ber under the physical shear imposed at the sham Biosciences), followed by a gel-filtration column spinneret. fi (HiLoad 16/60 Superdex 75 prep grade; Amersham In summary, our results provide the rst struc- Biosciences) equilibrated with 20 mM Tris–HCl (pH 8.0) tural insights into the assembly mechanism of B. and 100 mM NaCl, and then eluted in the same buffer. The mori silk at the atomic level. Revealing FibNT as an resulting fractions were analyzed by 15% SDS-PAGE. Site- essential module in mediating pH-responsive as- directed mutagenesis was performed using the Quick- sembly, we find that the functions of N-terminal Change site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA), with the plasmid encoding wild-type FibNT as domains in B. mori and spiders are strikingly similar, fi despite the very distinct sequences and three- template. The mutant proteins were expressed, puri ed, dimensional structures. The studies on the assembly and stored in the same manner as the wild-type protein. mechanism of B. mori silk will not only complement Crystallization and data collection our understanding of how the remarkable silk fiber forms but also provide structural insights for FibNT was crystallized at 289 K used hanging-drop designing artificial silk materials. vapor diffusion. The crystals of FibNT were grown in a Structure of N-terminal Domain of Fibroin 205 drop of 5 mg/ml protein in 20 mM Tris–HCl (pH 8.0) and Table 1. Data collection, phasing, and refinement statistics 100 mM NaCl with an equal volume of reservoir solution [1.2 M ammonia sulfate and 0.1 M sodium acetate (pH 4.6); SAD peak the actual pH in the drop is 4.7] within 7 days. The SeMet Data collection statistics derivative crystals were grown under the same conditions. Wavelength (Å) 0.9794 The crystals were transferred to a cryoprotectant (reservoir Space group P6522 solution supplemented with 25% glycerol) and flash- Unit cell parameters a, b, c (Å) 74.52, 74.52, 208.23 frozen in liquid nitrogen. Multiwavelength anomalous α β γ dispersiondataforaSeMetderivativecrystalwere , , (°) 90, 90, 120 Resolution limit (Å) 50.00–3.00a (3.05–3.00) collected with an MX225 CCD (MARresearch, Germany) Unique reflections 7427 (328) at the Shanghai Synchrotron Radiation Facility at a Completeness (%) 99.4 (94.5) b radiation wavelength of 0.9794 Å using 17 U at 100 K. Rmerge (%) 11.2 (65.4) All diffraction data were indexed, integrated, and scaled I/σI 40.2 (3.3) with HKL2000.23 Redundancy 31.9 (19.1) SAD phasing Structure determination and refinement Heavy-atom sites 6 Mean factor of merit after SOLVE 0.35 Mean factor of merit after RESOLVE 0.68 The crystal structure of FibNT from a SeMet-substituted protein crystal was determined to a maximum resolution Refinement statistics of 3.0 Å using the SAD phasing method. The SHELX Resolution limit (Å) 37.26–3.00 24 R-factor (%)c 31.1 program suite was used to locate heavy atoms, and six d selenium atoms were identified. The phase was calculated R-free (%) 33.6 RMSD bond length (Å)e 0.008 and further improved with the program SOLVE/ ‡ 25 RMSD bond angles (°) 1.143 RESOLVE . Electron density maps calculated from Average of B-factors (Å2) 101.8 fl f solvent- attened experimental phases lack side-chain Ramachandran plot features and cannot be traced, possibly due to a strong Most favored region (%) 89.9 anisotropic diffraction effect along the a and b axes. When a Additionally allowed region (%) 7.9 B-factor of −50 was applied to the data,26 the electron Protein Data Bank entry 3UA0 density was greatly sharpened, and an initial model was a The values in parentheses refer to statistics in the highest- built automatically with the BACCANEER program resolution bin. 27 b ∑ ∑ −〈 〉 ∑ ∑ implemented in the CCP4 program suite. A complete Rmerge = hkl i|Ii(hkl) I(hkl) |/ hkl iIi(hkl), where Ii(hkl)is model for FibNT was finally built manually from the initial the intensity of an observation, and 〈I(hkl)〉 is the mean value for model using the Coot program,28 and side-chain assign- its unique reflection. Summations are over all reflections. c ∑ − ∑ ments were confirmed by the positions of SeMet. The R-factor= h|Fo(h) Fc(h)|/ hFo(h), where Fo and Fc are the fi observed and calculated structure factor amplitudes, respectively. model was then re ned with the Refmac5 program, and d the final model was evaluated with the programs R-free was calculated with 5% of the data excluded from the 29 30 refinement. MolProbity and Procheck. The data collection and e fi RMSD from ideal values. structure re nement statistics are listed in Table 1. f Categories were defined by MolProbity. Sequence alignment was performed using the programs CLUSTAL W31 and ESPript.32 Structural comparison was carried out using the DALI server§. All structure figures τ 33 an ALV/DLS/SLS-5022F spectrometer with a multi- were created using the program PyMOL. digital time correlation (ALV5000) and a cylindrical 22- mW UNIPHASE He–Ne laser (λ=632 nm). The intensity– CD spectroscopic analysis intensity time correlation function G(2)(t,q) was measured to determine the linewidth distribution G(Γ). For diffusive The CD spectra of FibNT and mutants were recorded at relaxation, Г is related to the translational diffusion 18 °C with a spectropolarimeter (Jasco, 810) using a quartz coefficient D of the scattering object (macromolecules or cuvette with a path length of 0.1 cm. The protein samples colloid particles) in dilute solution or dispersion by 〈Γ〉 2 were dissolved to 0.1 mg/ml in the buffers of 20 mM D=( /q )C → 0,q =0 and to the hydrodynamic radius – πη potassium phosphate and 100 mM ammonium bisulfate at from the Stockes Einstern equation Rh = kBT/6 D, η pH 3.0, 4.6, 6.0, and 7.0, respectively. All samples were where , kB, and T are the solvent viscosity, the Boltzmann centrifuged at 16,000 rpm for 10 min at 18 °C. The protein constant, and the absolute temperature, respectively. was omitted from the control sample, and each spectrum Hydrodynamic radius distribution was calculated from was corrected by subtracting the baseline (control). All the Laplace inversion of a corresponding measured G(2)(t, experiments were performed in triplicate. q) using the CONTIN program. All DLS measurements were conducted at a scattering angle θ of 45° at 37 °C. DLS and scanning electron microscopy Protein samples (at approximate concentrations of 1 mg/ ml) were prepared in Tris–HCl buffer (20 mM Tris–HCl and 150 mM NaCl) in the pH interval 7.0–4.0. The effect of pH on the hydrodynamic diameter (Rh)of the N-terminal domain was measured at 25±0.1 °C with For scanning electron microscopy imaging, the FibNT sample (1 mg/ml) at pH 5.0 was absorbed onto a carbon- coated copper grid by floating the grid on a drop of FibNT ‡ http://www.solve.lanl.gov solution for 30 s. The excess solution was removed by filter § http://www2.ebi.ac.uk/dali/ paper blotting. The samples were dried and covered with 206 Structure of N-terminal Domain of Fibroin a gold layer using a Hummer gold sputtering system and 8. Shao, Z. Z. & Vollrath, F. (2002). Materials: surprising imaged with a Sirion200 electron microscope (FEI Co., strength of silkworm silk. Nature, 418, 741. Hillsboro, OR, USA) operated at 20 kV. 9. Marsh, R. E., Corey, R. B. & Pauling, L. (1955). An investigation of the structure of silk fibroin. Biochim. Biophys. Acta, 16,1–34. Accession number 10. Dobb, M. G., Fraser, R. D. B. & Macrae, T. P. (1967). Fine structure of silk fibroin. J. Cell Biol. 32, 289. fi The nal coordinates and structure factors were 11. Chen, X., Knight, D. P., Shao, Z. & Vollrath, F. ∥ deposited in the Protein Data Bank under accession (2002). 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