Crystal Structure of Human Type III Collagen Gly991–Gly1032

Cystine Knot-containing Peptide Shows Both 7/2 and 10/3Triple Helical Symmetries*

Received for publication, July 16, 2008, and in revised form, September 16, 2008 Published, JBC Papers in Press, September 19, 2008, DOI 10.1074/jbc.M805394200

Sergei P. Boudko‡, Jurgen Engel§, Kenji Okuyama¶, Kazunori Mizuno‡, Hans Peter Bachinger‡1,and Maria A. Schumacher�2

From the ‡Research Department, Shriners Hospital for Children, Portland, Oregon 97239, §Biozentrum, University of Basel,Klingelbergstrasse 70, CH-4056 Basel, Switzerland, the ¶Department of Macromolecular Science, Graduate School of Science,Osaka University, Toyonaka, Osaka 560-0043, Japan, and the �Department of Biochemistry and Molecular Biology,M. D. Anderson Cancer Center, Houston, Texas 77030

Type III collagen is a critical collagen that comprises extensi-ble connective tissue such as skin, lung, and the vascular system.Mutations in the type III collagen gene, COL3A1, are associatedwith the most severe forms of Ehlers-Danlos syndrome. A char-acteristic feature of type III collagen is the presence of a stabi-lizing C-terminal cystine knot. Crystal structures of collagentriple helices reported so far contain artificial sequences like(Gly-Pro-Pro)n or (Gly-Pro-Hyp)n. To gain insight into thestructural properties exhibited by the natural type III collagentriple helix, we synthesized, crystallized, and determined thestructure of a 12-triplet repeating peptide containing the natu-ral type III collagen sequence from residues 991 to 1032 includ-ing the C-terminal cystine knot region, to 2.3 A resolution. Thisrepresents the longest collagen triple helical structure deter-mined to date with a native sequence. Strikingly, the Gly991–Gly1032 structure reveals that the central non-imino acid-con-taining region adopts 10/3 superhelical properties, whereasthe imino acid rich N- and C-terminal regions adhere to a 7/2superhelical conformation. The structure is consistent withtwomodels for the cystine knot; however, the poor density forthe majority of this region suggests that multiple conforma-tions may be adopted. The structure shows that the multiplenon-imino acids make several types of direct intrahelical aswell as interhelical contacts. The looser superhelical struc-ture of the non-imino acid region of collagen triple helicescombined with the extra contacts afforded by ionic and polarresidues likely play a role in fibrillar assembly and interac-tions with other extracellular components.

Collagens are the most abundant proteins in animals, com-prising an estimated one-third of the total protein by weight(1–3). At least 27 collagen types, which are formed from 42distinct polypeptide chains, exist in vertebrates (1–2). In addi-tion, more than 20 additional proteins adopt collagen-likestructures such as collectins, ficolins, and scavenger receptors(2). Collagen is an essential molecule in vertebrates, because itplays the dominant role in maintaining the structure of tissues.However, collagen and collagen-like proteins have many otherimportant roles, such as cell adhesion, chemotaxis, cell migra-tion, and the regulation of tissue remodeling during cell growth,differentiation, morphogenesis, and wound healing (2).All collagen molecules consist of three polypeptide chains,

called � chains, which contain at least one region of repeatingGly-Xaa-Yaa sequences (1–2). In the collagen molecule, thethree � chains each fold into a polyproline II-like left-handedstructure, and the three polyproline II-like chains twist aroundeach other to form a right-handed superhelix, called the colla-gen triple helix (4–7). Critical to the formation of the triplehelix is the presence of a glycine residue at each third position inthe chain because this residue is the only one that can exist inthe small space at the center of the triple helix (8). Each of thethree chains therefore has the repeating structureGly-Xaa-Yaa,in which Xaa and Yaa can be any amino acid but are frequentlythe imino acids proline in the Xaa position and hydroxyproline(Hyp) in the Yaa position. Because both proline and Hyp arerigid, cyclic imino acids, they limit rotation of the polypeptidebackbone and thus contribute to the stability of the triple helix.Collagen polypeptides that lackHyp can fold into a triple helicalconformation at low temperatures, but the triple helix formedis not stable at mammalian body temperature (8).The number of Gly-Pro-Hyp repeats is the main, but not

exclusive, factor in determining collagen thermostability (9).Approximately 90% of collagen tripeptide units contain at leastone non-imino acid residue in the Xaa and/or Yaa position, andthese residues likely play a role in collagen structure, stability,and function (5–7). Indeed, a notable feature of the collagentriple helix is that the amino acids occupying Xaa and Yaa posi-tions are solvent-accessible. Because of this, these residueswould be predicted to play important roles in interactions withother molecules, such as extracellular matrix proteins. In addi-tion, these residues are predicted to be important in collagen

* This work was supported by a grant from the Shriners Hospital for Children,in part by Burroughs Wellcome Career Development Award 992863 (toM. A. S.), and in part by United States Department of Energy ContractDE-AC03-78SF00098. The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

The atomic coordinates and structure factors (code 3DMW) have been depositedin the Protein Data Bank, Research Collaboratory for Structural Bioinformat-ics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

1 To whom correspondence may be addressed. Tel.: 503-221-3433; Fax: 503-221-3451; E-mail: hbp@shcc.org.

2 To whom correspondence may be addressed: Dept. of Biochemistry &Molecular Biology, Unit 1000, University of Texas M. D. Anderson CancerCenter, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-834-6392; Fax:713-792-7448; E-mail: maschuma@mdanderson.org.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 47, pp. 32580 –32589, November 21, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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triple helix self-association leading to fibril formation. The bestcharacterized and most common collagen fibril form is the67-nm (D) periodic fibril, which is observed inmost connectivetissues (10, 11). Collagen types I, II, III, V, and XI self-associateto form these characteristic fibrils. Studies suggest that the axialstaggering of collagen molecules that give rise to these fibrils isdue to electrostatic and hydrophobic interactions betweenneighboring molecules (12–14). Specifically, oppositelycharged residues in the Xaa and Yaa position of the collagentriple helix are predicted to play a role in determining the axialstagger of the fibril (11). Such charged residues have also beenimplicated in the interactions between macrophage scavengerreceptor and other molecules and the hexamer formation ofthe serum complement protein C1q (15–17).Type III collagen contains multiple charged residues in the

Xaa and Yaa positions of its chain (18). Type III collagen is thesecond most abundant collagen in human tissues after type Iand is primarily found in tissues exhibiting elastic properties,such as skin, blood vessels, and internal organs. Type III colla-gen is common in fast growing tissue, particularly at the earlystages of wound repair (19, 20). Mutant mice have been gener-ated by gene targeting in which the type III collagen gene(COL3A1) has been knocked out (20). These knock-out miceexhibit irregularly sized collagen fibers in the skin dermis aswell as the aortic adventitia, which indicates the importance oftype III collagen in regulating collagen fiber size. Notably, typeIII collagen null mice die perinatally, usually because of bloodvessel or intestinal rupture. Thus, these data demonstrate thattype III collagen is necessary for proper organ and tissue func-tion, especially in distensible organs. These findings are con-sistentwith the fact thatmutations of type III collagen cause themost severe form of Ehlers-Danlos syndrome, EDS IV,3 whichaffect internal organs, arteries, joints, and skin. Indeed, EDS IVcan result in sudden death when the large arteries rupture (20).The most severe forms of EDS IV are correlated with pointmutations that substitute a residue for a glycine near the C-ter-minal end of the triple helix, including G1012R, G1018V, andG1021E (21).Structurally, type III collagen is a homotrimer composed of

three �1(III) chains and resembles other fibrillar collagens. Akey feature in the formation of type III collagen is a so-calleddisulfide or cystine knot, which is located between the triplehelical region and the C-terminal telopeptide (22, 23). The knotis formed by three interchain disulfide bonds, and it signifi-cantly stabilizes the triple helical structure. The stabilityimparted by the disulfide knot has been successfully used toproduce collagenous peptides that otherwise would be toounstable to study (24, 25). Production of these peptides involvesthe C-terminal extension by the bis-cysteinyl-sequenceGPCCG, followed by air or glutathione oxidation at lower tem-perature under slightly basic conditions.In the 1950s two structuralmodels for collagenwith different

fiber periods and helical symmetries were proposed based onthe fiber diffraction pattern of native collagen. These were the

7/2 helical model with a 20-Å axial repeat (26, 27) and the 10/3helical model with a 30-Å axial repeat (27). These single-helicalmodels were discarded after the proposal of a triple helicalstructure with 10/3 helical symmetry and a 28.6-Å axial repeatby Ramachandran andKartha (28). The first crystal structure of(Pro-Pro-Gly)10 showed a triple helixwith 7/2 helical symmetrywith a 20-Å axial repeat (29). Recently, the fiber diffractionanalysis of native collagen was performed based on theadvanced diffraction data acquisition techniques and revealedthat the x-ray diffraction data can be explained not only by theprevailing 10/3 helical model but also by the 7/2 helical model(7). Almost all of the high resolution structures of model pep-tides adopt a 7/2 helical symmetry, and the conformation closeto the 10/3 helix appears only in the guest region of host-guestpeptides, like the T3-785 peptide and the integrin-binding pro-tein complexed with integrin (7).Crystallization and structure determination of collagen pep-

tides is a challenging task. All of the available crystal structuresare of either artificial mimics (like (GPP)n or (GPO)n, whereO� 4(R)-hydroxyproline) or host-guest peptides where a shortstretch of one to three native tripeptide units are flanked bythree to five GPO repeats.Unfortunately, these structures give only limited insight into

how side chains other than imino acid residues in the Xaa andYaa positions contribute to collagen structure and stability(30–43). The length of a collagen peptide is an importantdeterminant in crystallization. Longer fragments impart higherflexibility, which may impede crystallization, whereas the pro-duction of a thermally stable triple helix actually requires longersequences and the addition of artificial stabilizing tripeptideunits, like GPO. These factors have limited the length of colla-gen fragments that have crystallized to only nine to eleven trip-eptide units. Furthermore, this has restricted the number ofintegrated native tripeptide units to only one to three. Onestrategy used to obtain structural information on a longer triplehelix was to fuse a stable trimeric molecule, foldon, which is atrimerization domain of fibritin, to a (GPP)10 collagen mimic(44). The resulting crystal structure revealed that there was adramatic kink between the (GPP)10 triple helix and the foldondomain, which permitted adaptation to the mismatch betweenthe 3-fold rotation symmetry of the foldon domain and the oneresidue stagger of the collagenous structure (45). Here wedescribe another approach to stabilize a long collagen triplehelix structure by the use of a native type III collagen C-termi-nal disulfide knot (46–49). Specifically, we crystallized a42-residue collagen peptide containing the C-terminal type IIIcollagen sequence from residues 991 to 1032, which containsthe residues most commonly mutated in very severe forms ofEhlers-Danlos syndrome IV. This peptide also contains thenative C-terminal cystine knot region. Thus, we demonstratethat utilization of the cystine knot opens doors to crystallizationof lengthy and native collagen fragments.

EXPERIMENTAL PROCEDURES

Peptide Sequences—The sequence of the type III collagenpeptide used in this study is as follows: (GPI GPO GPR GNRGERGSEGSOGHOGsMOGPOGPOGAOGPCCGG)3. (O is4(R)-hydroxyproline, and sM is L-selenomethionine). These

3 The abbreviations used are: EDS, Ehlers-Danlos syndrome; MAD, multi-wavelength anomalous diffraction; HPLC, high pressure liquid chromatog-raphy; Fmoc, N-(9-fluorenyl)methoxycarbonyl; ASU, asymmetric unit.

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residues correspond to residues 991–1032 of the human collagenIII a chain, with exception of the L-selenomethionine, which wassubstituted in place of the wild type glutamine for use in multiplewavelength anomalous diffraction (MAD) phasing.Peptide Synthesis and Purification—The peptide was synthe-

sized on an ABI433A peptide synthesizer with 0.25 mM Fmoc-Gly-PEG-PS resin, a 4-fold excess of Fmoc amino acids andO-(7-azabenzotriazol-1-yl)-N,N,N�,N�-tetramethyluroniumhexafluorophosphate as activating agent. The Fmoc aminoacids carried the protection groups Cys-Trt, Hyp-t-Bu, Gln-Trt, His-Trt, Ser-t-Bu, Glu-O-t-Bu, Arg-Pbf, Asn-Trt, and Tyr-t-Bu. The peptide was cleaved off the resin and deprotected for4 h at room temperature with 90% trifluoroacetic acid, 5% thio-anisole, 3% 1,2-ethanedithiol, 2% anisole. Subsequently, thepeptide was precipitated in cold ether, redissolved in H2O, andlyophilized. The reduced peptide was then purified by reversephase HPLC using a C18 column (Vydac, Hesperia, CA; 50 �250 mm, 10–15-�m particle size, 300-Å pores) with an aceto-nitrile/water gradient and 0.1% trifluoroacetic acid as ion-pair-ing agent. Finally, the peptide was characterized by electro-spray/quadrupole/time-of-flight mass spectrometry (Q-tofmicro; Waters Associates) and amino acid analysis.Peptide Folding, Oxidation, and Purification of Disulfide-

linked Trimer—The lyophilized, reduced peptide was dissolvedin degassed and N2 saturated 50 mM sodium acetate buffer, pH4.5, under N2 atmosphere and was kept at 4 °C for 24 h to allowtriple helix formation prior to oxidation. Two strategies of oxi-dation were used: exposure to atmospheric O2 or addition ofreduced (10mM) and oxidized (1mM) glutathione and exposureto atmospheric O2. In both cases, the pH was raised to 8.3 witha saturated solution of Tris. Oxidation was carried out for 5–7days, and the peptide mass was periodically analyzed by liquidchromatography-mass spectrometry. The maximum yield ofcovalently linked trimeric peptide was �60–70% and wasslightly higher when glutathione was used for oxidation. Toseparate covalently linked trimeric peptide from other oli-gomers, the oxidized crude material was dissolved in deionized8Murea solutionwith 0.1% trifluoroacetic acid to prevent disul-fide exchange and applied to a sieve column. Trimer-contain-ing fractions were pooled out and further purified by reversephase HPLC using a C18 column.Peptide Crystallization, Data Collection, Structure Deter-

mination, and Refinement—The purified and lyophilizedcovalently linked trimeric collagen III peptide, Gly991–Gly1032, was dissolved at a concentration of 15 mg/ml in 5mM acetic acid. The peptide was crystallized at 22 °C usingthe hanging drop vapor diffusion method. For crystalliza-tion, 2 �l of the peptide solution was mixed with 2 �l of thereservoir solution of 20% polyethylene glycol monomethylether 550. The crystals appeared as very thin plates in aperiod of 1–5 days and are monoclinic, space group P21 witha � 31.98 Å, b � 21.52 Å, c � 68.97 Å, and � � 92.58°.Although the crystals diffracted beyond 2.5-Å resolution,they displayed extremely high mosaic spread (�3.0°) evenwhen x-ray intensity data were collected at room tempera-ture. Thus, several strategies for cryoprotection were tried inan attempt to improve the quality of the diffraction. The datawere collected at ALS Beamline 8.2.1. Good quality data

were obtained for only one crystal. For this data collection,glycerolwas first added to the drop containing the crystal to a finalconcentration of 10%. The drop sat for 8 h, and the crystal wasplaced directly in the cryostream. However, at this point themosaic spread was still unacceptably high. Thus, the crystal wasannealed several times by removing the crystal from the cryo-stream and placing back in the drop solution. After two annealingcycles the diffraction, although still highly mosaic (1.7°), was ofsufficient quality to collect data. A complete three wavelengthMADdata set was collected on this crystal andwas used for struc-ture determination.The positions of the three selenomethionines in the triple

helix were obtained using SOLVE. The phases obtained fromthese positions were improved by density modification in CNS,and the resulting density modified map was used for modelbuilding in O (50–52). After two-thirds of the structure wasbuilt, phase combination using phases from the partial modelwas used to improve themap. This permitted the current struc-ture, which consists of one complete triple helix in the crystal-lographic asymmetric unit (ASU) to be built. Multiple cycles ofsimulated annealing, xyzb refinement, and rebuilding in Oresulted in an Rwork/Rfree of 24.3%/27/4% to 2.30 Å resolution.The currentmodel has excellent stereochemistry (Table 1) (53).Multiple omit maps were calculated throughout the process toconfirm the correctness of themodel. Nonetheless, the electrondensity of the cystine knot residues remained poor, and theC-terminal residues that precede the cysteine residues displayhigh B-factors consistent with this region being disordered orconsisting of multiple conformations.Analysis of Triple Helix Geometry—Helical parameters were

calculated based on the method of Sugeta and Miyazawa (54)

TABLE 1Selected crystallographic data for Gly991–Gly1032 structure

Wavelength (�) 0.9797 1.0200 0.9796Resolution (Å) 68.84-2.30 68.78-2.30 68.84-2.30Overall Rsym(%)a 7.5 (24.6)b 6.2 (24.5) 7.6 (24.5)Overall I/�(I) 20.5 (2.5) 20.4 (2.3) 20.4 (2.5)No. of total reflections 19,657 19,640 19,658No. of unique reflections 3964 3962 3960Multiplicity 5.0 5.0 5.0Overall figure of meritc 0.490Crystal parametersSpace group P21Cell parameters (Å) a � 31.98, b � 21.52,

c � 68.97, � � 92.58°Resolution (Å) 68.8-2.30Overall Rsym(%)a 6.2(24.5)Overall I/�(I) 20.4 (2.3)No. of total reflections 19640No. of unique reflections 3962

Refinement statisticsResolution (Å) 68.8-2.30Rwork/Rfree(%)d 24.3/27.4Root mean square deviationBond angles (°) 1.92Bond lengths (Å) 0.008B values (Å2) 1.5

Ramachandran analysisMost favored (%/no.) 92.9/52Additionally allowed (%/no.) 7.1/4Generously allowed (%/no.) 0.0/0Disallowed (%/no.) 0.0/0

aRsym � ���Ihkl � Ihkl(j)�/�Ihkl, where Ihkl(j) is the observed intensity and Ihkl is thefinal average value of intensity.

b The values in parentheses are for the highest resolution shell.c Figure ofmerit� ���P(� )ei�/�P(� )� �, where� is the phase andP(� ) is the phaseprobability distribution.

d Rwork � ���Fobs� � �Fcalc��/��Fobs� and Rfree � ���Fobs� � �Fcalc��/��Fobs�, where all ofthe reflections belong to a test set of 5% randomly selected data.

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for every amino acid residue using the program, PHEL (6). Theinput data for the calculation of the jth triplet consist of threesets of nine parameters for the jth, (j 1)th, and (j 2)th aminoacid residues. Here, nine parameters of the jth amino acid res-idue are bond lengths ofN(j)-C�(j), C�(j)-C�(j), andC�(j)-N(j1), bond angles of C�(j � 1)-N(j)-C�(j), N(j)-C�(j)-C�(j) andC�(j)-C�(j)-N(j 1), and dihedral angles of C�(j � 1)-N(j)-C�(j)-C�(j), N(j)-C�(j)-C�(j)-N(j 1), and C�(j)-C�(j)-N(j 1)-C�(j 1).

RESULTS AND DISCUSSION

Folding and Oxidation of the Gly991–Gly1032 Peptide—Previ-ous studies demonstrated that the correct folding andoxidation

of the disulfide knot requires preformation of the collagen triplehelix (47, 48). Triple helix formation of the reduced Gly991–Gly1032 peptide was confirmed by thermal unfolding as moni-tored by the change of CD signal at 225 nm. The midpointtransition temperaturewas 12 °Cmeasured at the 1mg/ml pep-tide concentration in 0.1 M sodium acetate buffer, pH 4.5. Pro-nounced hysterisis was observed for the reduced peptide tran-sitions, as expected (48). Oxidation was performed at 5 mg/mlpeptide concentration and 4 °C, which is the required temper-ature for triple helix formation. Notably, the effectiveness oftrimer formation was �60–70%, which is similar to what haspreviously been reported (47). The Tm of 30 °C measured forthe oxidized peptide was substantially higher in the samebuffer. Crystallization was performed at 22 °C, at which tem-perature the triple helix is stable.Overall Structure of Collagen Type III (Gly991–Gly1032)

Peptide—The structure of the Gly991–Gly1032 peptide wasdetermined by MAD. For simplicity the residues in each chainhave been numbered from 1 to 42 (i.e. residue 1 corresponds to991 and residue 42 corresponds to 1032 in the native sequence).For MAD analysis, the Gln, corresponding to Gln16 wasreplaced by selenomethionine (see “Experimental Proce-dures”). The crystallographic ASU consists of one complete tri-ple helix (Figs. 1 and 2). As has been observed in other collagenpeptide crystal structures, the N- and C-terminal ends of eachof the three triple helical chains display weak electron densityand appearmostly disordered. TheC-terminal residues are par-ticularly disordered and C-terminal residues that are presentdisplay significantly elevated B-factors (Fig. 1B). The final

FIGURE 1. Overall structure of the Gly991–Gly1032 peptide structure. A, theoverall triple helix is shown for the Gly991–Gly1032 structure and coloredaccording to superhelix symmetry: region 1 is colored yellow, region 2 is red,region three is colored yellow, and region 4, the cystine knot region, is green.Residue 4 from each chain is numbered (to indicate stagger), and the lastresidues that are observed in each chain are also labeled. B, the Gly991–Gly1032

triple helix shown in the same orientation as A but colored according to B-fac-tor, with the gradation of blue to red reflecting increasing B-factors (i.e. bluerepresents low B-factors, and red represents high B-factors). This figure wasmade using PyMOL (55).

FIGURE 2. 2.30 Å resolution composite omit map of the Gly991–Gly1032

structure. Composite omit map contoured at 1.0 � showing the density forthe center region of the ASU and surrounding symmetry-related molecules.The individual chains in each triple helix are colored magenta, pink, andyellow.

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model consists of residues 3–39 of chain A, residues 4–40 ofchain B, and residues 2–38 of chain C. There are also 61 watermolecules in the structure. The final Rwork/Rfree is 24.3%/27.4%to 2.3-Å resolution. The packing of triple helices in the crystalappears to be pseudotetragonal (Fig. 3).The Disulfide Knot—The Gly991–Gly1032 peptide contains a

GPCCGG sequence at the C terminus, which has been shownto form a so-called cystine knot. The connectivity of the cystineknot has not been determined despite the fact that it has beenextensively studied by several techniques including NMR (47).There are eight possible ways to connect the six cysteines

to form the disulfide knot. Twomodels were previously suggestedbased on steric compatibility (47,56). According to these modelingstudies, the three collagen chains inour structure are designated as A, B,and C, where chain A has a one-res-idue stagger toward the N terminus,followed by the B chain, and finallythe C chain (Fig. 1A). Based on thistype of stagger, the first model pro-posed by Bruckner et al. (56) has aconnectivity as follows; A1-B1/A2-C1/B2-C2 (Fig. 4A). The secondmodel proposed by Barth et al. (47)has the connectivity, A1-B2/A2-C1/B1-C2 (47) (Fig. 4A). The two mod-els share the A2-C1 disulfide bridgeyet differ in the other disulfide con-nectivities. These two models alsodiffer in the final conformation that

would be adopted by the peptides containing the cysteines. Spe-cifically, in the first model, the cystine knot residues adopt �/�dihedral angles consistent with a collagen triple helix, whereasthis is not the case for the second model.We see weak electron density for this region of the structure.

Clear density is only observed for one disulfide bond, between theA1 and B2 cysteines (Fig. 4B), which is in conflict with the firstmodel. In addition, the �/� dihedral angles of the cysteines in ourstructure deviate significantly from those observed in a collagentriple helix. Thus, although the electron density for the two

FIGURE 3. Views of the crystal packing and stagger of the collagen III Gly991–Gly1032peptide. A, close-up lateral view of the triple helices that are within

contact distance of the single helix in the ASU, which is numbered 1 and colored yellow. B, side view of the interacting helices. C, crystal packing. Carbon,nitrogen, and oxygen atoms are colored yellow, blue, and red, respectively. The collagen peptide in the ASU is numbered 1. A and B were made with PyMOL (55),and C was generated with O (52).

FIGURE 4. Cystine knot conformation. A, the two previously suggested cystine connectivities (models 1 and2) (47, 56) and a new model 3 containing the A1–B2 bond. Cysteine connectivities are indicated by colored linesfrom one cysteine to the next (magenta connectivities for model 1, blue for model 2, and green for model 3). Thestagger of the triple helix that corresponds to the Gly991–Gly1032 structure is indicated at the left side (A to B toC) in red. B, composite omit map contoured at 1.0 � showing the density for the cystine knot region. Cleardensity is only observed for the A1-B2 connection, which is consistent with model 2 and 3. The map is shownas a blue mesh. The structure is shown as sticks and colored according to atom type with carbon, nitrogen,oxygen, and sulfur shown in yellow, blue, red, and green, respectively.

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remaining disulfide bonds are poor, these combined data are sup-portive of themodel proposed by Barth et al. (47) or a thirdmodelwith the connectivity A1-B2/A2-C2/B1-C1 (Fig. 4A).The poor electron density we observed in the disulfide knot

region might be due to several reasons. One may be the flexi-bility of the last residues of the polypeptide chain. Indeed, thereare no crystal contacts to the C-terminal region, which couldstabilize these residues (Fig. 4B). However, the interchain disul-

fide bridges formed in the disulfide knot region are expected toproduce an extremely rigid structure and thus might beexpected to be visible in electron density maps. If this is thecase, another possible explanation for the lack of electron den-sity is the existence of multiple disulfide connectivities, whichare averaged over the whole ensemble of molecules in the crys-tal. Indeed, aswas noted only 60–70%of the peptide is correctlyoxidized into trimers, whereas the rest are trapped intocovalently linked dimers with two interchain disulfide bridgesand monomers with intrachain disulfide bonds. Therefore, itseems highly probable that even the covalently linked trimersmight containmore than one possible structure of the disulfideknot. The presence of multiple disulfide connectivities wouldexplain why NMR also failed to delineate a single structure forthe cystine knot (47).Multiple connectivities are highly unlikelyin the real collagen structure. It is possible that the formation ofthe correct disulfide connections may require other regions ofthe collagen III chain not present in our structure. Indeed, thepeptide used in our studies does not include the C-terminaltelopeptide sequence, which normally follows the cystine knot,nor does it contain the C-terminal propeptide that initiates tri-merization of the triple helix. These regions could play impor-tant roles in selecting the native conformation of the disulfideknot.The Gly991–Gly1032 Structure Contains Regions of Distinct

Superhelical Symmetry—The Gly991–Gly1032 peptide can bedivided into four main regions based on amino acid sequencetype: an N-terminal imino acid-containing region, a middlestretch that contains non-imino acids in the Xaa and Yaa posi-

FIGURE 5. Helical twist values for the three chains. The helical twist valuesare indicated for the three chains (A chain in red, B chain in blue, and C chain ingreen). The calculate averages for the three distinct regions are given by blackbars.

FIGURE 6. Intrachain, interchain, and interhelical contacts from non-imino acids. A, stick showing close up of direct intrachain and interchain ionicinteractions and hydrogen bonds as mediated by arginine, glutamate, and serine residues within a single Gly991–Gly1032 triple helix. The three strands arecolored pink, magenta, and yellow. Contacts are indicated by dashed lines. B, close-up of the interhelical stacking interaction between histidine 23 residues onchains B and C. One triple helix is colored green and the other yellow. C, close-up view showing both interchain and interhelical network between Arg15, Ser17,and Ser20 on one chain with Ser17 and Arg15 on a second triple helix. The two triple helices are labeled. This figure was made with PyMOL (55).

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tions, a C-terminal imino acid rich region, and finally, the cys-tine knot region from residues 37 to 42, which contains theGPCCGG motif (Fig. 1A). Strikingly, our helical analysesrevealed that these regions display significantly different con-formations. Specifically, residues in the imino acid-rich zonesfrom peptide positions 3 to 5 and 18 to 35 display standard 7/2superhelical conformation, whereas the imino acid-poor areafrom residues 6–17 adopts 10/3 superhelical symmetry (Fig.1A). As a result, residues 6–17 display a loose helical twist of34.7°, whereas residues 3–5 and 18–36 have much tighter hel-ical twists of 53.0 and 48.3°, respectively (Fig. 5). Other looserhelical twist values were found for the Gly-Phe-Hyp-Gly-Glu-Arg sequences in the integrin-binding protein (44.4°) and theIle-Thr-Gly-Ala-Arg-Gly-Leu-Ala-Gly sequence of the T3-785peptide (42.0°).As noted, the residues in the cystine knot region show strong

deviation from any type of triple helical symmetry. The fact thatregion 3, residues 18–36 (corresponding to collagen III resi-dues 1010–1027), is a hot spot for mutations leading to themost severe forms of EDS IV suggests that the formation of thistighter triple helix symmetry is important in maintaining thestability of the collagen III triple helix structure. Indeed, thestretch of residues from 6 to 17 that precede region 3 adopts alooser conformation. The looser conformation observed in thisregion is likely attributed to the presence of non-imino acids in

the Xaa and Yaa positions of the triple helical chains as similaralterations in collagen superhelical symmetry have beenobserved in the T3-785 peptide, which contains a short stretchof biologically relevant sequence (37).Intra- and Interstrand Contacts—The Gly991–Gly1032 struc-

ture contains a number of non-imino acid residues includingcharged and polar residues and therefore provides examples ofseveral kinds of intrachain, interchain and interhelical contactsthatmay aid collagen stability and self-assembly. Previous stud-ies examining collagen stability at different pH values indicatedthat ion pairing interactions increase the stability of the triplehelix (57). In the Gly991–Gly1032 structure there are numerousintra- and interchain ion pair and hydrogen bonding interac-tions observed between charged and polar residues (Fig. 6)(Table 2). There are multiple examples of interchain contacts,but the only intrachain ion pairing interaction is that betweenthe side chains of Arg15(A) and Glu18(A). Interchain ion pairsinclude those between Arg9(C) to Glu14(A) and Arg12(A) toGlu14(B) (Fig. 6, A and C). In addition to ion pairing interac-tions, there are numerous interchain hydrogen bonds. Interest-ingly, many of the contacts involve serine residues. For exam-ple, Ser17(B) contacts Glu18(A), Ser17(A) interacts with bothArg12(C) and Arg15(C),and Ser20(A) hydrogen bonds withArg15(C) (Fig. 6, A and C). These hydrogen bonds form a con-tinuous stretch of interactions: Arg12(C) to Ser17(A) to

TABLE 2Intra/interchain and interhelical side chain interactionsEach residue in the peptide is indicated at the left and found three times in the triple helix (chains C, A, and B). The amino acids contacted by this residue are shown in thetable. Contacts between residues fromdifferent triple helices are indicated by the residues and the chainwith a prime designation (i.e.A�, B�, or C�). CK, cysteine knot region;NP, residue is not present in structure; Wat indicates that the residue is involved in water contacts; Sol indicates that the residue is exposed to solvent but not observed tobe making direct contacts to any water molecule; — indicates glycine residue; C�O, carbonyl; SeMet, L-selenomethionine.

Superhelical symmetry Chain C Chain A Chain BIle3 7/2 Sol Sol NPGly4 7/2 — — —Pro5 7/2 Sol Sol SolHyp6 10/3 Wat Wat WatGly7 10/3 — — —Pro8 10/3 Wat Wat WatArg9 10/3 Glu14(A) Wat WatGly10 10/3 — — —Asn11 10/3 Hyp24(C�)/Arg12(B) Wat Hyp27(A�)Arg12 10/3 Glu14(A)/Ser17(A)/Hyp21(C�) Glu14(B)/Gly31(C�O)C� Asn11(C)/His23(A�)Gly13 10/3 — — His23(A�)Glu14 10/3 Wat Arg9(C)/Arg12(C)/Hyp30(C�) Arg12(A)/His23(A�)Arg15 10/3 Ser17(C�)/Ser20(A) Arg15(C�O)(B)/Hyp30(B�)/Glu18(A) Ser20(A�)/Arg15(A)Gly16 10/3 — — —Ser17 10/3 Arg15(C�) Arg12(C)/Arg17/Arg15(C) Glu18(A)Glu18 7/2 Wat Arg15(A)/Ser17(B) Arg15(C�)Gly19 7/2 — — —Ser20 7/2 Glu18(B)/Hyp21(B) Arg15(C)/Arg15(B�) Hyp21(A)Hyp21 7/2 Arg12(C�) Ser20(B) Ser20(C)Gly22 7/2 — — —His23 7/2 His23(B�)/Hyp24(B) Arg12(C�O)(B�)/Gly13(C�O)(B�)/Glu14(B�) His23(C�)Hyp24 7/2 Asn11(C�) Wat His23(C)Gly25 7/2 — — —SeMet26 7/2 Sol Sol SolHyp27 7/2 Wat Asn11(B�) SolGly28 7/2 — — —Pro29 7/2 Sol Sol SolHyp30 7/2 Glu14(A�) Wat Arg15(A�)Gly31 7/2 Arg12(A�) — —Pro32 7/2 Sol Sol SolHyp33 7/2 Wat Wat WatGly34 7/2 — — —Ala35 7/2 Sol Sol SolHyp36 CK Wat Wat WatGly37 CK — — —Pro38 CK Sol Sol SolCys39 CK Sol Sol SolCys40 CK NP NP Sol

Structure of Human Type III Collagen (Gly991–Gly1032)

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Arg15(C) to Ser20(A). On the other side Arg12(C) forms an ionpair with Glu14(A), which ion pairs with Arg9(B). Interchainhydrogen bonds between Hyp residues and charged and polarresidues are also observed (Table 2). When the X position isoccupied by a residue other than proline, there is a buriedwatermolecule coordinated by at least three hydrogen bonds as foundin the T3-785 peptide (34).Interhelical Contacts—As found in other collagen peptide

structures, in the Gly991–Gly1032 structure water moleculesplay a key role in mediating interactions between residues indifferent triple helices. Such water-mediated contacts arethought to be important in fibril formation. However, theGly991–Gly1032 structure differs from other peptide structuresin that there are numerous direct ionic and polar contactsbetween triple helices as well as direct nonpolar interactions.One type of direct hydrogen bonding contact between collagentriple helices that has been observed in structures of other col-lagen triple helices is that between twoHyp residues located ondifferent triple helices. However, in our Gly991–Gly1032 peptidestructure there are no such contacts. Instead, direct hydrogenbonding interactions between non-imino acid side chains dom-inate and in fact, appear critical to the formation of the stag-gered stacking of collagen molecules observed in the crystal.The staggered axial packing is between helices arranged in anantiparallel manner. However, in vivo helices primarily pack ina parallel fashion to form fibrils. Nonetheless, data suggest thatthese types of interactions must play crucial roles in helicalpacking and fibril formation in vivo (12, 13).One extensive hydrogen bonding network is comprised of

arginine and serine residues. In this Arg-Ser network there arehydrogen bonds from Ser17(A) to Arg15(C) to Ser20(A) on onetriple helix to Ser17(C)� and Arg15(B)� (where prime indicatesresidues from a different triple helix) (Fig. 6C andTable 2). Thiscomplicated arrangement of interactions serves to stabilize thepacking of themolecules in the crystal. This is supported by thefact that the atoms of the residues involved in this networkdisplay among the lowest B-factors in the structure (Fig. 1B).Interestingly, another contact that appears crucial for stabiliza-tion of the staggered packing is a unique His(C)-His(B)� stack-ing interaction between residues on different collagen triplehelices (Fig. 6B). This appears to be the first instance of a hydro-phobic interhelical stacking interaction observed in a collagenstructure; yet the importance of such contacts in fibril forma-tion has been indicated by previous studies (12, 13).The finding that non-imino acid side chains can contribute to

thestackingof triplehelices intostaggeredarrays is consistentwithdata fromDoyle et al. (11) indicating that such residuesparticipatein determining the axial stagger of collagen fibrils in vivo (7, 58).However, the variability of the polar and ionic interactionsobserved from the same residue in different chains suggests thatthe interactionswe observe are likely an interchangeable subset ofmany possible interactions (Table 2). Indeed the lack of highlyspecific side chain-side chain interactions supports data frommagnetic resonance studies, which suggested that collagen mole-cules in fibrils experience a large degree of rotational freedomabout the helical axis, and therefore interhelical contacts are notcomprised of a single set of interactions (59). T

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Structure of Human Type III Collagen (Gly991–Gly1032)

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Gly991–Gly1032 Peptide �/� Dihedral Angles Show Deviationfrom Typical Collagen Structures—In the collagen structuressolved to date, the �/� dihedral angles of residues in the pep-tides all fall within a very narrow range of values (3, 30–41).This likely reflects the fact that these dihedral angles are criticalin preorganizing the formation of the triple helix while alsoallowing for the formation of optimal Gly-NH-OC-Pro Xaa inter-chain “signature” hydrogen bonds. As seen in Table 3, the averageGly-NH-OC-Pro Xaa interchain hydrogen bond distances andangles observed in our structure (3.03Åand167° for region 2; 2.97Å and 164° for region 3) are essentially the same as those in otherhigh resolution collagen triple helical structures (Table 3) (30–41). Moreover, the �/� dihedral angles of residues in region 3 ofour structure are also essentially the same as those observed inother high resolution collagen peptide structures.However, examination of the �/� dihedral angles of the res-

idues in region 2 of the Gly991–Gly1032 peptide shows signifi-cant deviations of� for glycine andXaa position residues (Gly�value of�163° comparedwith 175° and anXaa� value of�154°compared with 163°) as well as the dihedral angles of the Yaaposition arginine residues (Table 4). Although region 1, theN-terminal region of the structure, also shows some divergencefrom optimal triple helical geometry, similar deviations havebeen observed in the extreme N-terminal and C-terminal endsof other collagen peptide structures and appear to be caused byend fraying.Moreover, the small number of residues in region 1makes these averaged �/� dihedral angles values statisticallyinsignificant.Thus, unlike the notable departure of region 2 from triple

helical geometry, the deviations in region 1 are likely not signif-icant. Interestingly, the multiple deviations from ideal dihedralangles observed for residues in region 2 are consistent with thelooser conformation of the triple helical conformation in thisarea. These data indicate that region 2 likely does not form asstable a triple helix as does region 3 or other (GPP)n-containingstretches. It should be noted that the low B-factors of the resi-dues in region 2donot reflect on the stability of the triple helicalconformation in this region but rather are indicative of thenumerous interactions mediated by these residues to symme-try-related molecules, which act to stabilize their positionswithin the crystal. Thus, it appears that the presence of non-imino acid residues plays an important role in collagen struc-ture/function by providing a looser overall conformation of thetriple helix while also permitting the formation of importanthydrogen bonding and ionic interactions that stabilize not onlythe individual triple helix but may also mediate interhelicalcontacts that are important in collagen packing. However,more examples of structures of collagen molecules with non-imino acid residues in the Xaa and Yaa positions and concom-itant biochemical studies of such peptides are needed to eluci-date the importance of such contacts and the energetic trade offof a looser structure versus the added intrachain, interchain,and interhelical interactions.In conclusion, the structure of the collagen III peptide,

Gly991–Gly1032, opens the possibility of obtaining structuralinformation for native sequences of collagen peptides that hadpreviously been too unstable to crystallize. More importantly,the structure, which is the first of a long, non-imino acid-con- T

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Structure of Human Type III Collagen (Gly991–Gly1032)

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taining sequence of a native collagen triple helix, reveals prop-erties imparted by such amino acid residues thatmay be impor-tant in collagen folding, fibril formation, and interactions withother proteins.

Acknowledgments—We thank Kerry Maddox and Jessica Hacker forpeptide synthesis, amino acid analysis, and mass spectroscopy. Wethank the Advanced Light Source and their support staff.

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Structure of Human Type III Collagen (Gly991–Gly1032)

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Bächinger and Maria A. SchumacherSergei P. Boudko, Jürgen Engel, Kenji Okuyama, Kazunori Mizuno, Hans Peter

Knot-containing Peptide Shows Both 7/2 and 10/3 Triple Helical Symmetries Cystine1032Gly−991Crystal Structure of Human Type III Collagen Gly

doi: 10.1074/jbc.M805394200 originally published online September 19, 20082008, 283:32580-32589.J. Biol. Chem. 

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