In Situ D-periodic Molecular Structure of Type II Collagen

Abstract

Collagens are essential components of extracellular matrices in multicellular animals. Fibrillar type II collagen is the most prominent component of articular cartilage and other cartilage-like tissues such as notochord. Its in situ macromolecular and packing structures have not been fully characterized, but an understanding of these attributes may help reveal mechanisms of tissue assembly and degradation (as in osteo- and rheumatoid arthritis). In some tissues such as lamprey notochord, the collagen fibrillar organization is naturally crystalline and may be studied by x-ray diffraction. We used diffraction data from native and derivative notochord tissue samples to solve the axial, D-periodic structure of type II collagen via multiple isomorphous replacement. The electron density maps and heavy atom data revealed the conformation of the nonhelical telopeptides and the overall D-periodic structure of collagen type II in native tissues, data that were further supported by structure prediction and transmission electron microscopy. These results help to explain the observed differences in collagen type I and type II fibrillar architecture and indicate the collagen type II cross-link organization, which is crucial for fibrillogenesis. Transmission electron microscopy data show the close relationship between lamprey and mammalian collagen fibrils, even though the respective larger scale tissue architecture differs.

Introduction

Type II collagen fibrils are major components of cartilage, intervertebral discs, and the vitreous humor of the eye and are vital to the normal development of bones and teeth. These fibrils are formed from highly ordered 67-nm staggered arrays of collagen molecules, producing the characteristic D-periodic structure of fibrillar collagens (supplemental Fig. 1). Because the length of the type II collagen molecule (∼300 nm) is not an integer multiple of 67 nm, the fifth molecular segment does not extend across the whole D-period. Therefore, a gap region is defined, where there are only four molecular segments and an overlap region where there are five segments. This arrangement is known as the Hodge-Petruska scheme (see packing/microfibril in supplemental Fig. 1) (1). Each D-period is composed of arrays of five fragments of five different collagen molecules, which between them contain the complete sequence of one collagen molecule. The type II collagen molecule has three identical α1-polypeptide chains of 1,060 amino acid residues each, with a large uninterrupted triple-helical region and relatively short, nonhelical telopeptides (19 amino acid residues in the N-telopeptide, and 27 amino acid residues in the C-telopeptide) (2) that do not possess the Gly-Xaa-Yaa-repeating primary structure found in the triple-helical region. The lengths of the α-chains are the same, but they are displaced from one another by one residue in the triple-helix to allow for its proper super coiling (2).

In vitro, the polymerization of collagen molecules into fibrils is an entropy-driven self-assembly process (3), whereas in living tissues, it also involves cellular and specific extracellular matrix interactions (4). Proteoglycans (PGs),³ such as decorin, fibromodulin, and biglycan, bind type II collagen fibrils to stabilize the larger fibril bundles (fibers composed of multiple fibrils). The diameter of the latter being regulated through interactions with the anionic glycosaminoglycan chains and protein cores of the PGs (5). However, the internal order and stability of fibrils are chiefly maintained by intermolecular cross-linking between collagen molecules. Intrafibrillar links are formed through covalent bonds between lysine and hydroxylysine (Hyl, a modified amino acid specific to collagen; single letter abbreviation U) through a Schiff base reaction (6, 7). These bonds are formed between Lys and Hyl residues within both the helical and nonhelical telopeptide domains of collagen molecules within each D-period. Thus, it is apparent that although the telopeptides do not have triple-helical structure, they are very important for collagen cross-linking and the assembly of collagen molecules into fibrils (8). Proper cross-link formation ensures the structural integrity and stability of fibrils and the tissue. Abnormal cross-linking, either enhanced or inhibited, leads to connective tissue diseases such as Ehlers-Danlos syndrome, Marfan syndrome, glaucoma, and skin fragility in normal aging (9). Furthermore, the proper conformation of telopeptides has been shown to be important for normal fibril structure (10–13). Accurately defining the structure of the D-periodic, Hodge-Petruska (1) arrangement of type II collagen, that is, gap and overlap ratio, telopeptide conformations, and cross- link locations, may facilitate our understanding of these conditions.

Structural studies of type II collagen packing has been complicated by the technical limitations its fibrillar nature presents. The insolubility and size of mature collagen molecules makes nuclear magnetic resonance (NMR) inviable, and only small fragments of the collagen molecule or synthetic, short collagen-like peptides have been crystallized and studied by (single-crystal) x-ray crystallography thus far (14). Furthermore, disruption of the fibril structure to study the constituent collagen molecules in isolation compromises the goal of understanding the molecular packing within fibrils and the native collagen structure within the D-period. Electron microscopy offers significant capabilities for studying fibrillar collagens but does not provide sufficient resolution for characterizing the telopeptide conformation, and sample preparation may disturb subtle, small features such as the telopeptides.

Type II collagen fibrils have an intrinsically crystalline D-periodic structure, even though their lateral arrangement is less well ordered (15, 16). This means that x-ray diffraction experiments on whole intact tissue samples can provide information concerning the axial organization of collagen. Such structural data have been obtained previously (15, 16) but were less detailed than that seen recently for collagen type I (obtained by our group) (17–19). In this study, we applied techniques developed during earlier investigations of type I collagen structure (17, 19–21), to better define the D-periodic structure of type II collagen fibrils in situ. We present here the axial D-periodic structure of native type II collagen in lamprey notochord, determined by multiple isomorphous replacement to 1.9 nm resolution.

EXPERIMENTAL PROCEDURES

Materials

Adult female lampreys were kindly provided by the Ludington Biological Station of the U.S. Fish and Wildlife Service and J. Ellen Marsden of the University of Vermont.

Fiber Diffraction

The notochords of adult lampreys were carefully harvested and dissected from their sheaths, nearby muscle, and other tissues and stored at pH 7.5 in phosphate-buffered saline (8 mm Na₂HPO₄, 2 mm KH₂PO₄, 137 mm NaCl) or Tris-buffered saline (25 mm Tris, 150 mm NaCl) for 1–6 h at 4 °C before each experiment. The notochord samples were cut longitudinally into pieces (1 × 2 × 25 mm) along their longitudinal axis and mounted in custom made sample holders that provided for 1–2% stretching and preservation of their hydrated state. Isomorphous staining was performed as described previously (17, 20). Briefly, samples were treated with iodine (KI) and platinum (PtCl₃) for 1 h, followed by 15 min of washing in a large excess of buffer. I⁻ ions covalently bind to tyrosine and in some cases histidine, whereas Pt³⁺ ions associate with the sulfur group of methionine.

X-ray diffraction studies were performed primarily using the small and wide angle fiber diffraction instruments on the BioCAT Beamline 18ID at the Advanced Photon Source, Argonne National Laboratory. Some preliminary studies used the wide angle instrument on the BioCARS beamline 14BMC at the Advanced Photon Source. The sample-to-detector distance was 2,000 mm for small-angle x-ray scattering (1–7 meridional orders) and 150 mm for wide-angle x-ray scattering and microfocus wide-angle x-ray scattering (5–37 meridional orders, medium to high resolution fiber crystallography), and the wavelength was 1.033 Å. Diffraction images were recorded on a custom made “Brandeis” detector (48 × 48 micron pixel size) (22–24)) for wide-angle x-ray scattering, and on an AVIEX PCCD 16080 detector (38.4 × 38.4 micron pixel size) (22) for small-angle x-ray scattering. The exposure time was limited to 1 s to safeguard against radiation/heat damage to the portion of the sample being shot.

Data Analysis

Diffraction images were processed using CCP13 Fiberfix (25) and Fit2D (26) software for background subtraction, and CCP13 software was used for measurements of meridional intensities (17). Measured intensities (supplemental Fig. 2) (squares of amplitudes), were used for calculation of difference Patterson maps and a one-dimensional electron density map of the collagen type II axial unit cell (the D-period) by reverse Fourier transformation (in-house software). The scaling of the experimental amplitudes and the solution of the phases were performed as described previously (17, 19, 20).

Lamprey Collagen Type II Amino Acid Sequence

The entire collagen type II sequence is known for human, rat, and mouse, and partial sequences are known for lamprey (the primary source for this study) (ExPASy Sequence Data Bank codes P02458, P05539, P28481, Q2I8Y0, and Q2I8X9). Unlike collagen type I, collagen type II is highly homologous across the species (27), and its full sequence may therefore be reasonably estimated by homologous comparison (N-terminal half from rat, mouse, and human; C-terminal half from the lamprey sequence, Q2I8X9) (28).

Model Electron Density Map

The model electron density map was calculated (17, 20) using the residue level scattering factors of Hulmes et al. (21), the amino acid sequence described previously, the Hodge-Petruska scheme, and assuming a regular unit height spacing of the molecular helix (residue-to-residue distance). The telopeptides were initially modeled as elongated structures extending straight from the ends of the triple-helical region. After comparison with the difference Patterson and Fourier maps, the telopeptides were modeled as folded structures, similar to that of type I collagen C-telopeptide (although the N-telopeptide is required a double-folded conformation to fit the experimental data; see “Results”).

Determination of the Gap/Overlap Ratio

In this study, we took the approach that the gap/overlap ratio is determined by the extents of the telopeptides (17) and along the lines of the height/depth ratio approach of Bradshaw et al. (20). Hence, the overlap start and end points were determined by considering the following points. 1) The position of the iodine peaks for the telopeptides that give the location of the telopeptide ends: the distance from the telopeptide termini to the fold can be estimated from the number of known amino acids spanning the distance. 2) The point of the overlap region above average height versus gap region below average depth: the low resolution of the study means that the influence of the first few orders (particularly the first) produces a tail effect that makes the overlap appear larger than it is. This was partly countered through the calculation of the native electron density map with the amplitude of the first order reduced ten times (supplemental Fig. 3). 3) The model parameters that fit the difference Fourier heavy atom positions and the difference Patterson peak separation values and that best fit the native electron density, provided there is compliance with points 1 and 2. 4) Examination of the transmission electron microscopy (TEM) images of type II fibrils to confirm that the values determined from points 1–3 were reasonable: we hold that the x-ray data are the most accurate approach due to the use of native samples and better resolution and due to the multiple lines of approach used to reach the gap/overlap estimate.

TEM

Collagen type II fibrils were studied at the University of Chicago Electron Microscopy Center. Images were examined under 300 kV using an FEI Tecnai F30 microscope with a Gatan CCD digital micrograph (4k × 4k) as detector. Sample preparation was the same as for the diffraction experiments, followed by fixation, embedding, and sectioning into 90-nm sections on a Reichert-Jung Ultracut E microtome. Sections were stained with uranyl acetate and lead citrate to enhance the image contrast. We found that suspensions greatly enhanced the clarity of the human type II collagen fibrils. For these preparations, cartilaginous tissues were homogenized in Tris-buffered saline on ice for 1 min to release collagen fibrils and other matrix components. These samples were then placed on grids and stained with uranyl acetate.

The D-period from the x-ray structure was confirmed against the TEM data by sampling the gap/overlap ratio in the clearest TEM image (i.e. such as that shown in supplemental Fig. 4F) >10 unit cells. The S.D. was 0.0049, and the percentage error (as measured by the mean discrepancy between the sum of gap and overlap measurements versus the single D-band measurement was <0.05%), arriving at a value of 0.419:0.579D for the gap/overlap. The scale bars showing the gap/overlap and D-band extents were then superimposed on the other TEM images (see “Results”).

Telopeptide Structure Prediction and Minimization

The extent of folding or compression required of the telopeptides to fit the native electron density map was determined from the difference Patterson and Fourier maps. In addition, structure prediction calculations were performed for the conformation of the telopeptides. Both N and C termini were examined using the Chou-Fasman (29) and self-optimized prediction method with alignment (30) structure prediction methods, and the results were compared with the diffraction and electron microscopy data (D-period gap/overlap ratio, position of heavy atom peaks). The results were found to be in agreement; therefore, atomic coordinates of the telopeptides for one α-chain were generated de novo from the amino acid sequence and energy-minimized using NAMD (31) as a further check of the hypothesis that the telopeptides are folded.

Molecular Model and Surface Rendering

The molecular surfaces were calculated using “spock” (32–34) with the default options, except the surface polygon parameter, which was set to 120 for improved surface definition. The display option was set to “mesh” to allow the underlying bonds of the model to be seen. The calculated mesh is not an electron density map but simply a rendering of the molecular surface.

RESULTS

X-ray Diffraction Data

A medium wide angle diffraction pattern from native (unstained) type II collagen is shown in Fig. 1, with the central section containing the meridional series indicated. Diffraction images from native lamprey notochord and two heavy-atom derivatives were recorded, showing meridional reflections up to the 37th order or ∼18 Å. (Integrated and scaled intensities of orders 1–35 for native samples and derivatives are presented in supplemental Fig. 2.)

FIGURE 1.

Medium-wide angle x-ray diffraction pattern of collagen type II fibrils from lamprey notochord with 15–20 Å resolution of meridional reflections series.

Location of Heavy Atom-binding Sites and Difference Fourier and Patterson Maps

The amplitudes of the native samples were subtracted from the amplitudes of the derivative samples, and the resulting differences for each order, together with the calculated phases, were used in a reverse Fourier transformation. This operation produces an electron density map, whose peaks correspond to the positions of heavy atoms within the native-like unit cell (the D-period) of the collagen type II fibril. The iodine derivative difference Fourier map is shown in Fig. 2. Although the majority of these heavy atom-binding positions appear to correspond to the expected collagen amino acid residue labeling positions, some appear to have bound isomorphously at an unexpected location (no liable amino acid residues from the collagen sequence), and, given the corresponding native electron density, this may indicate the presence of the small leucine-rich repeat protein, biglycan, or a related small leucine-rich repeat protein at this location. It is known that small leucine-rich repeat proteins are attached to the surface of type II collagen fibrils at ∼0.7–0.8D (see “Discussion”).

FIGURE 2.

A, experimental and model electron density maps of native type II collagen D-periodic axial structure. B, difference Fourier map. Positive peaks show the attachment positions of iodine heavy atoms within the D-period; 0% is toward the beginning (N-terminal end, beginning of overlap) of the D-period, and 100% is toward the end (end of gap region). The positions of the iodine-labile residues are shown in black for the refined model. Red shows the positions of tyrosine residues were the telopeptides to adopt an extended, linear conformation. C, difference Patterson function for iodine derivative (black), and model Patterson function (red) based on the iodine labeling positions determined from A. D, difference Patterson function as for C and model Paterson function with the telopeptides adopting an extended and linear conformation. The scaled amplitudes (supplemental Fig. 2) were also used to calculate difference Patterson maps for the iodine derivative). The peak position in a difference Patterson plot corresponds to the distance between two points within the unit cell (plotted from 0 to 0.5-unit cell length in fractional coordinates). The peak height is a function of the relative electron density at the termini of each distance. In the present case, a peak represents the distance between heavy atoms that have labeled the unit cell contents isomorphously.

The presence of tyrosine residues in the telopeptides and telopeptide region, allows the detection of the ends of the telopeptides (difference Fourier, Fig. 2B; and Patterson maps, Fig. 2C and 3D). From these data, it was clear that the telopeptides are highly contracted or folded (Figs. 3 and 4), a possibility supported by the native electron density map (see below and Fig. 2A).

FIGURE 3.

A modeling of the N-telopeptide of collagen type II is shown in its extended conformation (A), possible cross-linking pattern of hexagonally packed type II collagen monomers with the N-telopeptide carrying monomer 1 marked yellow (B), and folded telopeptide conformation (C) supported by hydrogen bonds. Lys residue is shown in blue. The Hyl-9 shifts toward the triple helix (to the right) making the possibility of forming a covalent bond with Lys-949 of monomer 5 more favorable (D, two α-peptides are shown extended (blue), one is shown folded (green)); the cross-link is indicated by a light-red arrow. Black bars alongside blue bars in d show the large axial distance cross-links would need to reach if the telopeptide is extended (blue, see also A). Red bar shows short axial distance spanned by folded telopeptide (green, see also C). Monomers are ∼1.3 nm distant from each other in lateral space (B). Hydroxylysine is defined as single letter code U. Mesh displayed around model shown in A and B is a molecular surface rendering to show the model outline (see “Experimental Procedures”) and not electron density.

FIGURE 4.

Shown is a modeling of the C-telopeptide of collagen type II in extended (A) and folded (B) conformations, supported by hydrogen bonds; Hyl is shown in blue. The possible cross-linking pattern of hexagonally packed type II collagen monomers is shown with the C-telopeptide carrying monomer 5 marked in yellow (C). Lys-1050 shifts toward the triple-helix (left) making its covalent bonding with Hyl-106 of monomer 1 more favorable (D, two α-peptides are shown extended, blue; one is shown folded, green); the cross-link is indicated by a light-red arrow. Black bars alongside blue bars in D show the large axial distance cross-links would need to reach if telopeptide is extended (blue, see also A). Red bar shows short axial distance spanned by the folded telopeptide (green, see also E). Monomers are ∼1.3 nm distant from each other in lateral space (B). Hydroxylysine is defined as a single letter code (U). A and C is a molecular surface rendering to show the model outline (see “Experimental Procedures”) and not electron density.

Collagen Type II One-dimensional Electron Density Profile

The native electron density map was calculated using reverse Fourier transformation of the experimentally derived structure factors (Fig. 2). This map represents the sum of the five collagen molecular segments in lateral projection (perpendicular to the fiber axis) in the Hodge-Petruska scheme (supplemental Fig. 1) and is a direct function of the axial D-periodic structure of the type II collagen fibril. This map allows deductions to be made concerning the conformation of the collagen molecules, the collagen fibril parameters, and the telopeptide conformations (in conjunction with the heavy atom labeling data).

From the one-dimensional electron density map, the overlap is measured to be 0.42D, which is shorter than that of the collagen type I fibril, and the gap is ∼0.58D (Fig. 2). This overlap to gap ratio of 0.42:0.58 is confirmed by the TEM data (Fig. 5 and supplemental Fig. 4). Compared with type I collagen, these parameters might appear confusing because the type II molecule is four amino acid residues longer than the type I molecule, which would suggest that the overlap to gap ratio should be closer to 1:1 than the type I ratio of 0.46:0.54 (17, 35). After considering the difference Patterson and Fourier data, it is reasonable to assume that the shorter collagen type II overlap region is a consequence of the telopeptides adopting a conformation that makes them shorter in linear projection (see supplemental Fig. 3) (17). The telopeptides would have to be either highly compressed (“contracted”) or tightly turned to fit the measured gap/overlap ratio and difference Fourier and Patterson data. The compressed model does not seem plausible due to peptide bond constraints; compressed telopeptides would require a height translation of ∼0.8 Å to fit within the 0.42D overlap region.

FIGURE 5.

Cross-species comparison of TEM images of collagen type I (left) and type II (right) fibrils. Human quadriceps tendon (A), rat tail tendon (B), lamprey notochord (C), bovine articular cartilage (D), and human articular cartilage (E).

One-dimensional Model of Collagen Type II D-periodic Structure and Structure Prediction

To aid the analysis and interpretation of the one-dimensional electron density map, an electron density model of one D-period was calculated from the collagen type II amino acid sequence and the individual whole amino acid residue structure factors (Fig. 2) (21). In the model, the collagen molecules are staggered by 234 residues (∼67 nm), and the individual peptide α-chains are staggered by one amino acid residue within each triple-helix segment (17, 19). The initial model assumed a conformation of the telopeptides where they were straight and relaxed (Figs. 3 and 4 and supplemental Fig. 3). This model provided parameters for the D-period that disagreed with the one-dimensional map obtained from the experimental data (supplemental Fig. 3 and Fig. 2). The model overlap region (0.53D) was longer than the experimentally determined value (0.42D), and the gap region was shorter (theoretical, 0.47D; experimental, 0.58D).

Assuming that the telopeptides may form turns, unstructured coils, and relaxed strands (17, 21, 36), their amino acid sequences were examined, and possible conformations were predicted using the Chou-Fasman and self-optimized prediction method with alignment methods (supplemental Tables 1 and 2). Both these methods and the difference Fourier and Patterson data suggested tight turns in the N- and C-telopeptides, which would change the gap/overlap ratio in the model to better fit the experimentally determined values.

Telopeptide Conformations

According to the structure predictions (Fig. 3 and supplemental Table 1), the N-telopeptide can form two turns, one at position Gly-11 to Gly-12 and another at position Gly-4 to Gly-5. The telopeptide section between the triple-helix and the first turn has the greatest propensity to form β-strands. The peptide chains of the triple-helix itself are considered to be in almost a relaxed β-strand conformation, which is close to the polyproline II helical conformation. Given that the telopeptide is directly connected to the triple-helical regions, the N-telopeptide may have some “structural memory” (17, 37) of the polyproline II helix, despite not having a triple-helical conformation until it makes a 180° turn. A second turn is predicted after a straight section. The fragment between the two turns and the rest of the telopeptide after the second turn has a greater propensity to form a “random coil” (or turn), forming a high electron density area relative to the rest of the triple-helix, between amino acid positions 11 and 15 (Fig. 3). This N-telopeptide conformation makes the overlap region shorter and also brings the Hyl-9 residue closer for cross-linking to the Lys-949 in monomer five (a close molecular packing neighbor in type I collagen). If the N-telopeptide was simply straight and relaxed, Hyl-9 would be too distant from Lys-949 and the alternative cross-linking partners Lys-238 (monomer 2), Lys-703 (monomer 4), and Lys-937 (monomer 5) are even more distant. The average spacing between collagen type II monomers in the fibril is ∼1.3 nm, based on equatorial diffraction data (data not shown) and collagen type I parameters (19). There are eight C–C and two C–N bonds in the lysyl-hydroxylysine cross-link between the two peptide backbones. The C–C bond is 1.54 Å, and the C–N bond is 1.47 Å (38). Hence, the length of the whole lysyl-hydroxylysine link from peptide backbone to peptide backbone is maybe ∼1.5 nm at a maximum. Therefore, both the lysine and the hydroxylysine have to be in approximately the same lateral plane or no more than 0.8 nm apart in the axial direction (within three amino acid residue positions) to form the cross-link.

The C-telopeptide shows a propensity to form one sharp turn at the Gly-13 position of the telopeptide (Gly-1046 of the full molecular sequence) (supplemental Table 2 and Fig. 4). The fragment of the telopeptide between the turn and triple-helical region has a predicted structure close to a relaxed helix due to methionine clusters (supplemental Table 2), disturbed by glycine residues. The rest of the C terminus after the turn is more likely to be a random coil (Fig. 4 and supplemental Table 2). This folded conformation causes additional shortening of the overlap and extension of the gap region in the electron density model. It also moves Lys-1050 by approximately eight amino acid positions, moving it much closer to Hyl-106 for covalent cross-linking (Fig. 4) (see previous discussion of Lys-Hyl cross-link length).

The structures of the collagen type II telopeptides, as determined from the experimental data (difference Patterson and Fourier maps) and supported by the predicted conformations, were incorporated into the refined one-dimensional model of the D-periodic molecular packing (Fig. 2 and supplemental Fig. 3a). This model electron density map provided a much better approximation to the experimental data. Structure factors were calculated from the refined model and used to generate a model electron density map at the same resolution as the experimental map (35 meridional orders, Fig. 2). This allowed the simple R-factor (19) to be calculated, as an estimate of error between the two maps. The calculated R-factor was 0.19.

TEM Data: Mammalian and Lamprey Fibrils Are Homologues but Tissue Architecture Differs

Micrographs of collagen type I (rat tail tendon and human quadriceps tendon) and type II fibers (lamprey notochord, human articular cartilage, and bovine articular cartilage) were examined (Fig. 5 and supplemental Fig. 4). The collagen fibers in these images show the typical pattern for fixed collagenous tissues: black and white bands with ∼64 nm periodicity (D-period shortened during fixation and embedding (15, 39)). These images also confirm previous observations that collagen type II fibrils from lamprey notochord do not show any detectable differences from those of mammalian tissues (15, 16). The lamprey notochord fibrils have similar diameters of ∼35 nm, and the typical positive staining pattern is observed for mammalian fibrils (Fig. 5 and supplemental Fig. 4).

Despite the similarities between fibril morphology, the architecture of collagen fibril arrangement differs between mammalian and lamprey tissues. There are also some differences in cellular and PG content in these tissues. Lamprey notochord has a very specific cell distribution, and most cells were mechanically removed during sample preparation, whereas cartilage cells are embedded in the collagen meshwork and can be seen throughout the whole tissue (40, 41). Lamprey notochord principally contains the PGs, biglycan types I and II, which are similar in sequence to the bovine and human biglycans (comparison of lamprey sequence fragments Q9DE00 and Q9DDZ9 with human P21810 and bovine P21809, (42)), and hence are most likely to be structurally related to decorin and fibromodulin. In contrast, mammalian cartilage contains several different types of PGs, glycoproteins, and other types of collagen present (27, 42). The specific composition of the extracellular matrix plays a crucial role in the formation of collagen fibrils and the organization of the fibrillar meshwork. This influence appears to give rise to the complexity of the articular cartilage matrix, as observed by TEM (supplemental Fig. 4d), in comparison with the more “primitive” architecture of notochord tissue (supplemental Fig. 4b).

DISCUSSION

TEM images of collagen type I and II fibers from the same tissues used in x-ray diffraction experiments provided complementary information that correlated well with the results of the x-ray diffraction experiments and with the derived one-dimensional model of the D-period structure of collagen type II (supplemental Fig. 3 and Figs. 2 and 5). The experimental one-dimensional electron density map (Fig. 2) indicated that a previous observation reported for type I collagen (19, 20, 43) also applies to type II collagen: there are 234 amino acid residues in the molecular segments within the Hodge-Petruska D-periodic fibril-packing structure, which is 67-nm-long in hydrated samples, and 64–65-nm-long in dehydrated, fixed samples (Fig. 5). However, the overlap is 0.42D ± 0.03 (28.14 nm or 98-amino acid residues long), and the gap is 0.58D (38.86 nm or 136 amino acid residues) in type II collagen, whereas in type I collagen, the overlap is 0.46D and gap is 0.54D. The overlap/gap ratio for type II collagen has previously been suggested to be closer to 0.4D/0.6D than type I (39, 40, 44, 45) and is also confirmed from the TEM data (Fig. 5 and supplemental Fig. 4). This indicates that although there are some obvious similarities, collagen types I and II differ significantly at the level of molecular packing. These differences may arise due to the specific telopeptide conformation of each collagen type.

Experimental and Model Electron Density Maps of the Type II Collagen D-period

Fig. 2 shows both the one-dimensional map of the collagen type II D-periodic structure (blue) and the model constructed from the amino acid sequence and residue-scattering factors (green). These maps have similar features, although some peaks are not common to both. The initial theoretical model and experimental peak density positions differ significantly, indicating that the real molecular conformation is not simply linear, particularly in the configuration of the telopeptides (Figs. 2–4) and supplemental Fig. 3). A major peak in the gap region of the electron density map for native type II collagen that is not present in the model (see below) may correspond to dermatan sulfate PG binding sites, which, in the case of lamprey notochord, is biglycan or bigylcan-like protein; see above (40, 46). This is in agreement with the principal attachment sites for decorin in the e/d-band of type I collagen, as determined by TEM (5, 35, 47). Other peaks on the map may correspond to electron dense side chain residues of the collagen molecules or other extracellular matrix molecules ligated to the surface of the collagen type II fibril, and/or differences in the amino acid residue-to-residue spacing. The latter may arise in part from differences in helical symmetry (e.g. 10/3 versus 7/2 symmetry). Other significant differences between the experimental and model maps may be explained by the presence of other PGs and fibronectin and aggrecan molecules that may be associated with collagen fibers in lamprey notochord. Some of these peaks may be present in the type I collagen electron density map (17) but, if present, are much more subtle. Their greater prominence in the type II collagen one-dimensional map is due to the fact that type II fibrils are much thinner than type I fibrils (∼35 nm and 100–200 nm, respectively, although larger fibers, i.e. bundles of fibrils, may also be present), thereby increasing the effective occupancy and electron density contrast of PG molecules attached to the outside of the fibril.

Informative Differences between Model and Experimental Electron Density Maps

The model of the collagen type II D-periodic structure has several peaks in both the gap and overlap regions that correspond to clusters of amino acids with electron dense side chains, and in general, these peaks are also present in the experimental electron density map. However, there are two major differences between the experimental electron density map and the model: a trough in the map at 0.275D and a peak at 0.8D that are not readily explained by the collagen amino acid sequence or electron scattering density alone. As already noted, the data for the experimental electron density map were obtained from native tissues, which contain not only collagen, but also other extracellular matrix molecules. Biglycan may bind to several sites on the collagen monomer, but the electron density map indicates that one of these attachment sites on fibrillar type II collagen at 0.8D (Gly-905 to Pro-915, monomer four) is occupied in a highly ordered fashion. This is in agreement with the principal attachment sites for the biglycan homologue, decorin, in the e- and d-staining bands of type I collagen (48, 49). The presence of the model peak, in contrast to the experimental electron density map trough at 0.275D may suggest a significant and common molecular inflection among the five molecules in the overlap region, before and after this position. This would mean that the molecular sections passing through 0.275D would be straight but tilted on either side of this position, which would account for the greater electron density on either side of the 0.275D position, but the lower relative density at 0.275D. This type of inflection is also present in the type I collagen microfibril (19). Neither of these two differences between the experimental map or model affect the measurement of the gap-overlap ratio, however.

Telopeptide Conformations

The conformations of the telopeptides are important for collagen fibril assembly due to the covalent Lys-Hyl cross-links formed between the collagen molecules within these nonhelical domains (50, 51). Lysine or hydroxylysine is deaminated by lysyl oxidase, and a covalent link is formed spontaneously; this may occur at two places in the collagen type II molecule (7) (Figs. 3 and 4). The distribution of Lys and Hyl residues within the collagen molecule (Figs. 3 and 4) determines the pattern of cross-links and therefore the molecular stagger within the fibril. The N- and C-telopeptides are involved in cross-linking, and their conformations affect the axial position of the cross-linking residues, Hyl-9 and Lys-1050, which bind to Lys-949 and Hyl-106 in the triple-helical region, respectively, thereby influencing cross-link formation and therefore fibrillogenesis.

Previous investigations of the structure of fibrillar collagen indicated that the telopeptides form contracted and folded structures (9, 17, 21, 39). Electron microscopy studies of human collagen type II fibers suggested that the folding of the telopeptides is similar to that proposed here (39), but the 20 nm resolution of those studies was not sufficient to provide conclusive evidence (the stretched N- and C-telopeptides are 4.5 nm and 5.4 nm in length, respectively). Similarly, studies with synthetic or ex vivo collagen N- and C-telopeptides indicated an axial compression of the fragment(s), with possible folding (52–54). However, the specific telopeptide conformations were not determined. Subsequently, in x-ray diffraction investigations of native rat tail tendon collagen type I in situ, we showed the one-dimensional packing structure of the collagen molecules in the D-period. The conformation of the C-telopeptides showed a sharp turn around residues Pro-13 and Gln-14 and the specific locations of the lysine-hydroxylysine cross-links (17). These results were later confirmed when the full three-dimensional, higher resolution structure was determined (19). In this study, the conformations of collagen type II telopeptides were inferred from the native and difference Fourier density maps and predicted by the Chou-Fasman method and confirmed by SOPMA (supplemental Tables 1 and 2). Both telopeptides have a propensity to form a sharp turn in the middle of their sequences (Figs. 3 and 4), which make the whole collagen molecule 2% (5.7 nm) shorter and change the gap/overlap ratio of the model. These predictions agreed well with the observation that the overlap region must be shorter than linear telopeptide structures would allow and also with the peak distributions in the difference Patterson and Fourier maps because the heavy atom binding locations included positions within the telopeptides themselves.

Significantly, the N-telopeptide structure of type II collagen appears to differ from that of type I collagen, which does not have any sharp turns (17, 19). In contrast, the proposed collagen type II C-telopeptide structure is much closer to that observed for the collagen type I C-telopeptide and to the prediction of Ortolani et al. (39). It has tyrosine (Tyr-1058) and methionine (Met-1055) residues at the end that have been moved deeper into the overlap region, closer to the N terminus. This conformation is also supported by the difference Patterson and Fourier maps.

Implications of Telopeptide Structure for Cross-linking

The derived conformation of the collagen type II telopeptides suggests a specific, well ordered cross-linking pattern in the fibril that is different from that of collagen type I. It may also be the molecular basis of the differences in fibril diameter and fibril bundle organization between two of the most prominent fibrillar collagens, types I and II. The type II collagen molecule has more candidate residues for cross-linking than the type I molecule. At the C terminus, there are three potential cross-link forming Lys residues, due to the homotypic chain composition of type II molecules. In contrast, cross-linking at the C terminus of the type I molecule appears to occur primarily through its two α1-chains. Similarly, at the N terminus of collagen type II, Hyl-9 may form three covalent bonds with Lys residues on monomer five, whereas the potential for N terminus cross-linking in collagen type I is diminished in comparison (19). Therefore, the greater potential for cross-linking in collagen type II may lead to greater numbers of supporting covalent bonds within and possibly even between the fibrils (within fibril bundles) than in collagen type I. Thus, the type II fibril is likely to be more stable than the collagen type I fibril due in part to a higher cross-link content. The greater potential for interfibrillar cross-linking in type II collagen may also result in a more complex and possibly more stable network-like tissue organization. At the same time, because the type II N-telopeptide is significantly bulkier than the type I collagen, it may also function in sterically inhibiting the formation of type II fibrils as large as those observed for type I. This, in turn, would lead to more loosely organized fibril bundles (or thick fibrils) composed of these small and relatively thin fibrils (55).