The Collagen Fibril: The Almost Crystalline Structure

JOURNAL OF STRUCTURAL BIOLOGY ARTICLE NO. SB983976

122, 111–118 (1998)

The Collagen Fibril: The Almost Crystalline Structure Darwin J. Prockop and Andrzej Fertala Center for Gene Therapy, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19102 Received: January 22, 1997, and in revised form February 16, 1998

nm in diameter seen in collagen fibrils with growth and development (Craig and Parry, 1981); (f ) electron microscopic evidence that many collagen fibrils contain a substructure of microfibrils in which monomers are in a 1D stagger and folded into a pentameric helix (Piez and Trus, 1981); (g) informative X-ray diffraction patterns can be obtained from rat tail tendon and the sheath of the lamprey notochord (Eikenberry et al., 1984), but not from many other collagen fibrils (Brodsky and Eikenberry, 1982); and (h) in the best X-ray diffraction patterns obtained from rat tail tendon, the peaks used to define the crystal structure account for no more than 5 to 10% of the data and the rest of the information is lost in background scatter (Fratzl et al., 1993). It is likely that there are relatively simple, and perhaps even trivial, explanations for some of these observations. Here, we would like to focus on just two features of collagen fibrils that remain difficult to reconcile with the structure defined by X-ray diffraction: the roundness of fibrils and their growth from paraboloidal tips.

The structure of collagen fibrils has intrigued many investigators over the years. A crystal structure has been available for some time, but the crystal structure has been difficult to reconcile with other observations about collagen fibrils such as their roundness and their growth from paraboloidal tips. Several alternative models recently have been suggested, but none of them fully account for all the data. One recent approach to solving the fibrillar structure is to define specific binding sites on the collagen monomer that direct self-assembly of monomers into fibrils. r 1998 Academic Press Key Words: collagen fibrils; fibril structure; fibril models; protein-binding sites.

THE PARADOX

The structure of the collagen fibril presents a paradox. Most proteins are noncrystalline in vivo but crystallization solves the structure. In contrast, the collagen fibrils seen in vivo are seemingly crystalline. However, the complete structure of the collagen fibrils still eludes us. Thanks to the work of a number of superb scientists over several decades, particularly the contributions of Miller, Fraser, and their associates (Hulmes and Miller, 1979; Fraser et al., 1987; Wess et al., 1995), a crystal structure based on quasi-hexagonal packing has been developed from X-ray diffraction of collagen fibrils. However, the crystal structure presents with a challenge because it is difficult to reconcile with several observations about collagen fibrils (see Chapman, 1984; Prockop and Hulmes, 1994). These observations include: (a) the roundness in cross-section of all fibrils that are seen in vivo, including fibrils that are as small as 40 nm; (b) the growth of collagen fibrils from highly symmetrical and paraboloidal tips (Kadler et al., 1990b); (c) the parallel axial plates of hydroxyapatite seen in mineralized turkey tendon and in some bones (Traub et al., 1989; Landis et al., 1996); (d) a radial lattice spacing seen in cross-sections of rat tail tendon (Hulmes et al., 1985); (e) the incremental increases of about 8

THE PROBLEM OF ROUNDNESS

The problem of accounting for the roundness of collagen fibrils was first pointed out by Ramachandran (1967) 30 years ago who pointed out that uniform hexagonal packing of monomers must give rise to hexagonal fibrils (Fig. 1). Therefore, he suggested a cylindrical structure or a modified cylindrical structure in which the monomers were in a spiral orientation. Almost two decades later, Galloway (1985) addressed the same problem and presented drawings of a model in which the monomers were in cylindrical arrays (Figs. 2 and 3). In the Galloway model, the outer layers become lattice-like and allow packing of some monomers as quasi-hexagonal unit cells. However, our own examination of the model indicated that there are large discontinuities between the unit cells in the outer layers of the model (D. Silver, J. Miller, and D.J. Prockop, unpublished observations). 111

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THE GROWTH OF FIBRILS FROM POINTED TIPS

Our own interest in the structure of collagen fibrils initially arose from studies on collagen biosynthesis (see Prockop and Kivirikko, 1984). Subsequently, we pursued the problem in trying to understand how mutations that altered the amino acid sequence of the protein produced devastating diseases such as osteogenesis imperfecta (see Prockop and Kivirikko, 1995; Kuivaniemi et al., 1997). We focused our efforts on developing a pure system for assembly of collagen monomers into fibrils in vitro. Self-assembly of collagen monomers into fibrils in vitro was examined extensively by a large number of previous investigators beginning over four decades ago (Nageotte, 1927; Gross and Kirk, 1958; Wood, 1960; Williams et al., 1978; Veis and Payne, 1988; Trelstad and Hayashi, 1979; Silver et al., 1979; Holmes et al., 1986; Na, 1989). Essentially all the experiments, however, were carried out with collagen monomers extracted from tissues with cold acidic solutions, and fibril assembly was initiated by both warming and neutralizing the solutions. Assembly of monomers into fibrils was observed, and a number of important observations were made. However, it was apparent that the fibrils formed from acid-extracted collagens did not form round and tightly packed fibrils under even optimal conditions. Under physiological conditions they formed gels instead of fibrils. In retrospect, it is apparent that the difficulties encountered with self-assembly of acid-extracted collagens were explained by changes in conformation of the globular telopeptides of 11 to 26 amino acids each that are found at the ends of the three chains of the monomers. In our own experiments (Miyahara et al., 1984;

FIG. 1. Possible modes of packing of collagen monomers into fibrils as suggested by Ramachandran (1966).

FIG. 2. Super-coiled helical model for collagen fibrils as proposed by Galloway et al. (1985). The molecules are arranged in concentric cylindrical layers.

Kadler et al., 1987, 1990a,b; Romanic et al., 1992), we first developed procedures for isolating adequate amounts of type I procollagen, the soluble precursor of type I collagen. We then developed procedures for preparing relatively homogeneous preparations of the two enzymes, procollagen N-proteinase and procollagen C-proteinase, that process the procollagen to collagen (Hojima et al., 1985, 1989). We used the N-proteinase to cleave the N-propeptides and isolated the intermediate that is known as pCcollagen and that remains soluble under physiological conditions at about 1 mg/ml. The final system for fibril assembly consisted of pCcollagen and procollagen C-proteinase in a physiological buffer system. Cleavage of pCcollagen with C-proteinase in the system generated collagen monomers that had a solubility of less than 1 µg/ml and that spontaneously assembled into tightly packed collagen fibrils under physiological conditions. In agreement with previous observations, the assembly of monomers into fibrils was entropy driven and demonstrated features typical of a crystallization process (Kadler et al., 1987). Specifically, cleavage of the pCcollagen produced a supersaturated solution of monomers that then assembled into fibrils with a lag phase, a propagation phase and finally a critical concentration at equilibrium that was independent of the amount of protein in the solid phase. The diameter of the fibrils assembled varied with temperature so that at about 30°C, thick and

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FIG. 3. Transverse section through the Galloway (1985) cylindrical model for collagen fibril. Each successive sheet contains a further five molecules. As pointed out by Galloway (1985), the molecules are equivalently arranged within each sheet, but molecules are not arranged equivalently with respect to molecules in neighboring sheets. In the outer layers, the arrangement of the molecules becomes lattice-like and ‘‘reminiscent’’ of the unit cell suggested by Hulmes and Miller (1979). However, large gaps between the unit cells appear as successive layers are added to the model (D. Silver, J. Miller, and D. J. Prockop, unpublished observation).

seemingly crystalline fibrils were formed. At 37°C, the fibrils were of about the same diameter and flexibility as type I collagen fibrils seen in tissues (Kadler et al., 1990a). The relatively large fibrils formed at about 30°C facilitated a critical observation by Kadler when he was still a member of our laboratory group (Kadler et al., 1990b). Using darkfield light microscopy, Kadler saw that the first structures formed had a blunt end and a pointed end. With time, the fibrils grew by extension of the pointed tips without any apparent change in the diameter of the initial structure. He also observed that the monomers in the tips were oriented so that all the N-termini were directed toward the tip itself. After he returned to the University of Manchester, Kadler, together with Holmes and Chapman (Holmes et al., 1992), used STEM analysis to demonstrate that the pointed tips showed a linear increase

in mass with length. Therefore, the tips were paraboloids (Fig. 4). They also demonstrated that the initial pointed tips that formed generally had a finer taper than the secondary tips that subsequently formed at the opposite end of the same fibrils. A MODEL FOR GROWTH FROM POINTED TIPS

The paraboloidal shape of the collagen fibrils (Fig. 5) prompted considerable speculation as to how the symmetry of the tips was maintained as the fibrils grew. In our own thinking, we were struck by the analogy to other structures in nature that assemble symmetrically by use of a helical packing mode. In particular, we were struck by the fact that many crystals that grow in nature assemble from solutions that are not highly supersaturated, and they assemble by a process of face dislocation that produces

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FIG. 4. STEM analyses of the pointed fibrils of collagen fibrils assembled de novo by enzymic cleavage of type I pCcollagen (Holmes et al., 1992). (A) Darkfield STEM image of a more pointed a-tip that is the first tip to appear on a growing fibril. (B) Display of a scan from the boxed area in A demonstrating the distribution of mass along the fibril. (C) Plot of mass of the fibril as a function of distance from the end of an a-tip. (D) Darkfield STEM image of a b-tip of a fibril that forms later at the opposite end from the a-tip. (E) Scan from the boxed area in D. (F) Plot of mass of the function of distance from the end of the b-tip.

spiral growth (Boistelli and Astler, 1988). In addition, we are struck by the smooth and highly symmetrical shape of the most highly tapered tips (Fig. 5). Intuitively, it seemed difficult to explain how the highly symmetrical shape of the tips was maintained as the tips grew unless the growth was spiral or helical. We, therefore, developed a model for tip growth based on a helical structure (Silver et al., 1992). The model began with the assumption that the monomers first assembled in 1D staggers (or 3D overlaps). As indicated by many previous investigators (see Prockop and Hulmes, 1994), the 1D stagger monomers can readily be seen to assemble a pentameric microfibrillar structure. Once the helical structure is closed by addition of a fifth monomer, the microfibril was readily extended by addition of further monomers in 1D staggers. In trying to generate a paraboloidal tip, it was necessary to begin another

layer of monomers on the microfibrillar core. Here we arbitrarily chose addition of monomers in a 4D stagger (0.4D period overlap) at more or less random sites on the microfibrillar core. After the layer was initiated by addition of a monomer in a 4D stagger, the layer was extended by monomers adding in 3D overlaps. Computer simulation of the model made it possible to generates symmetrical and paraboloidal tip with a large number of equivalent binding sites for new addition of monomers through 3D staggers. Assuming that monomers were added at about the same rate to each of the binding sites, the tip readily grew symmetrically and the initial the paraboloidal morphology of the tip was maintained (Fig. 6). The growth of the tip was, therefore, a tiling process. One critical feature of the model was that growth of the fibrils was dependent on just two specific binding steps. The slope of the tip was readily defined by

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assuming that the two binding steps had different first-order binding constants. A high-affinity rate constant (k1 ) controlled the rate of addition of monomers in 3D overlaps. The k1 rate constant, therefore, controlled the longitudinal extension of the fibril by addition of monomers to equivalent binding sites in the helical tip. The second and lower rate constant (k2 ) controlled the 0.4D overlap step that initiated each new layer and controlled axial growth. ALTERNATIVE MODELS

After our model was proposed, Parkinson et al. (1995) proposed a drastically different model to

FIG. 6. Computer simulation of the helical model proposed by Silver et al. (1992) and a similar spiral model proposed by Hulmes (see Hulmes et al., 1995). Side views of the tips are shown with monomers represented as relatively thick rectangles. The top three figures are versions of the Silver et al. (1992) model. The taper of the tips was varied by varying the ratio of the highaffinity rate constant (k1 ) and the second and lower rate constant (k2 ). The spiral model (bottom frame) has an indeterminate core from which the spirals grow by equivalent additions of monomers. The taper of the tip is determined by the number of separate start sites for spirals that form on the core.

FIG. 5. (Top) Schematic representation of the growth of fibrils from pointed tips. The vertical line indicates the site of apparent change in polarity of monomers in the fibrils. Reprinted from Silver et al. (1992) with permission. (Middle) Perspective view of a-tip, showing the parabolic contours and changing slope expressed in terms of molecular diameters (md) per D period. (Bottom) Perspective view of b-tip, showing parabolic contours and changing slope. From Prockop and Hulmes (1994).

explain growth of collagen fibrils from paraboloidal tips. Parkinson et al. (1995) based their model on the principle of limited diffusion that is seen in electrochemical depositions or perhaps the formation of snowflakes. They assumed no specific binding interactions among monomers except for the tendency of monomers to bind through D period overlaps. The aggregates created in the model displayed several features in common with collagen fibrils, including an elongated morphology, a preference for tip growth, and a linear relationship between mass and distance from the tip. The Silver et al. (1992) and alternative models were discussed extensively at the last Alpbach meeting on Coiled Coils in 1993. The discussions led to the concerted effort by Hulmes et al. (1995) to compare the theoretical X-ray diffraction patterns generated by each of the competing models for fibril structure. Hulmes et al. (1995) found that the singlecrystal model as developed by Fraser, Miller, and associates (Hulmes and Miller, 1979; Fraser et al., 1987) gave rise to sharp peaks that did not readily correspond to the observed X-ray diffraction pattern of rat tail tendon. They found that the limited diffusion model proposed by the Manchester group

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(Parkinson et al., 1995) or a hard disk model proposed by Fratzl and associates gave no two-dimensional order. However, a combination of the singlecrystal and limited diffusion/hard disk model produced a theoretic X-ray diffraction pattern that began to resemble the observed X-ray diffraction pattern. The Silver et al. (1992) model did not generate a pattern consistent with the observed pattern. The model favored by Hulmes et al. (1995) was one in which they assumed a cylindrical model that underwent energy minimization and contained a partial liquid-like disorder in the gap region similar to the hard disk model. As indicated in Table 1, the suggested six or so different models for collagen fibril structure vary in extent to which they fit a number of observations about collagen fibrils. However, the results do not provide any obvious and definitive way of resolving the question of which model is the most accurate. ONE TEST OF THE MODELS: SPECIFIC BINDING SITES

In our own considerations, we (Fertala and Prockop, J. Biol. Chem., in press) have tried to search for testable hypotheses to differentiate among the different models. Here we focused on the fact that our own model (Silver et al, 1992) requires specific binding steps, whereas the other models

TABLE I Comparison of Models for Collagen Fibrils and Data on Structure of the Fibrils Fit to criteria

Models A. Single crystal (Fraser/Miller/ Wess) B. Hard disk/liquid-crystal/limited diffusion (Fratzl/ Manchester group) C. Spiral (Hulmes) D. Cylindrical 1. Galloway (1985) 2. Silver et al. (1992) 3. Hulmes et al. (1995) a

X-ray of round Growth Stepwise Micro fibril from increase in fibrillar (theor.) tips diameters substructure

No

No

No

Yes

No (Yes) a

Yes (Yes) a

No No

No Yes

No b

No

Yes

Yes

No b

Yes

Yes

Yes

Yes

No

Yes

Yes

With qualification (see Hulmes et al., 1995). Not energy minimized or partially disordered as in the Hulmes et al. (1995) model. b

either explicitly exclude such binding steps or do not specifically require them. Based on these considerations, we have tested the possibility that the presence of peptides with sequences found in varying parts of the collagen monomer may compete for specific binding sites and thereby inhibit fibril assembly. Our results demonstrate that several peptides with sequences from many different regions of the major triple helix of the protein have no affect on fibril assembly. However, peptides with sequences from the two N-telopeptides of the a1(I) and a2(I) chains and the two C-telopeptides of the two chains can inhibit fibril assembly. In examining the kinetics of fibril assembly, we found that addition of one of the peptides in the lag period completely prevented fibril assembly. Addition of the same peptide early in the propagation phase had a minor effect on the total amount of fibrils assembled, whereas addition later in the propagation phase has no effect. Therefore, the peptide appeared to inhibit an early nucleation step. A second observation was that the telopeptides bind specifically to one region of the a1(I) chain. The peptides bind to the CB7 cyanogen bromide peptide of the a1(I) chains that contain amino acids 552 to 821. They also bind to the collagenase B fragment of the a1(I) chain that contains sequences that begin at amino acid residue 776. Therefore, the results indicate the peptides bind specifically to a sequence between residue 776 and 821 of the a1(I) chain. Competitive binding assays with biotinylated Ctelopeptide defined the target region as amino acid residues 776 and 797. Moreover, examination of shorter fragments of the C-telopeptide of the a2(I) chain demonstrated that a sequence of only nine amino acids is sufficient to observe binding. Mutating the two aspartate residues in the nine amino acid sequence to serine had no effect on inhibition of fibril assembly. However, mutating the two tyrosine residues and one phenylalanine residue to serine in the same sequence eliminated the effect on fibril assembly. Based on these observations, we used the Sybill program on Silicon Graphics Computer to model the binding interactions. There appeared to be a good fit of both hydrophobic residues and electrostatic residues between the two putative binding sites (not shown). Surprisingly, all four telopeptides bind to the same region of the a1(I) chain. However, the binding constants differ. The C-telopeptide of the a1(I) chain has a dissociation constant of 4.1 3 1026 M. The binding constants for the other telopeptides are one to two orders of magnitude higher. In examining models of collagen assembly, it is apparent that binding of the C-telopeptides to the target site in the a1(I) chain places the monomers in the exact register

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In summary, we can offer several conclusions about the structure of the collagen fibril that may or may not reflect a consensus in the field: (a) Collagen fibrils are assembled by an entropy-driven process that involves specific binding sites on the monomers and that closely resembles crystallization. (b) The collagen fibril has some of the properties of both a crystalline structure and a liquid crystal. Some regions of thick fibrils may be crystalline but others more flexible and liquid-like. Also, the degree of crystallinity of a fibril may vary with changes in temperature, tension, and other conditions. (c) The available data do not exclude the possibility that monomers can perhaps assemble into fibrils by more than one route and into more than one structure. (d) One promising approach to resolving the apparent paradoxes about the structure of collagen fibrils is to define specific binding sites and the affinities of these binding sites in collagen monomers. This work was supported in part by NIH grant AR-43366. REFERENCES

FIG. 7. Scheme suggesting how binding of the a1-C-telopeptide to the region between amino acid 776 and 797 of the a1(I) chain can allow collagen monomers to assemble in register into a microfibril and then allow the microfibril to grow longitudinally. The model is based on the data of Prockop and Fertala (J. Biol. Chem., in press ).

of a 1D stagger (Fig. 7). In effect, the binding of the a1-C-telopeptide brings the two monomers into the appropriate register. After the monomers are aligned in register, lateral association of the monomers apparently recruits additional binding forces from hydrophobic and electrostatic interactions along the length of the molecules. If it is assumed that the monomers add by 1D staggers and then fold into a pentameric microfibril, the registration established by binding the a1-C-telopeptides allows both the assembly of the fibril and the longitudinal growth to the fibril (Fig. 7). The binding of the two N-telopeptides to the same site in the a1(I) chain is not consistent with a D-period stagger of monomer binding. One possibility is that the binding of the N-telopeptides provides the second binding constant required by the Silver et al. (1992) model for axial growth from the microfibrillar core. We are currently in the process of testing this possibility.

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