Molecular packing in type I collagen fibrils

J. Mol. Biol. (1991) 218; 815-823

Molecular

Packing in Type I Collagen Fibrils

A Model with Neighbouring Collagen Molecules Aligned in Axial Register A. V. Kajava Institute of Protein Research Academy of Sciences of the U.S.S.R 142292 Pushchino, Moscow Region, U.S.S.R. (Received 1 August

1990; accepted 20 December 1990)

A detailed stereoehemical analysis of intermolecular interactions of collagens made wit.h molecular models and summarized experimental data resulted in a new three-dimensional structural model for collagen fibrils. In this model collagen molecules aligned in axial register form a bunch. The bunches are aligned head to tail and penetrate by 300 A into each other, forming microfibrils; these in turn assemble into fibrils. The new model differs from all the others in that its characteristic axial regularity, with a period of 670 A, results from staggering of the adjacent microfibrils formed by unstaggered molecules rather than from the axial staggering of neighbouring collagen molecules.

anadysis of intermolecular interactions in collagen. In our model, not only is there a well-defined microfibrillar level of organization but also molecules in the microfibrils are packed on a slightly distorted hexagonal lattice. This allows us to explain the whole set of experimental data within the framework of only one model.

1. Introduction There is independent evidence, mainly from electron microscopy, for a well-defined microfibrillar substructure in native collagen fibrils (Olsen, 1963; Gelman et al., 1979; Parry & Craig, 1979). Several types of structural model with two-stranded (Woodhead-Galloway et al., 1975), four-stranded (Veis & Yuan, 1975), five-stranded (Smith, 1968; Miller & microfibrils Parry, 1973) and eight-stranded 1973) have been (Nemetschek & Hosemann, proposed on the basis of these data and the information provided by the medium angle X-ray diffraction patterns of fibrils. Divergence of these models from X-ray diffraction data and from measurements of fibril density led to the suggestion of a new type of model based on quasi-hexagonal packing of collagen molecules (Hulmes & Miller, 1979; Trus & Piez, 1980; Fraser et al., 1983). Models with this molecular packing were accepted and widely used for interpretation of X-ray diffraction data. However, these models cannot substitute entirely for models with microfibrils. For example, there is no well-defined microfibrillar substructure in the model with a quasi-hexagonal arrangement of molecules, and up to now the results of electron microscopy of collagen fibrils (Na et al., 1986) have been explained by models with five-stranded microfibrils (Smith, 1968; Miller & Parry, 1973). Here, we describe a new three-dimensional model structure for collagen fibrils. It is based on experimental data and on the results of stereochemical

2. Stereochemical Analysis of Intermolecular Interactions of Collagen It is known that the hydrophobic effect plays the main role in self-association of collagen molecules into fibrils (Cassel, 1966). For example, differential scanning calorimetry shows that the enthalpy of denaturation for native fibrils is virtually identical with that of collagen solutions (McClain & Wiley, 1972). Therefore the stability of collagen fibrils is determined mainly by changes in solvent entropy, i.e. by the hydrophobic effect. It is also known that the ability of collagen triple helices to aggregate with each other decreases with decrease in temperature (Fessler, 1960). This result confirms that hydrophobic interactions are the main factor determining self-organization of collagens into fibrils, since a decrease in temperature is always accompanied by a reduction in the hydrophobic effect. Estimation of hydrophobic interactions is a complicated and still unsolved problem: it is necessary to consider simultaneously the van der Waals’ contacts, hydrogen bonds and ion pairs of a whole system containing collagen triple helices and 815

oo~~-~s3s/9l/ososlri-o9

$03.00/O

0 1991 Academic Press Limited

816

A. V. Kajava

the water molecules surrounding these helices. At present computer calculations and other approaches are unable reliably to reproduce the real pattern of collagen intermolecular interactions. However, such problems often do not require explicit energy calculations and knowledge of the main effects at a qualitative level may be sufficient. Stereochemical a.nalysis using a precision molecular model is the most suitable tool for solving such multiparameter operations. Here, we describe a stereochemical analysis performed with molecular models to elucidate hydrophobic interactions between collagen molecules. The hydrophobic effect forces out water molecules from the intermolecular spaces of a-helices and P-sheets that consist of non-polar residues and are assembled into aggregates. Aggregates of collagen molecules, unlike those of cx-helices a,nd P-sheets, have water between triple helices. The presence of water molecules in fibrils is due t’o the structure of the triple helices of collagen. The triple helix consists of extended polypeptide chains, most of the donor-acceptor hydrogen bond groups of which interact with water. If all water molecules were removed from the intermolecular spaces of collagen molecules, the donor-acceptor groups would be dehydrated. Thus, the least possible amount of intermolecular water favours the hydrophobic effect, while the absence of water leads to dehydration of hydrogen bond donors and acceptors. As a result of these counter-directed forces an intermediate situation is realized in collagen fibrils: some of the ordered water molecules are released when triple helices aggregate with each other, and the rest are retained in the int,ermolecular space (Fig. 1). Most of the water is forced out from the fibril when molecules of collagen are packed in a hexa-

gonal array. Therefore, during stereochemical analysis, we placed triple helices of collagen with t’he struct,ure proposed by Rich & Crick (1961) (built with Corey-Pauling-Koltun models and skeletal wire models) in the axial projection at points of a hexagonal lattice. The helices were placed at a distance of 13 to 14 A (1 A = 0.1 nm) from each other. This distance between collagen molecules had been determined in an X-ray diffraction study of collagen fibrils (Miller & Wray: 1971). We then examined the network of collagen-wat,er-collagen hydrogen bonds, the interactions of charged sidegroups and the van der Waals’ contacts between collagen molecules. Stereochemical analysis has shown that the bulky side-chains of t’he nearest-neighbour molecules of collagen can be in direct contact with each ot’her (Fig. 1). Moreover, side-chains with donoracceptors of hydrogen bonds, as well as non-polar residues, can form contacts between their non-polar parts, while their polar parts can form hydrogen bonds with water. However, side-chains do not pack tightly in the intermolecular space and the distance between the triple helices of collagen is not determined by van der Waals’ contacts bet,ween side-chains. Analysis with molecular models ha,s shown that this distance. and therefore the amount of intermolecular water. i.e. the value of the hydrophobic interaction. depends on distortion of the network of hydrogen bonds in t’he peptide group-water-peptide group complex (Fig. 2) rather t,han on the side-chain interactions. It should be ment’ioned that variat,ion 01 the triple helix pitch does not affect this result. Inasmuch as the hydrophobic int’eraction between collagen molecules is determined by peptide groups, the triple helix is hydrophobically homogeneous

(a) lb) Figure 1. Axial project’ion project’ion of (a) separate collagen molecules and (b) molecules forming a complex. The Figure is drawn to scale. Triple helices of collagen assembled into aggregate ar,,0 at about 14 A from each other. Thin lines denote hydrogen bonds between water molecules ordered around collagens.

Molecular Packing in Collagen Fib&

817

A B C A B C h

A

Figure 2. Schematic representation of 3 fragments of triple helices of collagen (left). In the process of coiling the mutual arrangement of collagens changes periodically in the axial projection: A, 3, C, A, B . Three characteristic orientations of hexagonal packing of collagen molecules occurring in I period are shown in the middle. A part of the intermolecular space with collagen triple helices located closer to each other is represented on the right. Atoms with Z-co-ordinates within f 1.5 A from the plane of reference perpendicular to the helical axis that passes through the C’=O groups are shown in this intermolecular space. The Figures on the right are drawn to a larger scale. Open circles correspond to oxygen atoms, filled circles to carbon atoms, hatched circles to nitrogen atoms. Contours around the atoms demonstrate van der Waal’s volumes. Alanine was taken as a side-chain. Hydrogen bonds are shown by broken lines.

along the axis. Thus, the hydrophobic effect is greatest when the area of contact between the interacting molecules is maximal, i.e. when the molecules are in the axial register. The only obstacle for this arrangement of collagen molecules may be electrostatic repulsion of charged residues of the same sign that are placed opposite each other when triple helices are in register. However, stereochemical analysis has shown that these groups can avoid each other by movement of the side-chains. The smallest distance between them is more than 10 A, and the water molecules between the groups considerably reduce charge repulsion. Moreover, it is known that charged groups (Lys, Arg and Glu, Asp) are not distributed randomly along the molecule of type T collagen. Charged residues of opposite sign frequently pair in the sequence (Doyle et al., 1974). Previous

studies

have shown

(Hulmes

et al., 1973);

and our stereochemical analysis has confirmed, that the tendency of negative and positive charges to be close together in the collagen sequence provides the greatest number of intermolecular ion pairs when

neighbouring molecules of collagen are colinear in register. Thus, interaction of charged side-chains does not prevent, but more probably promotes, the unstaggered arrangement of collagen molecules. Stereochemical analysis of intermolecular interactions between collagen molecules also reveals the following axial regularity. It is known that the and common, most conservative, sequence Gly-Met-Hyl(GalGlc)-Gly-His-Arg of type T collagen molecules from a wide variety of vertebrates is located at about 300 A from the N terminus (and sometimes at 300 A from the C terminus). This is the site of the enzyme-catalysed process leading to glycosylation of hydroxylysine (Aguilar et al., 1973; Dixit et al.; 1978). Molecular model building has shown that the distance between the molecules in the region where 2-&X-D-glyCOSylO-/?-n-galactosylhydroxyiysine is located must increase at least 1.3 times because of the bulk of the carbohydrate groups (Fig. 3). An increase in the distance between collagen molecules aligned in the longitudinal register should also be expected at

A. 8. Kgjava

818

the ends of the molecules as there are, at the N and C termini of collagen non-triple helical telopeptide which regions, 16 and 25 residues long, respect’ively. do not contain a glycine residue at every third 1984). Substitution of bulkier position (Kuhn; residues for glycine leads to thickening of collagen molecules and, consequently, increases the intermolecular dist’a.nce at the N and C termini at least 1.3 times. Thus, stereochemical analysis leads to the conclusion that the formation of aggregates consisting of collagen molecules in the longitudinal register is most probable at the initial stage of type I collagen self-organization into fib&. Such bunches of collagen molecules are about 1.3 t,imes thicker at the ends of the molecules and at the site of attachment of carbohydrate groups. On the basis of these results, as well as electron microscopic data on selfassembly of collagen fibrils (Veis & Yuan, 1975; Trelstad et al., 1976; Bruns et al.; 1979) and the data on cross-linking between collagen molecules in fibrils (Light & Bailey, 198Oa,b; Mechanic et al., 1987) that confirm the conclusion on the unstaggered arrangement of neighbauring molecules, a new scheme of molecular packing of collagen in the fibril was proposed. This general scheme of packing was then refined to a new model of the three-dimensional structure of the collagen fibril, conforming to the X-ray diffraction data.

3. Formulation

of the Structural Collagen Fibrils

(a) Qeneral

Model

for

scheme of packing

Collagen fibrils may be represented by a cylinder with a 670 A periodic cross-striated surface. All the models of fibrils suggested earlier explain this crossstriated pattern of collagen by axial staggering of neighbouring collagen molecules by 670 A relative to each other. The essential difference in the new model of collagen fibrils suggested here is that the characteristic axial regularity is the result of staggering of adjacent microfibrils formed by unstaggered molecules (see Fig. 4). In our model, several closely packed collagen molecules placed colinearly and in register form a bunch. The bunches are 3000 A long. The bunches are lined head to tail and penetrate into each other at a distance of 300 A, forming a microfibril. The microfibrils thicken by $-fold in the areas of bunch junctions, repeating every 2680 A along the axial direction. Then the microfibrils assemble into fibrils and, when closely packed, are arranged so that zones with different densities appear within the collagen fibril. These zones alternate along the fibril axis and, as a result, the fibril acquires axial regularity with a, period of 670 A (see Figs 4 and 5). Tt is clear from the proposed scheme how the interaction between molecules ahd microfibrils can lead to the very regular stagger required for the well-defined band pattern of collagen fibrils. First, as shown, interchain interactions can promote the

unstaggered, colinear arrangement of collagen moiecules. The collagen molecule bunched are thicker at, the ends because of carbohydrate groups a.nd teiopeptides. The more molecules in the bunch, the stronger the externa.1 molecules bend. Hence, the bunch size could be controlled by the tendencv of the collagen triple helixes to be in a straight o;ientation. The distance between the collagen molecules is greater at the bunch ends than in the central part which facilita,tes bunch interpene’tration. Then the carbohydrate groups block penetration of the bunches by more than 300 A (Fig. 4). Finally, closepacking of the microfibrils, which thicken in the area,s of bunch junctions, dictate a 670 A periodic cross-striated pattern of collagen fibrils. (b) &‘odel derived from

X-ray

data

The X-ray diffraction pattern of the collagen fibril exhibits a series of Bragg reflections that indicates the presence of a three-dimensional lattice. It follows from the suggested scheme of molecular packing (Figs4 and 6) that the three-dimensional lattice has a unit cell with angles a, p and y roughly equal to 90”, 90” and 120”, respectively. The unit cell edges a and b are approximately equal and c is 2680 A. Analysis of the diffraction patterns of collagen fibrils shows that the positions of nearequatorial Bragg reflections observed and predicted by the unit cell are in good agreement with each other if a and b are in the range 80 to 90 A. When the unit cell has such dimensions, the bunches of collagen molecules forming a microfibril are about, 40 A thick. Stereochemical analysis has shown that this bunch is a,ble to contain six to seven closely packed collagen molecules. Fanning of the near-equatorial reflections in the X-ray diffraction pa,ttern indicates that the collagen molecules are slightly inclined to the fibril axis, either by the tilting of straight molecules or by supercoiling. Thus, from t,he analysis of molecular packing and the X-ray diffraction pattern we conclude that six different ways of arrangement of collagen molecules are possible within the framework of the new collagen fibril model with the unit cell a, b = 80 to 90 A, c E 2680 A, a, /? zz 90”; y z l20”. For a bunch wit’h either six or seven collagen molecules: (I) The molecules wind around the central one in the bunch. (2) The molecules are inclined to the fibril axis. (3) The molecules wind and are inclined. The final choice for a collagen fibril struct,ural model requires detailed calculation of the X-ray diffraction pattern from each of the six variants of molecular packing. We ca’lculated the X-ray diffraction pat.terns as follows. The computations were carried out with a set of mathematical programs et al., 1989). When caleulat’ing FROG (Urzhumtsev these models a collagen molecule was approximated by a cylinder of radius 4.9 8; as in the calculation of the X-ray pattern from the quasi-hexagonal model (Miller & Tocchetti, 1981). To study distribution of

Molecular

Packing

in Collagen

Fibrils

819

Figure 3. A space-filling model of triple helical domains of collagen with (right) and without (left) carbohydrate groups. The Figure demonstrates the relation between the diameter of collagen molecules, size of the carbohydrate group and intermolecular distance. The fragments of collagen molecules at the top of the Figure are placed at the usual distance (14 A). At the bottom the intermolecular distances increase due to bulky carbohydrate groups. In reality it is not so much that the intermolecular distance increases as that the glycosyl galactosyl moieties can deflect from the plane between collagen molecules. Hydrogen atoms of the left-lower triple helix that have no carbohydrate groups are dotted.

(a)

(b)

(c)

Figure 4. Schematic representation of the levels of collagen fibril arrangement. From left to right: (a) collagen hbril with a characteristic cross-striated pattern; (b) collagen fibril pattern demonstrating the arrangement of microfibrils determining the axial periodicity (D) of 670 a. Numbers 1, 2, 3, 4 denote microfibrils staggered with respect to that at When microfibrils are arranged in this way, the the left-hand side by 0, 1 x D; 2 x D, 3 x D (D = 670 A), respectively. zones of closer packing of collagen molecules (shaded in the left Figure) alternate with zones of smaller density. (c) The region of bunch overlap. Collagen molecules are in the plane of the Figure. In reality the molecules are packed hexagonally.

820

A.

V. Kajava

Figure 6. Srrangement of microfibrils in the axial projection of collagen fibrils. Numbers 1, 2, 3, 4 correspond to the same number as in Fig. 4 and denote staggering microfibrils along the fibril a,xis. La.rge circles designate overlapping regions. The structure unit cell with the parameters a: b and the angle y is outlined with a dotted line. A mirror-reflection arrangement is possible too.

Figure 5. Model of the three-dimensional structure of collagen fibrils. Every wire represents a microfibril. White regions of the wire denote the place of penetration of 2 bunches.

the electron density within the unit cell we generated a three-dimensional point lattice with a step of 2.3 8. The electron density at lattice points located molecules designated by within the collagen cylinders is equal to one. The points placed outside the molecules have a zero electron density. Before calculating the new structural model we determined t.he X-ray diffraction pattern from the quasi-hexagonal model. Our results are in conformity with those obtained by Miller & Tocchetti (1981), and therefore our calculation procedure is acceptable for analysis of such types of structure. We then calculated for the collagen fibril structural model each of the six variants possible within the framework of the new scheme of molecular packing. Calculations show that the X-ray diffraction pat,tekn obtained from the model with the bunch containing six st,raight molecules inclined to the

fibril axis has the best coincidence with the observed X-ray pattern. The unit cell is a = 86.5 ,q. 5 = 86.5 A, c = 2690 A, SI = 946”, p = 88.05”: y = 120”. In the axial projection of this ceil the cent,res of microfibrils were placed in positions with (2,~) co-ordinates (l/4: l/4) (3/4, l/4) (l/4, 3/4) (3/4, 3/4). Collagen molecules were packed closely in bunches (Fig. 7). The overlapping regions of bunches have 12 molecules packed hexagonally, so that’ the microfibril transverse section has a thirdorder axis of symmetry. The distance between the cent.res of neighbouring collagen molecules is about 14 A. The molecular orient’ation is parallel to the c axis, i.e. the molecules are tilted t,o 4~6”. There is a less ordered segment along the microfibril bet$ween t,he main thin segment and the segment where bunches penet’rate int’o each other. For simplicity of calculation this segment wa,s represented a,s a prolongation of the thin part of the microfibril, i.e. by six closely packed molecules.

Figure 7. Axial projections of a collagen fibril with 5 molecules in a bunch. Points denote collagen molecules.

collagen

Packing

Molecular

Table 1 Observed-f and calculated

I(R, Z) for

the equatorial in the medium angle native rat tail tendon

and near equatorial rejlections X-ray diffraction pattern from

t-2, 2) (0; 2) (2, 0)

027

0.26

0019

(-3, (-3, (-2, C-1, (1,

035

0.38

0.004, 0.019

046

0.41

0.019

o-53

0.53

0004

0.58

057

0,004, 0022

0.71

@73 075

0.037 @056

080

079

0.004

1) 2) 3) 3)

O-022

33

45

27

20

2) (2, 1)

(-4: 2) (2: 2) (-4, (0, (4, (-5, (-5, (-3, (-2, (2, (3,

4) 4) 0) 2) 3) 5) 5) 3) 2)

(-6, (-4, (-2; (2, (4,

4) 6) 6) 4) 2)

t-6, 0) t-6; ‘3) (0, 6) 05 0)

t Observed by Miller ~4,Tocchetti

002m

- O-05 E

0.055 0.065

E

18

20

28.5

25

1.5

25

45 465

78 79

435

81

(1981).

In the new model the unit cell is larger than in all the previously proposed models, and; consequently, the X-ray diffraction pattern predicted by our model could display additional reflections that were not observed experimentally. However, a detailed comparison between the observed and calculated intensity distribution within the X-ray diffraction pattern confirmed the validity of the proposed model. Table 1 lists the calculated and observed (Miller & Tocchetti, 1981) intensities of equatorial and near-equatorial reflections in the medium angle X-ray diffraction pattern from rat tail tendon collagen fibril. The equatorial reflection nearest to the origin of the X-ray pattern is predicted by the unit cell of the model to be at about l/80 8-l (not shown in Table 1). However, the intensity of this reflection calculated from our model is considerably less than, say, the intensity of the next reflection, l/38 8-l. This result is in good agreement with the experimental data: it is known that the reflection l/SO 8-l is observed only after special treatment of the specimen (Nemetschek et al., 1979) and is rarely seen under usual conditions. Judging from X-ray data obtained by several independent researchers, the general features of the intensity distribution within the X-ray diffraction pattern for practical purposes do not change;

in Collagen

Fibrils

821

however, the positions of equatorial reflections. especially with R > l/15 A-’ (where R and is are the radial and axial co-ordinates, respectively, of the collagen diffraction pattern) vary depending on the analysed specimen and the methods of preparation. It is known that some research groups (North & Hosemann, 1973) et al.: 1954; Nemetschek revealed only one spacing in the l/13.6 8-l region of the X-ray diffraction pattern of collagen fibril while others (Miller & Parry, 1973; Fraser & MacRae; 1981) revealed two spacings. Our model predicts in this region two scarcely resolved reflections with different 2 values and the same R value. As the reflections observed in the region l/13.6 8-l (see Table 1) are hardly resolved, the total intensity in this region was divided by 2, and each half was assigned to the reflections estimated by Miller Oz Parry (1973). Figure 8(a) represents photowriter diffract’ion patterns from the proposed model for collagen fibril. The interference function was convoluted with a Gaussian function of 0.003 A-’ half-width around each point of the Bragg reflection and then projected onto R. The intensities obtained in this way were then convoluted with a Gaussian function of 0.005 8-l in Z to simulate the observed breadth of the reflection. A two-dimensional (R, Z) array was constructed thus and then a photograph was generated by computer, in which brightness at any point corresponds to the intensity in the twodimensional array at that point. The R-factor defined for our model was:

c C~&-Jh2 x 100% c 1‘3 >

= 26%,

where I,, I, are the calculated and observed intensities, respectively, and k is the optimal scale coeffcient. This value is greater than that in the quasihexagonal model (11%; Miller & Tocchetti, 1981); however, it is sufficient to confirm the validity of the new model. In the preceding discussion it has been assumed that the row lines are parallel to the meridian of the X-ray pattern. The data for native fibers, however, show a small angular splitting for several row lines (Miller & Wray, 1971; Fraser & MacRae, 1981). They form an angle of 87” with the equatorial plane and are parallel to the near-meridional or zero-order row line. This means that in our model the angle between the unit cell base and the equatorial plane is 3”. The normal of the unit cell base should be located on the plane of one of the vectors of the base and fiber axis. (c) Agreement microscopic

of the new Jibril model with electron data and chemical cross-linking of collagen

molecules

Agreement with X-ray data is not a conclusive argument in favour of the new structural model, the more so as the quasi-hexagonal model has a better conformity (for example, the R-factor) with the

A. V. Kajava

822 z&-“1 4

0.003 (a 1

-0.003

(b)

Figure

8. Calculated medium angle X-ray diffraction pattern from the proposed model of co!lagen fibrils. diffraction pattern. (b) / Diffraction pattern with intensity contours. Dotted line 15. broken line 20 and continuous line 30 arbitrary units. (a) Photowritten

observed X-ray pattern. However, considerat’ion of X-ray data along with electron microscopic data and chemical cross-linking of collagen molecules in the native state shows that the suggested model compares well with the quasi-hexagonal model. As mentioned above, there is electron microscopic evidence for a well-defined microfibrillar substructure of the colla,gen fibril. It is also known that the fibril diameter is a discrete value equal to n x 80 A (Parry & Craig, 1979). This is another argument in favour of the microfibrillar substructure of the collagen fibril. Thus, morphological findings do not support quasi-hexagonal models having molecular crystal features. An attempt to reconcile this conflict was made by Trus & Piez (1980), who proposed the existence of compressed microfibrils on a pseudo-hexagonal lattice. However, the compressed microfibril model is not satisfactory if we take into account the results of electron microscopic investiga’tions of the self-organization of collagen fibrils in vitro and in viva (Veis & Yuan, 1975; Trelstad et al., 1976; Bruns et al.; 1979). These data show that collagen molecules assemble into bunches in which they are in register at the first stage of self-organization of collagen fibrils. The bunches then interact at their ends and form microfibrils. Microfibrils form collagen fibrils having a characteristic axial regularity with a period of 670 A. The new scheme of molecular packing of collagen in fibrils suggested here readily explains the observed pathway of fibril self-organization. Our model, unlike most of the previous ones, complies with the data on cross-linking between neighbouring collagen molecules in the fibril. From these data it follows that there are two main types of contact between molecules in collagen fib&: those between molecules unstaggered with respect to each other and those between molecules overlapping by 300 A (Light & Bailey, 1980a,h; Hankel et

al.: 1987). Our arrangement of molecules in coliagen fibrils is in agreement with this pattern of covalent cross-links as, in the model, molecules overlapping by 300 a and those unstaggered are the nearest neighbours and can form covalent bonds. A trifunctional intermolecular cross-link connecting t’wo unst,aggered molecules and a staggered one with a 300 A overlap has been recently identified in skin

Figure 9. Lateral arrangement, of collagen molecules in t’he structure proposed by Fraser et al. (1983). Kumbers 0, I,4 denote 0, 1 x D, 4 x II staggering of collagen mo!ecales along the fibril axis (where D is the axial periodicity). The large circles represent the radial positions of the p-carbon atoms of the triple hehx and denote the van der Waals’ contours of the molecule. The small circles represent the possible radial positions of the a-carbon atoms. The shaded triangle denotes schematically the van der W’aals’ contour of the trifunetional chemical cross-link. Apexes of the t’riangle are the points of connection of the cross-link with the a-carbon atoms. This cross-link would be possible in the quasi-hexagonal model if all the apexes could contact simultaneously with small circles of 0, 0 and 4 molecules. The figure is drawn to scale.

Molecular Packing in Collagen Fibrils collagen fibrils (Mechanic et al., 1987). This trifunctional cross-link is incompatible with quasi-hexagonal models of collagen fibrils (Fig. 9), but agrees well with our model. In 1987, Nakamura succeeded in finding a chemical cross-link between collagen molecules staggered by 1 x D, 2 x D, 3 x D (where D is 670 A). Within the framework of our model such contacts are possible between collagen molecules from neighbouring microfibrils. It should be mentioned that almost all X-ray diffraction patterns assumed as a basis for models of collagen fibrils were obtained on rat tail tendon. However, in contrast to the straight arrangement of microfibrils in rat tail tendon fibrils, interstitial collagen has a “helical” or “twisted” microfibrillar arrangement, as has been revealed by electron microscopic (Raspanti et al., 1989) and by X-ray (Folkhard et al., 1987) data. The “helical” and the “straight” fibrils have a cross-striated surface, so the principal scheme of packing in both types of collagen should be the same. However, in helical fibrils the microfibrils are better defined and twisted. It is difficult to imagine the three-dimensional structure of the helical fibril as a molecular crystal with quasi-hexagonal packing of collagen molecules. At the same time, in our scheme of molecular packing, the “straight” fibril is easily transformed into the helical one by slightly coiling the collagen molecules about the central one, which coincides with the microfibril axis. Microfibrils formed by coiled collagen molecules should become more pronounced and, moreover, unlike straight microfibrils, should be inclined to supercoil. I am grateful to Drs R. A. Abagyan, S. B. Malinchik and A. G. Urzhumtsev for consultations and valuable suggestions; to Drs N. S. Andreeva, A. V. Efimov, N. G. Esipova, V. I. Lim, P. L. Privalov, A. S. Spirin and V. G. Tumanyan for reading the manuscript, for discussions and for criticism.

References Aguilar, J. H.; Jacobs, H. G., Butler, W. T. & Cunningham, L. W. (1973). J. Biol. Chem. 248, 5106-5113. Bruns; R. R., Hulmes, D. J. S., Therrien, S. F. & Gross, J. (1979). Proc. Nat. Acad. Sci., U.S.A. 76, 313-317. Cassel, J. M. (1966). Biopolymers, 4; 989-997. Dixit, S. N., Seyer, J. M., Kang, A. H. & Gross, J. (1978). Biochemistry, 17, 5719-5722. Doyle, B. B., Hukins, D. W. L., Hulmes, D. J. S., Miller, A., Rattew, C. J. & Woodhead-Galloway, J. (1974). Biochem. Biophys. Res. Commun. 60, 858-864. Fessler, J. H. (1960). Biochem. J. 76, 452-471. Folkhard, W., Christmann, D., Geercken, W., Knoerzer, E., Koch, M. H. J., Mosler, E., Nemetschek-Gansler, 42c, H. & Nemetschek, T. Z. (1987). Naturforschung, 130331306. Fraser, R. D. B. & MacRae, T. P. (1979). J. Mol. Biol. 127, 1299133.

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Fraser, R. D. B. & MacRae, T. P. (1981). Int. J. Biol. Macromol. 3, 9918. Fraser, R. D. B., MacRae, T. P.; Miller: A. & Suzuki; E. (1983). J. Mol. BioZ. 167; 497-521. Gelman, R. A.; Williams, B. R. & Piez, K. 8. (1959). J. Biol. Chem. 254, 1809186. Hankel, W.; Glanville, R. W. & Greifendorf, D. (1987). Eur. J. Biochem.

165, 427-436.

Hulmes, D. J. S. & Miller, A. (1979). Nature (London), 282, 878-880. Hulmes, D. J. S.; Miller, A., Parry, D. A. D., Piaz; K. A. & Woodhead-Galloway, J. (1973). J. Mol. Biol. 79, 137-148. Kuhn, K. (1984). Collagen Rel. Res. 4: 309-322. Light, N. D. & Bailey, A. (1980a). Biochem. J. 185. 373-381.

Light, N. D. & Bailey, A. (19800). Biochem. J. 189, 111-124. Martin, G. R., Byers, P. H. & Piez, K. A. (1975). Advan. Enzymol. 42, 167-188. McClain, P. E. & Wiley, E. R. (1972). J. Biol. Chem. 247, 692-697.

Mechanic, G. L., Katz, E. P., Henmi, M., Noyes, C. & Yamanchi, RII. (1987). Biochemistry, 26, 3500-3509. Miller, A. (1976). In Biochemistry of Collagen (Ramachandran, G. N. & Reddi, A. N., eds), pp. 85-136, Plenum Press, New York. Miller, A. (1982). Trends Biol. Sci. January, 13-18. Miller, A. & Parry, D. A. D. (1973). J. Mol. Biol. 75, 441-447. Miller, A. & Tocchetti, D. (1981). Int. J. Biol. Macromol. 3, 9918. Miller, A. & Wray, J. S. (1971). Nature (London), 230, 437-439.

Na, G. C., Butz, L. J., Bailey, D. G. & Carroll; R. J. (1986). Biochemistry, 25, 958-966. Nakamura, Y. (1987). Int. J. Biol. Macromol. 9, 281-290. Nemetschek, Th. & Hosemann; R. (1973). Kolloid 2. 2. Polymere, 251, 1044-1056. Nemetschek, Th., Riedl; H. & Jonak, R. (1979). J. Mol. Bio2. 133, 67-83. North, A. C. T., Cowan, P. M. & Randall, J. T. (1954). Nature (London), 174, 1142-1143. Olsen, B. R. Z. (1963). 2. Zellforsch. 59, 184-191. Parry, D. A. D. & Craig, A. A. (1979). Nature (London), 282, 213-215. Raspanti, M., Ottani; V. & Ruggeri, A. (1989). Int. J. Biol. Macromol. 11, 367-371. Rich, A. & Crick, F. H. C. (1961). J. Mol. Biol. 3, 4822505. A., Benazzo, F. & Reale, F. (1979). Ruggeri, J. Ultrastruct. Res. 68, 101-110. Smith, J. W. (1968). Nature (London), 219; 157-158. Timpl, R., Wiedmann, H., van Delden, V., Furthmayr, H. & Kuhn, K. (1981). Eur. J. Biochem. 120. 2033211. Trelstad, R. L., Hayashi, K. & Gross, J. (1976). Proc. Nat. Acad. Sci., U.S.A. 73, 402774031. 286, Trus, B. L. & Piez, K. A. (1980). Nature (London), 300-301. Urzhumtsev, A., Lunin, V. & Vernoslova, E. (1989). J. AppZ. Crystallogr.

22, 500-506.

Veis, A. & Yuan, L. (1975). Biopolymers, 14, 8955900. Woodhead-Galloway, J., Hukins, D. W. L. & Wray. J. S. (1975). Biochem. Biophys. Res. Commun. 64, 1237-1244.

Edited by D. de Rosier