Conformation and restricted segmental flexibility of C1, the first component of human complement

J. Mol. Biol. (1983) 168, 563-577

Conformation and Restricted Segmental Flexibility of C1, the First Component of Human Complement PAK H. Poo.~, VERNE N. SCHUMAKER, MARTIN L. PHILLIPS AND CANDI('EJ. STRANG

The Department of Chemistry and Biochemistry and the Molecular Biology Institute University of California, Los Angeles, CA 90024, U.S.A. (Received 13 December 1982, and in revised form 13 April 1983) Seventy selected images of chemically crosslinked C1 are analyzed to illustrate structural details of the ClqClr2Cls z complex. From inspection of these images, the Clr2Cls 2 tetramer can be seen to be located in the region of the Clq arms, cleanly separated from the C1 q heads and from at least 90%, if not all, of the C1 q stem. From measurements made upon 65 images, the semicone angles formed between the spreading arms and the symmetry axis passing through the stem of C1 may be calculated. Unlike Clq, for which a wide variety of angles is found, the C1 complex appears to possess a restricted range of angular flexibility with an average value of about 50~. The volume inside the cone formed by the spreading arms of Clq is too small to contain the entire Clr2Cls 2 tetramer; at least some of the tetramer must lie outside the cone when it is bound to Clq to form C1. From our knowledge of the sizes and structures of its subunits, and from symmetry considerations, a model is proposed for the configuration of the C1 complex in which the middle portion of the Clr2Cls 2 tetramer is centrally located among the arms close to the stem of the Clq and with the two protruding ends of the tetramer wrapped around the outside of the cone. Functional implications of this more rigid structure are discussed with relevance to Clq-induced aggregation of latex beads and Cl-induced disaggregation.

1. I n t r o d u c t i o n Ii

C1, the first c o m p o n e n t of h u m a n complement, is the protein t h a t recognizes the presence of immune complexes and initiates the c o m p l e m e n t cascade. I t is. composed of five subunits: one Clq of 410,000Mr two C l r of 80,000Mr each, and two Cls, also of 80,000Mr each. The first of these, Clq, is a collageneous glycoprotein with a most unusual shape t h a t has sometimes been described as a " b u n c h of tulips". I t has six sites, which possess a specific affinity for binding to the Fc regions of I g G t or IgM. The function of Clq is to combine with soluble immune complexes or to clusters of a n t i b o d y r0olecules a t t a c h e d to membranebound antigens. Simultaneous binding at a n u m b e r of sites b y Clq is t h o u g h t to provide the recognition signal which, b y an u n k n o w n mechanism, triggers the t Abbreviation used: Ig, immunoglobulin.

563 0022-2836/83/230563-15 $03.00/0

9 1983 Academic Press Inc. (London) Ltd.

564

P. H. POON E T A L .

proteolytic cleavage of the p r o e n z y m e portions of the C1 complex. The C1 p r o e n z y m e is composed of two molecules of C l r and two of Cls. The two C l r become cleaved and a c t i v a t e d first, perhaps autocatalytically, and then the a c t i v a t e d C~r catalyzes the cleavage and a c t i v a t i o n of C]-s. The a c t i v a t e d forms of these enzymes are written C~r and C]-s. The function of C~s is to cleave C2 and C4, which are separate p l a s m a proteins, and by this cleavage to initiate the c o m p l e m e n t cascade, which involves over 20 peptides, and leads to an i n f l a m m a t o r y response, opsonization of t a r g e t cells, c h e m o t a x i s of phagocytic cells, and direct a t t a c k on m e m b r a n e s of the invading bacteria or virus. There are m a n y excellent reviews, of which we will mention three: P o r t e r & Reid (1979), Metzger (1978) and Muller-Eberhard (1975). A detailed presentation of the s t r u c t u r e of C l q is given b y P o r t e r & Reid (1979), following their earlier work proposing a model for this molecule (Reid & Porter, 1976). T h e y showed t h a t Clq is composed of six subunits of identical amino acid sequence, each forming a head, a r m and one-sixth of the stem. When examined with the electron microscope, C l q a p p e a r s to possess 6-fold s y m m e t r y ; however, the subunits are connected in pairs by a single disulfide bond, so t h a t the molecule could possess a perfect s y m m e t r y no greater t h a n three. The structure of the Clr2Cls2 t e t r a m e r , as seen with the electron microscope, is a long, rod-like molecule, as described b y T s c h o p p et al. (1980). These authors also performed h y d r o d y n a m i c studies, and the results of these were in a g r e e m e n t with the highly elongated structure seen with the electron microscope. One curious feature of the conformation of the molecule was a reversed " S " twist, and a likely explanation, as proposed b y t h e m , is t h a t " T h i s preferred adsorption indicates t h a t the c o m p l e x e s . . , have two different sides with different affinity to the carbon films". A 2-fold rotational s y m m e t r y axis located in the center of the flexible t e t r a m e r , perpendicular to the grid, would provide the complex with two distinct sides and would explain the 2-fold s y m m e t r y of the reversed " S " shape seen with the electron microscopet. A model has been proposed for the structure of C1 on the basis of electron microscope studies (Strang et al., 1982) in which the long, flexible Clr2Cls 2 t e t r a m e r i c subunit is w r a p p e d a m o n g and around the C l q arms, in the region lying between the Clq stem and the Clq heads:~. I n this paper, we show a substantial n u m b e r of selected images of chemically crosslinked C1 and the results of m e a s u r e m e n t s m a d e upon these, to allow us to provide additional structural detail, and to suggest, from volume and s y m m e t r y considerations, the p a t h followed b y the Clr2Cls 2 t e t r a m e r as it winds a m o n g the C l q arms. t Since the carbon-coated grids are held and viewed in an inverted position in the electron microscope, the reversed "S" is a proper "S" when the specimen is viewed from above the carbon film rather than through it. In order to distinguish between the morphologically distinct portions of Clq, we am using the term heads to refer to the globular, non-collageneous domains of Clq, which are thought to contain the binding sites for the antibody Fc; the term stem to refer to the rod-like bundle formed of 6 collagen triple helices that begins near the N-terminal e'hd and extends to between 37 and 40 residues from the N-terminal end, where the helices separate from each other: and the term arms to refer to the 6 separate collagen triple helices beginning at residues 37 to 40 and extending to residues 88 to 90, where the non-collagenous sequence of the heads begins. Thus, the arms connect the stem to the heads.

CONFORMATION AND FLEXIBILITY OFC1

565

Symmetry considerations are important in attempting to construct a model for the C1 complex in order to take advantage of the presence of multiple strong contact sites. At first, there would seem to be no way in which symmetry could be maintained in matching pairs of contact sites on a first object possessing 2-fold symmetry with triplets of sites on a second object possessing 3-fold symmetry. However, it is possible to match sites between objects possessing 2-fold and pseudo-6-fold symmetry if quasi-equivalent bonding is permitted. Quasiequivalence is the design principle used successfully to explain the structures for the large icosahedral virus capsids, as explained so elegantly by Casper (1965). In this paper we show how quasi-equivalence may be employed to construct a reasonable model for C1, compatible with electron microscope images, volume considerations, and symmetries of the subunits. The CIr2Cls 2 tetramer is suggested to pass between the Clq arms so that the 2-fold and pseudo-6-fold rotational symmetry axes coincide. Then the two ends of the protruding tetramer wrap around the outside of the cone formed by the spreading Clq arms and bind to three pairs of contact sites on the six arms of Clq. The principle of quasiequivalence is invoked to explain how the tetramer may bind to pairs of contact sites on opposites arms of Clq that may have slightly different orientations because Clq cannot have perfect 6-fold symmetry. Intramolecular flexibility of the Clq subunit is a property .that has been noted frequently (Shelton et al., 1972; Svehag et al., 1972; Knobel et al., 1975) and attributed to a limited degree of flexibility at a joint located at a position where the collagenous arms join to the stem of the molecule (Schumaker et al., 1981). Intramolecular flexibility of the Clr2C1 s2 tetramer is a feature of that molecule as well, since it has been seen in many different conformations on the electron microscope grid (Tschopp et al., 1980) and has been described as a flexible, rod-like structure (Stranget al., 1982). In this paper we show that the C1 complex formed between one molecule ef Clq and a single tetramer of Clr2Cls 2 is a more rigid structure than either the Clq or the Clr2Cls 2 from which it is formed. This extra rigidity would seem to be a consequence of the structure that is proposed here for the C1 complex in which the tetramer wraps around the Clq and binds to its arms. One consequence of a loss of flexibility of the Clq subunit might be a reduced capacity to form precipitates with immune complexes and IgG-coated latex beads. Thus, the capacity of Clq to form precipitates with immune complexes led to its original isolation by Muller-Eb~erhard & Kunkel (1961). Hallgren (1979) has shown that C1 inhibits the agglutination of IgG-coated latex beads by Clq; indeed, it promotes their dissociation. A difference in the flexibilities of C1 and Clq may account for the contrast in behavior. 2. Materials and Methods The work repol~ed here is an extension of the study presented by Stranget al. (1982). The preparation of Clq, the Clr2Cls2, and the reassembly of the C1 complex and its crosslinking, and the electron microscope grid preparation technique, were all described in detail in that study, and need not be repeated here. The method used in the present study for the analysis of the distribution of cone angles for C1 follows the method used in a previous study (Schumaker et al., 1981) for the distribution of cone angles in Clq.

566

P . H . POON E T AL.

Considerable pains have been taken to select '~good" pictures of C1, and about 1200 images of C1 molecule have been examined to find the 70 views presented here, and about 50 more not shown. Thus, approximately 10% of the molecules give well-defined images. The rest are overlapping or less well-resolved, although compatible with what is shown in Figs 1 and 2 below. 3. R e s u l t s

I n Figure 1 35 profile views of C1 are p r e s e n t e d from which it is possible to gain an impression of the region occupied b y the Clr2Cls 2 t e t r a m e r when a t t a c h e d to Clq. The rows and columns of Figure 1 are labelled with n u m b e r s and letters so t h a t particular images m a y be located. Individual Clq heads m a y be seen in m o s t of these images, although it is often difficult to c o u n t j u s t six. In one case, C4, the heads a p p e a r to be clumped together into two groups; in other cases, t h e y are spread in a fan-like structure, A3 ; occasionally, t h e y are jumbled, C3. The stem of the Clq subunit is exposed along m o s t of its length, and a line of stain is often seen along the stem, A3, A4, B5 and E3. The Clr2Cls 2 t e t r a m e r , which a p p e a r s to be a rod-like molecule with a length of 510 A to 590 A (Tsehopp et al., 1980 ; S t r a n g et al., 1982) would extend far beyond the limits of the fan if it were outstretched. Thus, the C l r 2 Cls2 m u s t be folded into a more c o m p a c t structure, and can be seen to be located in the region where the C l q a r m s come together to form the stem. The a p p a r e n t shape of the folded Clr2Cls 2 is not always the s a m e ; sometimes it a p p e a r s as a voluminous b u t poorly defined mass, B1, B5, C6 and C7, while in other images it becomes a narrow, often fan-shaped structure, A3, A4, B6 and D1. The collagenous a r m s are 15 A in d i a m e t e r and difficult to resolve ; even so, portions of the a r m s can sometimes be seen between the C l q heads and the region covered b y Clr2Cls 2, A3, A4 and B6. Thirty-five top views of C1 are shown in Figure 2. Again, the C l q heads are visible and individual heads m a y be counted; sometimes the c o u n t is six, D2, b u t occasionally more, C3, C4, or less, C7. We interpret a count of more t h a n six to indicate t h a t an end-poi~ion of the folded Clr2Cls 2 t e t r a m e r had become detached from the Clq a r m s and was lying unfolded and o u t s t r e t c h e d a m o n g the heads, increasing the a p p a r e n t count. The folded Clr2Cls 2 mass can be seen to be centrally located, often cleanly separated from the C l q heads. I t is found to h a v e an irregular shape, A1, B1, D1 and D2. I n order to provide q u a n t i t a t i v e m e a s u r e m e n t s , tracings were m a d e of the enlarged images. A sampling of such tracings is shown in Figure 3. F r o m the tracings of the profile views, the width of the C1 stem, the length of the C1 stem, and the projected area covered b y the Clr2Cls 2 complex could be measured. The results of these m e a s u r e m e n t s are s u m m a r i z e d in Table 1. Also included in Table 1 are similar m e a s u r e m e n t s of the length and width of the C l q stem, determined from similar tracings m a d e on nine profile views of C1 q found a m o n g the images of cross-linked C1. Fro. 1. Thirty-five profile views of chemically crosslinked CI show that the Clq heads and at least 90% of the stem are clearly separated from the Clr2Cls 2 tetramer, which is folded among the Clq arms. Individual images are discussed in the text. A water-soluble carbodiimide was used to chemically crosslink the complex; uncrosslinked preparations show only dissociated Clq and ClrzCls2 molecules attached to the grid. Magnification, 331,000 x.

CONFOI'CMATION

A

2

B

AND FLEXIBILITY

C

OFC1

D

567

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m m mmmm

3

4

6

m m 9 .,.

r'.

m mmm m FI(:. 1.

P . H. P O O N E T A L .

568

// // // // // // // // // // // // // // // // // // // // A

2

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B

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FI(~. 2.

D

CONFORMATION AND F L E X I B I L I T Y O F C I

569

The projected area covered b y the Clr2Cls 2 complex also could be measured from top views. F r o m the a v e r a g e d projections of the areas occupied by the Clr2Cls 2 as seen in the top views, the radius of a circle of equal area m a y be calculated as 78 A. F r o m the side views, a smaller average projected area is calculated. The folded t e t r a m e r occupies a volume roughly the shape of a cylindrical disk with an axial ratio of a b o u t 2 : 1. F r o m the top-view images of C1, the degree of spreading of the a r m s seems to be roughly the s a m e from molecule to molecule; t h a t is, the vertex angles formed b y the spreading a r m s do not a p p e a r to v a r y as m u c h a m o n g the C1 as they do a m o n g the Clq. In order to test this impression q u a n t i t a t i v e l y , the tracings of the enlarged images were used to d e t e r m i n e the radius of the circle t h a t comes closest to passing though the centers of all of the C l q heads. F r o m this radius, a n d from the average length of the Clq a r m s as d e t e r m i n e d from the profile images, the cone angle was calculated for each t o p view, as described b y S c h u m a k e r et al. (1981). The distribution of semi-cone angles, as d e t e r m i n e d from 65 top-view images of C1, is shown in Figure 4(b). I t is seen to p e a k sharply a t 50 ~ although 40% of the molecules do fall into the flanking bars of the histogram. I n contrast, the distribution for Clq, reproduced in Figure 4(a), has a m u c h b r o a d e r range of angles, although it still peaks at 50 ~. This value of 50 ~ is s o m e w h a t less t h a n the value of 60 ~ e s t i m a t e d from neutron scattering from C l q in solution (Gilmour et al., 1980).

4. D i s c u s s i o n

(a) Crosslinked C1 complexes I n a previous s t u d y (Siegel & S c h u m a k e r , 1983), it was shown t h a t C l q and Clr2Cls 2 formed a 1 : 1 complex with an association c o n s t a n t of 6.7 • l0 T M-1 and a s e d i m e n t a t i o n coefficient of 16 S. The crosslinked complexes reported here were prepared a t concentrations of C l q and Clr2Cls2 a t which essentially all the material would be present as the 1 : 1 complex; m o s t of these complexes s e d i m e n t a t e d at 16 S both before and a f t e r crosslinking. Clq, b y itself, was not crosslinked b y the s a m e t r e a t m e n t ( S t r a n g e t al., 1982). Therefore, the complexes shown in Figures 1 and 2 could not represent C l q dimers. I t is theoretically possible t h a t the crosslinking reagent has cause d d i s p r o p o r t i o n a t i o n a n d r e a s s e m b l y of 2Clr2C1 s2 -* C1 r 4 + C1 s4, which then recombined with 2C1 q to yield a m i x t u r e of C l q C l r 4 and ClqCls4 molecules and t h a t these were being studied ; however, there is no evidence to s u p p o r t such an a s s u m p t i o n . Moreover, we have seen very similar images to those shown in Figure 1 and 2 in uncrosslinked material, at a b o u t the frequency t h a t would be calculated from the dilution Fro. 2. Thirty-five top views of chemically eroselinked C1 show that the Ciq heads are clearly separated from the Clr2Cls 2 tetramer. The tetramer, which appears in electron micrographs as an elongated structure with a contour length of 510 or 590 A, is seen to be folded into a compact structure located in the center of these top view images, obscuring the stem of the Clq. As described in detail in a previous study of Clq (Schumaker et al., 1981), these top view images are thought to represent Cl molecules that have attached to the grid by the Clq heads, with the arms and stem raised above the grid "like the lunar lander on the surface of the moon." Magnification, 331,000 x.

570

P.H.

POON ETAL.

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9 FIC. 3. Representative examples of the tracings of C1 molecules used to compile the data presented in Table 1. The tracings were made from enlarged prints from the original electron micrograph plates, so t h a t each image filled a 3 in square. Tracings of Clq also were measured for the dimensions of the stem, as presented in Table 1. The areas of the central region occupied by Clr2Cls 2 and stem in the top views were measured with a planimeter.

CONFORMATION AND FLEXIBILITY

OF C1

571

TABLE 1

Measurement of dimensions and areas of C1 subunits on the assembled complex Molecule CI CI CI CI Clq C1 q

Measurement

Number of images

Stem length Stem width Projected area occupied by Clr2Cls 2 Projected area occupied by Clr2Cls 2 Stem length Stem width

26 26 24 66 9 9

View Profile Profile Profile Top Profile Profile

Dimension (A) or area (.aft)J118_+12 56 _+3 11,700_+4300 19,100_4200 131 __.6 63 +_3

1 standard deviation of the population, s. i

$,

i

i

i

I

i

I

5O

0

E 2O "5 ,5 z I0

-h-

~'0 50 40 50 60 70 80 20' 3'0 40 5 0" 60 70 ' 80 Cone angle (deg.) (o) (b)

FIC. 4. A comparison of the distributions of the cone angles for (a) Clq and (b) CI as determined from measurements of 81 and 65 top views. The data for Clq were taken from a previous publication (Schumaker et al., 1981). For C1, measurements were made of the radii of circles providing the best fitting through the centers of the Clq heads on the enlarged tracings. Cone angles were then determined from the known dimensions of the C1 subunit, as described by Schumaker et al. (1981).

required for the electron microscopic studies. Therefore, we believe the images s~hown here are crosslinked C1 complexes and, although these could be distorted in their fine details, both because of the presence of chemical modification and because of the forces of binding to the carbon film, they probably represent, on the average, the structures of C1 complexes at a leveF of resolution achieved in these electron micrographs. (b) C1 dimensions Fom the data presented in Table 1, it can be seen that the width of the C1 stem is a little less than the width of the Clq stem ; and that the length of the exposed C1 stem is 900//0 of the length of Clq. Although the difference in the average lengths would seem to be significant, we will assume that little or none of the Clr2Cls2 tetramer is located on the stem of the Clq subunit as it exists in solution, and that the apparent shortening is due to a portion of the tetramer occasionally becoming loose and overlapping the stem when the complex attaches to the carbon film and is dried in the stain.

572

P. H. P O O N E T AL.

(e) C1 structure : volume available for packing A cross-sectional view of the model used to estimate the volume available for packing of the Clr2Cls 2 subunit around or a m o n g the C l q a r m s is shown in Figure 5. The volume of a right circular cylinder located just below the stem of Clq is calculated for the average radius, r = 78 A, occupied b y Clr2Cls2. The calculated values presented in Table 2 indicate t h a t there is insufficient volume in the cage formed b y the spreading Clq arms, Vi, to place inside the entire Clr2Cls2 t e t r a m e r although, due to uncertainties in the dimensions, this s t r u c t u r e cannot be ruled out absolutely. However, there is sufficient volume available outside of the cage, V t - V i - V a - - 7 " 5 x 105 A 3, in which to place all of the h y d r a t e d mass. Moreover, the circumference of a circle with a radius of 78 A is 490 A, only a little less t h a n the contour length of 510 A for Clr2Cls 2, as e s t i m a t e d b y T s c h o p p et al. {1980). Therefore, a simple and plausible model for C1 would be a model in which the Clr2Cls 2 encircles the Clq, perhaps similar to the p a t h t a k e n by a strand of popcorn loosely draped around a holiday tree if the contour length of the t e t r a m e r is actually 590 A as e s t i m a t e d b y S t r a n g e t al. (1982). This model also has two other virtues. (1) The binding sites would be exposed on the outside of the cone m a k i n g t h e m readily accessible for the reversible binding t h a t is found to occur between the t e t r a m e r and Clq (Siegel & S c h u m a k e r , 1983); and {2) the binding sites on C~r2C~s 2 would be exposed, and thus readily accessible to interaction with the Ci-inhibitor. However, in a t t e m p t i n g to assemble this model, a s y m m e t r y problem develops, as will be explained next. (d) C1 structure: a problem of symmetry The s y m m e t r y problem in the assembly of C1 is illustrated in Figures 6 and 7. H o w can Clr2Cls 2, which a p p e a r s to exhibit 2-fold s y m m e t r y , bind to a structure

7T m

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_

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T

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Fro. 5. A cross-sectional view of Clq used to calculate the volumes available for the folded CI r2Cls2 subunit inside and outside of the cage formed by the Clq arms. The volumes of the rectangle, the truncated cone inside the arms, and of the arms are calculated from simple geometrical considerations, as a function of the radius r, the height h., and the semi-cone angle, 0 = 50~ These values are given in Table2, as well as volumes for the Clr2Cls 2 tetramer, assuming various amounts of water of hydration.

CONFORMATION AND F L E X I B I L I T Y O F C I

573

TABLE 2

Vol~tmes available for the folded ClrzCls2 tetramer inside and outside the cage formed by the Clq arms Parameter

Dimensions

Radius, r (,~) Height, h (.~) Total volume, Vt (A3) Inside volume, Vi (h 3) Arm volume, |~t (A3) Outside volume, (I"1- Vi- Va) (h a) Anhydrous volume of the C1,'2Cls2 (Mv/N)(A a) Hydrated volumeS (.~3) Hydrated volumew (.~3)

78 65 12-4 x l0 s 4.1 x l0 s 0'8 x l0 s 7"5 X l0 s 4-2 x l0 s 5-2 x l0 s 6"8 x l0 ~

t The total volume of all 6 arms is given as 6 x (7"5 h) 2 xlrx 115 3, = 1"22x 105 .~a. S Assuming 0"2 g H20/g Clr2Cls2. w Assuming 0"6 g H20/g Clr2Cls2. such as C l q , which c a n e x h i b i t o n l y perfect 3-fbld symmet~'y, a n d y e t a d v a n t a g e be t a k e n of t h e m u l t i p l e , i d e n t i c a l c o n t a c t sites t h a t m u s t e x i s t on each of t h e molecules? W e s u g g e s t t h a t t h e a n s w e r to this s y m m e t r y que.stion m a y lie in t h e c o n c e p t of " q u a s i - e q u i v a l e n c e " , which h a d b e e n e m p l o y e d successfully to e x p l a i n t h e s t r u c t u r e s of the icosahedral viruses. I d e n t i c a l c o n t a c t sites m a y be e m p l o y e d , u s i n g two s l i g h t l y d i f f e r e n t o r i e n t a t i o n s , to f o r m t h e capsids of t h e larger v i r u s e s from t h e i r s u b u n i t s . W e s u g g e s t t h a t flexible s t r u c t u r e s such as C l r 2 C l s 2 a n d C l q m a y be a s s e m b l e d in a n a n a l o g o u s m a n n e r u s i n g i d e n t i c a l c o n t a c t sites w i t h s l i g h t l y d i f f e r e n t o r i e n t a t i o n s . T h u s , C l q is a t r i m e r b e c a u s e t h e helices are j o i n e d in p a i r s b y a d i s u l p h i d e b o n d n e a r t h e N - t e r m i n a l e n d o f the s t e m . O t h e r t h a n this, t h e a r m s h a v e i d e n t i c a l a m i n o acid s e q u e n c e s a n d e q u i v a l e n t b i n d i n g sites s h o u l d be p r e s e n t , a l t h o u g h these m a y be o r i e n t e d i n - s p a c e a l i t t l e d i f f e r e n t l y o n o p p o s i t e a r m s . T h e s t r u c t u r e o of C l r 2 C l s 2, as s u g g e s t e d b y T s c h o p p et al. (1980),

I

5

2

I

(o)

(b)

Fro. 6. (a) The Clr2('ls2 tetramer is shown in this cartoon in the "S" shaped conformation with a vertical 2-fold axis and with tile contact sites all pointing in the same direction. (b) By rotating onehalf of the molecule about a horizontal axis, a circular structure can be formed, maintaining sites intact but pointing in opposite directions.

574

P. H. P O O N E T AL. A

B

B

A

B

(b)

(o)

!

! !

I

@ (c)

(d)

FIO. 7. Several different arrangements are shown of pathways for the backbone of tile Clr2('ls 2. folded around and through tile Clq arms, compatible with tile volumes for tile tetramer, as described in Table 1. The labels A and B distinguish alternate Clq arms joined by a disulfide bond in the noncollagenous region at tile end of tile Clq stem: A and B are quasi-equivalent orientations. Thus. Clq has 3-fold symmetry, and pseudo-6-fold symmetry. The ('lr2('ls 2 tetramer is assumed to have 2-tbld rotational symmetis,, through an axis passing through the center of the flexible molecule, when tile 2 halves are arranged identically. (a) and (b) Top views fi'om the bare end of tile stem. In (a). tile Clr2Cis 2 is wlupped around the outside of the Clq. Since this could be accomplished only if the binding sites were pointing in opposite directions, it cannot be a correct model. In (b), the binding sites are pointing in the same directions and. if A and B ale quasi-equivalent orientations of tile ('lq arms. all of the contacts may be formed. This is tile preferred model. A 4th pair of binding sites also may be located just beneath tile stem of the ('lq. and again these would be quasi-equivalent since the tetramer would pass between arms AB on one side, and between arms BA on the other. (c) and (d) Two possible profile views for the pathway of tile Clr2Cls 2 tetramer: (c) shows a planar tetramer located some distance away from the end of tile stem, (d) shows how the C1 r2Cls 2 could be bent in the middle if the 4th pair of contact sites beneath the stem were to be occupied (this is our'preferred model compatible with volume and symmetl T restrictions, and with most of the profile and top-view images of Figs ! and 2).

is p r o b a b l y a l i n e a l ' a r r a n g e m e n t o f s u b u n i t s in t h e o r d e r C l s - C l r - C l r - C l s . Most of the molecules exhibit a gentle, reversed "S" shape when attached to the electron microscope grid, implying the existence of a 2-fold axis lying perpendicular to and passing through the center of the molecule. For the Clr2Cls: tetramer to bind to all s i x a r m s o n C l q , e a c h h a l f o f t h e t e t r a m e r w o u l d h a v e t o p o s s e s s t h r e e s i t e s ,

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labeled l, 2 a n d 3 in Figure 6. These would bind to three pairs of c o m p l e m e n t a r y sites on t h e six C l q arms, which m a y be labeled l', 2' a n d 3'. O p p o s i t e a r m s o f C l q c o n t a i n i n g these sites are q u a s i - e q u i v a l e n t because o f the disulfide b o n d i n g p a t t e r n , and m a y be distinguished b y labels A a n d B. T h u s , in F i g u r e 7(b), it m a y be seen t h a t the c o n t a c t s are 1-1'A, 2-2'B a n d 3-3'A for o n e - h a l f o f the t e t r a m e r a n d 1 - 1 ' B , 2 - 2 ' A a n d 3 - 3 ' B for the o t h e r h a l f t . Since the A a n d B a r m s possess pairs o f q u a s i - e q u i v a l e n t sites, t h a t is, since t h e y possess identical s t r u c t u r e s with only small differences in o r i e n t a t i o n , a n d since b o t h the t e t r a m e r a n d C l q are flexible, all o f the c o n t a c t s m a y be f o r m e d readily to g e n e r a t e the model s h o w n in Figure 7(b). Thus, the model s h o w n in Figure 7(b) is suggested for C1, in which the t e t r a m e r passes b e t w e e n the a r m s a n d t h e n each h a l f w r a p s back a r o u n d the C l q on the outside o f the cone. I n this model, the 2-fold a n d pseudo-6-fold s y m m e t r y axes coincide a n d three pairs o f q u a s i - e q u i v a l e n t c o n t a c t s are f o r m e d a t the periphery. This w o u l d fulfil b o t h the s y m m e t r y r e q u i r e m e n t s a n d also be consistent with the images seen with the electron microscope. A d d i t i o n a l details c o n c e r n i n g the model are given in the legend to F i g u r e 7. Finally, t h e c o m p o n e n t p a r t s m a y be assembled into the c o m p l e t e d model illustrated in Figure 8. W e do n o t believe a basically different model can be c o n s t r u c t e d in which a flexible rod with 2-fold s y m m e t r y can bind to a cone o f a r m s with 3-fold s y m m e t r y , even utilizing the principle o f q u a s i - e q u i v a l e n c e . ' M o r e c o m p l i c a t e d p a t h w a y s of folding o f the C l r 2 C l s 2 t e t r a m e r could be d r a w n , b u t even these, we

FIG. 8. A profile view is shown of the model suggested for the completely assembled C1 structure, with tile Cl r2Cls 2 tetramer passing through the arms of the C1q, and attached to the stem of the'Clq in the middle. Both ends of the Clr2Cls 2 tetramer wrap around and are attached to the Clq arms on the outside. t The letter designations A and B are used to refer to different orientations of the C1q arms, and not contact sites. The numerical designations I. 2 and 3 refer to different contact sites on the Clr2Cls 2 tetramer. The complementary binding sites on the Cl q arms. which may be designated as 1', 2' and 3', may be different fl'om each other, or they may be identical or overlapping sites. Since each Clq arm will contain all 3 of tile sites l', 2' and 3'. the Cl r2Cls 2 may combine with the Clq in 6 equivalent ways. This may help to offset tile apparent difficulty that would seem to arise in threading the Clr2Cls 2 between the arms of the Clq.

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believe, are variations on the simple theme presented here, whereby the center of the Clr2Cls 2 tetramer passes through the arms close to the stem so that the 2-fold and pseudo-6-fold symmetry axes are brought into coincidence.

(e) Accessibility to Cf-inhibitor The model for the structure of the C1 complex proposed here should not be viewed as a static entity, but rather as a dynamic structure in which the Clr2Cls 2 and Clq subunits are in reversible equilibrium between the free and bound forms. Indeed, the dissociation constant in free solution increases by an order of magnitude when the activated C1 is formed (Siegel & Schumaker, 1983). I f this increase in dissociation constant is caused by an increased rate of dissociation, then the CTr2CYs2 tetramer would probably become readily accessible to the Ci-inhibitor at physiological concentrations. The release of the Clr2Cis 2 might be somewhat slowed if several of the Clq heads were tightly bound to an immune complex, but even in this case, reversible release of individual Clq heads and segmental flexibility might permit rapid dissociation of the activated CTr2C~s2 tetramer. (f) Restricted segmental flexibility of C1 As calculated from the histograms in Figure 4, the average cone angle is 49-75~ and the standard deviation is • ~ for the Clq population of 81 molecules, while the average cone angle is 49"85~ and the standard deviation is • 6"49~ for the C1 population of 65 molecules. Statistically, there is no significant difference between the means, but the differences in standard d.eviations are highly significant (P < 0"001). It is difficult to understand how differential absorption to the supporting carbon film could result in two symmetrical distributions with essentially identical means but with standard deviations that differ by a factor of almost two. We feel these data strongly support the plausible assumption that the addition of the Clr2Cls 2 tetramer to Clq greatly reduces the range of segmental flexibility of the C1 q arms. Some of the apparent loss in flexibility could be due to the presence of the chemical crosslinks; however, the water-soluble carbodiimide employed in this study is a "zero length" crosslinking reagent, which tends to join groups already in close proximity. Moreover, there is indirect evidence that C1 is inherently less flexible than Clq, as discussed next. The addition of Clq to IgG-coated latex beads causes aggregation of the beads. Prior addition of C1, on the other hand, not only fails to induce aggregation but it also prevents aggregation by subsequent addition of Clq (Hallgren, 1979; Hallgren et al., 1979). The latter authors state "The most probable basis of the activity is the competition between C1, with high affinity for IgG~coated particles, and Clq. The inability of C1 to induce particle aggregation might be caused by the Clq subunits, Clr and Cls, sterically inhibiting the subunit Clq to bridge between the particles". Since Figures 1 and 2 show that the Clq heads are exposed in C1, it does not seem likely that the Clr2Cls 2 could be "sterically inhibiting" by concealing some of the binding sites for IgG which are located on

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the Clq heads. The plausible explanation for this steric inhibition is a loss of flexibility and restriction of the arms to a cone angle of 50 ~ occasioned by the binding of Clr2Cls 2. Indeed, if the Clr2Cls 2 is wound first through and then around the Clq arms as suggested in Figure 8, we would expect a rather rigid structure to result. Moreover, this structure for C1 is well-adapted for multivalent binding to a single surface, since the arms form a cone with the heads, and thus the binding sites for Fc regions, located on the periphery and capable of making contact with a single surface. Clq, in contrast, can be widely spread with opposite arms extended at an angle of 180 ~ To serve most efficiently as a bridge between two large structures, a crosslinking reagent should have its binding sites pointing in opposite directions. This is possible for Clq, b u t a p p a r e n t l y not for C1. Thus, we believe t h a t the observed restricted segmental flexibility, compatible with the model for the structure of C1 presented above, is the most plausible explanation for the differences in aggregation behavior reported for Clq and C1. We express our gratitude to the National Science Foundation for its generous support of these studies through research grant no. PCM 80-21368. We also thank the Heart and Lung Institute, National Institutes of Health, for a training grant (HL 7386) for M.L.P. REFERENCES Casper, D. L. (1965). In Viral and Rickettsial Infections in Man (Horsfall, F. L. & Tamm, I., eds), 4th edit., p. 51, Lippincott, Philadelphia. Gilmour, S., Randall, J. T., Willan, K. J., Dwek, R. A. & Torbet, J. (1980). Nature (London), 285, 512-514. Hallgren, R. (1979). Immunology, 38, 529-537. Hallgren, R., Stalenheim, G. & Venge, P. (1979). Scand. J. Immunol. 9, 365-372. Knobel, H. R., Villiger, W. & Isliker, H. (1975). Eur. J. Immunol. 5, 78-81. Metzger, H. (1978). Contemp. Topics Mol.,Immunol. 7, 119-152. Muller-Eberhard, H. J. (1975). Annu. Rev. Biochem. 44, 697-724. Muller-Eberhard, H. J. & Kunkel, H. G. (1961). Proc. Soc. Exp. Biol. Med. 1~, 291-295. Porter, R. R. & Reid, K. B. M. (1979). Advan. Protein Chem. 33, 1-71. Reid, K. B. M. & Porter, R. R. (1976). Biochem. J. 155, 19-23. Schumaker, V. N., Poon, P. H., Seegan, G. W. & Smith, C. A. (1981). J. Mol. Biol. 148, 191197. Shelton, E., Yonemasu, K. & Stroud, R. M. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 65-68. Siegel, R. C. & Schumaker, V. N. (1983). Mol. Immunol. 20; 53-66. Strang, C. J., Siegel, R. C., Phillips, M. L., Poon, P. H. & Schumaker, V. N. (1982). Proc. Nat. Acad. Sci., U.S.A. 79, 586-590. Svehag, S.-E., Manhem, L. & Bloth, B. (1972). Nature.New Biol. 238, 117-118. Tschopp, J. W., Villiger, W., Fuchs, H., Kilchherr, E. & Engel, J. (1980). Proc. Nat. Acad. Sci., U.S.A. 77, 7014-7018. Edited by H. E. Huxley