Arrangement of subunits and domains within the Octopus dofleini

Transcription

1 Proc. Nadl. Acad. Sci. USA Vol. 87, pp , February 1990 Biochemistry Arrangement of subunits and domains within the Octopus dofleini hemocyanin molecule (protein assembly/subunits/octopus) KAREN I. MILLER*t, ERIC SCHABTACHt, AND K. E. VAN HOLDE* *Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR ; and tbiology Department, University of Oregon, Eugene, OR Contributed by K. E. van Holde, December 4, 1989 ABSTRACT Native Octopus dofleini hemocyanin appears as a hollow cylinder in the electron microscope. It is composed of 10 polypeptide subunits, each folded into seven globular oxygen-binding domains. The native structure reassociates spontaneously from subunits in the presence of Mg2+ ions. We have selectively removed the C-terminal domain and purified the resulting six-domain subunits. Although these six-domain subunits do not associate efficiently at ph 7.2, they undergo nearly complete reassociation at ph 8.0. The resulting molecule looks like the native cylindrical whole molecule but lacks the usual fivefold protrusions into the central cavity. Partially reassociated mixtures show dimers of the subunit that have a characteristic parallelogram shape when lying flat on the electron microscope grid, and a "boat" form in side view. Removal of the C-terminal domain from monomers results in the removal of two characteristically placed domains in the dimers. These observations allow the development of a model for the arrangement of the subunits within the whole molecule. The model predicts exactly the views seen in the electron microscope of both whole molecule and dimeric intermediates. Hemocyanins are copper proteins that serve to transport oxygen in many species of arthropods and molluscs (1, 2). Although arthropod and molluscan hemocyanins have long been assumed to be closely related proteins, recent sequencing studies indicate that these two classes of hemocyanins have probably evolved independently from copper proteins such as tyrosinase (3, 4). Pronounced differences in both primary and quaternary structure differentiate the two classes. The hemocyanins of arthropods are built up from subunits of moderate size (ca. 75 kda), each containing one binuclear copper oxygen-binding site. As a consequence of x-ray diffraction studies, the structures of these proteins are becoming well understood (5, 6). Much less is known concerning the structures of molluscan hemocyanins. These proteins exist in the hemolymph as very large molecules, in most cases assembled as 1O-mers or 20-mers of polypeptide chains. Each chain is immense, having a molecular mass kda (1, 2). Each of these subunits contains in turn a number of folded domains (or functional units), each of mass ca. 50 kda and carrying one binuclear copper site. No x-ray diffraction analyses on any of these molluscan hemocyanins or their subunits have been reported to date, although crystals and diffraction patterns have been obtained for the C-terminal domain of Octopus hemocyanin (7). For a number of years, our laboratory has been investigating the structure of the hemocyanin of the Pacific octopus, Octopus dofleini. We have shown that the functional molecule in the hemolymph is a decamer, of mass 3600 kda composed of 10 chains of mass 360 kda (8). Electron micro- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact graphs of the native molecule are shown in Fig. la] and lbl. The molecule is a hollow circular cylinder; the top view (Fig. la]) exhibits a fivefold symmetry with a highly reproducible pattern of five small projections into the central cavity. Diameter is about 320 A. The side view (Fig. lb]) shows a three-tiered structure, with no evidence of axial asymmetry. The decameric whole molecule requires divalent ions for stability and can be dissociated into subunits by dialysis against EDTA. This dissociation has been shown to be wholly reversible upon restoration of divalent cations to the solution (8, 9). The subunits obtained by dissociation exhibit the structure shown in Fig. le], each showing seven globular domains. Specific cleavage between these domains can be achieved by limited proteolysis; through such studies the domains have been shown to be immunologically distinct (10). Each contains one oxygen-binding site. The domains have earlier been designated by labels Odl-0d7, on the basis of immunological purification. Since the sequence of domains in the polypeptide chain has now been established (4) to be , we now wish to relabel them as domains a-g (Fig. le2). Sequencing of cdna clones has to date provided complete sequences for the three domains at the C terminus (e, f, g) (4, 11) Ȧ key to understanding the structure of the functional hemocyanin molecule lies in determining the roles played by different domains in the assembled structure. For example, we may ask: Which are in the cylinder wall, and which are involved in the inner projections? What is the relative orientation of the 10 subunits-do they array in a parallel or antiparallel manner? How is the three-tiered wall constructed? In recent studies we have been examining by electron microscopy the assembly of Octopus hemocyanin, using both native subunits and subunits from which a specific domain has been removed. We believe that these studies now allow unambiguous answers to the above questions. MATERIALS AND METHODS Hemocyanin was purified from whole blood by gel filtration on Bio-Gel A-Sm (Bio-Rad) in 0.1,I (ionic strength) Tris, ph 7.65/50 mm MgCl2/10 mm CaCI2. The C-terminal domain was removed by limited proteolysis with protease from Staphylococcus aureus strain V8 (EC ; Sigma) in 0.05 M ammonium carbonate at ph 8.0 (enzyme/substrate ratio, 10-4; 370C for 24 hr). A series of trial digestions was carried out for various times and with several enzyme concentrations to determine the optimal conditions for removal of the C-terminal domain with minimal further cleavage. The resultant digest was subjected to gel filtration on Bio-Gel A-0.5m at 40C in 0.1 ji Tris (ph 7.2) in order to separate the 6D fragment from undigested (7D) subunits and smaller Abbreviation: nd, n-domain. tto whom reprint requests should be addressed.

2 Biochemistry: Miller et al. al bi cl *: *S;-i W\~t *> 5;F t ;; - - a.4 Xrtr. w Proc. Natl. Acad. Sci. USA 87 (1990) ; '- Sa el,v.s v A t > > > s _ s sw Jkt r.-. t. _ IL~~~~ 1SG ~~ I'4f3.N FIG. 1. Electron micrographs of 0. dofleini hemocyanin and the proposed model ofthe three-dimensional structure. Column 1, seven-domain (7D) native hemocyanin; column 2, model structure; column 3, 6D modified hemocyanin; row a, top view of whole molecule; row b, side view of whole molecule; row c, top view of dimer; row d, side view of dimer; row e, subunit. (x345,oo0.) fragments. Reassociation of the 6D fragment was examined by rapid dialysis against 0.1,u Tris buffer containing 50 mm MgCl2 and was monitored by analytical ultracentrifugation in a Beckman model E equipped with a photoelectric scanner. Electron microscopy on both the intact (51S) molecule and the reassociating 7D and 6D subunits utilized negative staining with 0.5% or 0.8% uranyl acetate on thin carbon films supported on nets. Grids with support film were rendered hydrophilic by glow discharge shortly before use. Just prior to application to grids, preparations of the intact (SiS) molecule were diluted with 0.1 M Tris buffer containing 50 mm Mg2+ to concentrations suitable for staining. For reassociation studies of 7D or 6D subunits, small amounts of a 4 M solution of MgCl2 were added to give final concentrations between 30 mm and 100 mm to solutions of the subunits in 0.1 M or 0.05 M Hepes buffer. The reassociating mixture was sampled at timed intervals thereafter. Reassociations were carried out either at a protein concentration suitable for staining without further dilution or at higher concentrations with subsequent dilution with the appropriate buffer immediately before staining. All reassociations were done at C. Microscopy employed a Philips CM-12 electron

3 1498 Biochemistry: Miller et al. microscope operated at 80 or 100 kv. Low-dose procedures were used. Magnification was calibrated using crystalline catalase. RESULTS AND DISCUSSION In previous studies, we examined the Mg2+-activated reassociation of Octopus hemocyanin subunits into functional decamers, using light scattering, fluorescence, and sedimentation techniques (12). The sedimentation studies showed that the process was slow, and indicated the major components present during reassociation to be completely assembled molecules and unreacted monomer. Light scattering and fluorescence experiments showed that the rate-limiting step is second order in monomer. We now find, by electron microscopy of samples taken during reassociation, evidence for a number of intermediate species. The smaller of these take the form of crescents with two beads on the concave side (Fig. lcj); others appear to be small parallelograms (Fig. ldj). We believe these to be two views of a dimer of the subunit, for very similar structures can be isolated when Helix pomatia hemocyanin is taken to high ph; these have been demonstrated to be dimers by molecular weight measurement (13). The sedimentation coefficient of such dimers is found to be approximately 20 S. It is not the aim of this paper, however, to describe the reassociation process in detail; this will be the topic of future publication. Rather, we wish to focus here on the role played by the C-terminal functional unit, domain g, in forming the decameric structure. The results will in turn allow us to propose a model for the packing of subunits in the whole molecule. Earlier studies have shown that domain g can be removed from the whole subunit by mild digestion with either trypsin or S. aureus V8 protease (10). After exploratory studies with both enzymes, we found that the V8 protease was the more selective in cleavage, cutting first between domains f and g and later between domains c and d. Limited digestion followed immediately by gel filtration on Bio-Gel A-0.5m produced the elution pattern shown in Fig. 2. It has distinct peaks for the 6D, 3D, and 1D fragments. SDS/ polyacrylamide gel electrophoresis (Fig. 2 Inset) showed that peak 6 (lane 3 from left) was highly enriched in a fragment that migrated slightly more rapidly than the 7D native subunit. 2 Proc. Natl. Acad. Sci. USA 87 (1990) Calibration of the gel confirmed that this was a 6-unit residuum (data not shown). Further confirmation came from electron microscopic analysis of the material in peak 6, which showed predominantly 6D rather than 7D particles (i.e., Fig. le3). As in the case of the intact 7D subunit, we see here no consistent spatial arrangement of the domains in the isolated subunits. That the single unit first removed by V8 protease is in fact domain g has been demonstrated in earlier studies (10). Purification of the 6D fragment allowed us to ask the following questions. (i) Is domain g required for reassociation into decamers? (ii) If 6D subunits can reassociate, can we deduce from the structure of the reconstituted decamer the position of the g units in the molecule? Reassociation experiments were performed by rapid dialysis of the purified 6D fragment against buffer containing 50 mm MgCl2. Under these conditions, reassociation has been shown to go to completion with 7D fragments over the ph range (9). At ph 7.2, some reassociation of 6D fragments could be detected in the analytical ultracentrifuge, but it did not proceed much beyond the =20S dimer. At ph 8.0, however, at least 75% of the sedimenting protein had reassembled into a very homogeneous population of 46S particles. Fig. 3 shows the distribution of the sedimentation coefficient at each ph, calculated according to the method of van Holde and Weischet (14). These reassociated mixtures were then examined in the electron microscope. At ph 8.0 (Fig. 4a) we saw predominantly structures that appeared very similar to the native 51S whole molecule in side view (Fig. 1b3), but in the top view the cylinder lacked the five distinct projections into the central space (Fig. 1a3). Occasionally, a molecule was observed in which one central projection (or portion thereof) could be observed. We interpret these as molecules in which contaminating 7D fragments have been incorporated. As we have already observed for native hemocyanin (Fig. 1 al and a2), the diameter of this molecule when measured in face view is about 320 A; in side view the diameter is about 380 A and the thickness about 190 A. The larger diameter seen in side view is almost certainly an artifact due to the flattening and consequent elongation of the molecules when they are oriented "on edge" on the support film. At ph 7.2 (Fig. 4b) a few whole molecules could be seen but the major portion of the sample consisted of dimers and monomers. The dimer formed from 6D fragments was also similar in side view (Fig. 1d3) to the 7D version, but in top view (Fig > 0.6 a) ph 8 Z ' 0 Fraction FIG. 2. Elution profile of 0. dofleini subunits following digestion with S. aureus V8 protease and gel filtration on Bio-Gel A-0.5m, in 0.1,u Tris (ph 7.2). Peaks 6, 3, and 1 contain primary 6D, 3D, and 1D proteins. (Inset) SDS/7.5% polyacrylamide gel electrophoresis of, from left to right, native 7D subunit, total digested mixture, peak 6, peak 3, and peak s value, S FIG. 3. Distribution of sedimentation coefficient (s) calculated according to the method of van Holde and Weischet (14) for 6D 0. dofleini hemocyanin subunits reassociated in 0.1,u Tris/50 mm MgCl2 at ph 7.2 or 8.0. Reassociation is much more homogeneous and complete at ph 8.0.

4 Biochemistry: Miller et al. Proc. Nati. Acad. Sci. USA 87 (1990) 1499 FIG. 4. Electron micrographs of partially reassociated 6D subunits of 0. dofleini hemocyanin, showing many dimers at ph 7.2 (a) and many complete molecules at ph 8.0 (b). (x 104,400.) 1c3) it lacked the two small domains on the inner side of the crescent. The unassociated monomer (Fig. 1e3) had only six domains. The three-tiered structure of the side wall of the cylindrical whole molecule, and the fact that this view is unaltered in the reconstitute from 6D subunits, establishes that the first six of the seven domains in the Octopus subunit form the side wall. The seventh domain (g) projects into the interior space; these must be evenly distributed top and bottom to account for the symmetrical side views. Thus we conclude that the 10 subunits must be arranged in antiparallel pairs to form the cylindrical structure, a conclusion consistent with the apparent fivefold axis of symmetry in this decameric molecule. The effect produced on electron microscopic images of the dimer by removal of the two g domains gives further confirmation of the position of domain g. Also, the fact that two domains are removed from this image when 6D monomers are used gives additional proof that it does indeed represent a dimer. FIG. 5. Model of a dimer of two subunits of 0. dofleini hemocyanin, used to build the molecular models of Fig. 1 a2-d2. FIG. 6. Two fivefold rotational integration photographs of the top view of 0. dofleini hemocyanin. (x 1,075,000.) The question now remaining is: How are the other six domains arranged to form the three-tiered side wall? For evidence on this question we turn to the various intermediates found in reassociation kinetics experiments (15). The side views of the dimer have a characteristic parallelogram shape. If one considers the angles of this parallelogram and the dimensions of the cylinder, the diagram shown in Fig. 5 presents itself as a probable arrangement of the subunits within the side wall. It requires that the subunits be arranged diagonally with two domains from the subunit in each of the three tiers. Ten subunits folded in this way would give a cylinder 3 domains high and 20 domains in circumference, which is in good agreement with the molecular dimensions. A model was constructed on this basis and photographs (Fig. 1 a2-d2) predict exactly the molecular shapes and dimensions seen in the electron microscope (Fig. 1 al-di). It should be noted that such a model, with each domain g projecting into the central space, gives just the kind of fivefold image seen in Fig. lal. Fig. 6 shows fivefold rotational integration ("Markham rotation") (16) of two different face views of 51S molecules. These rotations give further support to the model structure. In the model (Fig. 1a2) domain g appears as five pairs of beads on the inner face of the cylinder. These are offset from each other rotationally by approximately 1 domain. The rotation photograph shows this offset arrangement clearly. The dimer in top view shows a crescent with two beads on the concave side (Fig. 1c2) and in side view a parallelogram with 3- and 4-domain-wide sides (Fig. 1d2). Occasionally one sees a trimeric parallelogram (not shown), but never a larger side view. Trimeric and larger top views are common. Perhaps side views of partially reassembled molecules larger than trimers are structurally unstable due to their curvature and break apart when flattened against the grid. There are several ways in which the subunit could be folded to fit six domains into the side wall in a parallelogram array. Fig. 5 depicts one of the simplest. Our model bears some resemblance to that proposed by van Breeman et al. (17) and by Berger et al. (18) for H. pomatia hemocyanin. However, the evidence that H. pomatia hemocyanin forms an asymmetric decamer from 8D subunits leads to a quite different structure. The Berger et al. model could, of course, give no information about the location of specific domains. Confirmation of specific details of the model we propose must await further studies using antibodies to individual domains. We especially wish to thank Dr. Fumio Arisaka, who originally suggested using the electron microscope to follow association of Octopus hemocyanin subunits. This work was supported by National Science Foundation Grant DMB (K.E.v.H. and K.I.M.). K.E.v.H. is the recipient of an American Cancer Society research professorship. The electron microscope was obtained with funds from National Institutes of Health Biomedical Research Support Shared Instrumentation Grant 1-S10-RR (E.S.).

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