Proc. Nadl. Acad. Sci. USA Vol. 87, pp. 1496-1500, 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 97331-6503; and tBiology Department, University of Oregon, Eugene, OR 97403 Contributed by K. E. van Holde, December 4, 1989 ABSTRACT Native Octopus dofleini hemocyanin appears graphs of the native molecule are shown in Fig. la] and lbl. as a hollow cylinder in the electron microscope. It is composed The molecule is a hollow circular cylinder; the top view (Fig. of 10 polypeptide subunits, each folded into seven globular la]) exhibits a fivefold symmetry with a highly reproducible oxygen-binding domains. The native structure reassociates pattern of five small projections into the central cavity. spontaneously from subunits in the presence of Mg2+ ions. We Diameter is about 320 A. The side view (Fig. lb]) shows a have selectively removed the C-terminal domain and purified three-tiered structure, with no evidence of axial asymmetry. the resulting six-domain subunits. Although these six-domain The decameric whole molecule requires divalent ions for subunits do not associate efficiently at pH 7.2, they undergo stability and can be dissociated into subunits by dialysis nearly complete reassociation at pH 8.0. The resulting molecule against EDTA. This dissociation has been shown to be wholly looks like the native cylindrical whole molecule but lacks the reversible upon restoration of divalent cations to the solution usual fivefold protrusions into the central cavity. Partially (8, 9). reassociated mixtures show dimers of the subunit that have a The subunits obtained by dissociation exhibit the structure characteristic parallelogram shape when lying flat on the shown in Fig. le], each showing seven globular domains. electron microscope grid, and a "boat" form in side view. Specific cleavage between these domains can be achieved by Removal of the C-terminal domain from monomers results in limited proteolysis; through such studies the domains have the removal of two characteristically placed domains in the been shown to be immunologically distinct (10). Each con- dimers. These observations allow the development of a model tains one oxygen-binding site. The domains have earlier been for the arrangement of the subunits within the whole molecule. designated by labels Odl-0d7, on the basis ofimmunological The model predicts exactly the views seen in the electron purification. Since the sequence of domains in the polypep- microscope of both whole molecule and dimeric intermediates. tide chain has now been established (4) to be 7-4-3-6-5-2-1, we now wish to relabel them as domains a-g (Fig. le2). Hemocyanins are copper proteins that serve to transport Sequencing of cDNA clones has to date provided complete oxygen in many species of arthropods and molluscs (1, 2). sequences for the three domains at the C terminus (e, f, g) (4, Although arthropod and molluscan hemocyanins have long 11). been assumed to be closely related proteins, recent sequenc- A key to understanding the structure of the functional ing studies indicate that these two classes of hemocyanins hemocyanin molecule lies in determining the roles played by have probably evolved independently from copper proteins different domains in the assembled structure. For example, such as tyrosinase (3, 4). Pronounced differences in both we may ask: Which are in the cylinder wall, and which are primary and quaternary structure differentiate the two involved in the inner projections? What is the relative ori- classes. The hemocyanins of arthropods are built up from entation of the 10 subunits-do they array in a parallel or subunits of moderate size (ca. 75 kDa), each containing one antiparallel manner? How is the three-tiered wall con- binuclear copper oxygen-binding site. As a consequence of structed? In recent studies we have been examining by x-ray diffraction studies, the structures of these proteins are electron microscopy the assembly of Octopus hemocyanin, becoming well understood (5, 6). using both native subunits and subunits from which a specific Much less is known concerning the structures of molluscan domain has been removed. We believe that these studies now hemocyanins. These proteins exist in the hemolymph as very allow unambiguous answers to the above questions. large molecules, in most cases assembled as 1O-mers or 20-mers of polypeptide chains. Each chain is immense, AND METHODS having a molecular mass 350-450 kDa (1, 2). Each of these MATERIALS subunits contains in turn a number of folded domains (or Hemocyanin was purified from whole blood by gel filtration functional units), each of mass ca. 50 kDa and carrying one on Bio-Gel A-Sm (Bio-Rad) in 0.1 ,I (ionic strength) Tris, pH binuclear copper site. No x-ray diffraction analyses on any of 7.65/50 mM MgCl2/10 mM CaCI2. The C-terminal domain these molluscan hemocyanins or their subunits have been was removed by limited proteolysis with protease from reported to date, although crystals and diffraction patterns Staphylococcus aureus strain V8 (EC 3.4.21.19; Sigma) in have been obtained for the C-terminal domain of Octopus 0.05 M ammonium carbonate at pH 8.0 (enzyme/substrate hemocyanin (7). ratio, 10-4; 370C for 24 hr). A series of trial digestions was For a number of years, our laboratory has been investi- carried out for various times and with several enzyme con- gating the structure ofthe hemocyanin ofthe Pacific octopus, centrations to determine the optimal conditions for removal Octopus dofleini. We have shown that the functional mole- of the C-terminal domain with minimal further cleavage. The cule in the hemolymph is a decamer, of mass 3600 kDa resultant digest was subjected to gel filtration on Bio-Gel composed of 10 chains of mass 360 kDa (8). Electron micro- 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 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: nD, n-domain. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 1496 Downloaded by guest on September 29, 2021 Biochemistry: Miller et al. Proc. Natl. Acad. Sci. USA 87 (1990) 1497 al .2 ; '- Sa bi cl *: ,vs.v A t > > > s _ s sW - a .4 Xrtr . w Jktr.-.t. _ IL~~~~ el 1SG ~~ I'4f3.N*S;-iW\~t *>5;F t ;; - FIG. 1. Electron micrographs of0. 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 mM Mg2+ to concentrations suitable for staining. For reas- by rapid dialysis against 0.1 ,u Tris buffer containing 50 mM sociation studies of 7D or 6D subunits, small amounts of a 4 MgCl2 and was monitored by analytical ultracentrifugation in M solution of MgCl2 were added to give final concentrations a Beckman model E equipped with a photoelectric scanner. between 30 mM and 100 mM to solutions of the subunits in Electron microscopy on both the intact (51S) molecule and 0.1 M or 0.05 M Hepes buffer. The reassociating mixture was the reassociating 7D and 6D subunits utilized negative stain- sampled at timed intervals thereafter. Reassociations were ing with 0.5% or 0.8% uranyl acetate on thin carbon films carried out either at a protein concentration suitable for supported on nets. Grids with support film were rendered staining without further dilution or at higher concentrations hydrophilic by glow discharge shortly before use. Just prior with subsequent dilution with the appropriate buffer imme- to application to grids, preparations of the intact (SiS) diately before staining. All reassociations were done at molecule were diluted with 0.1 M Tris buffer containing 50 22-24°C. Microscopy employed a Philips CM-12 electron Downloaded by guest on September 29, 2021 1498 Biochemistry: Miller et al. Proc. Natl. Acad. Sci. USA 87 (1990) microscope operated at 80 or 100 kV. Low-dose procedures Calibration of the gel confirmed that this was a 6-unit were used. Magnification was calibrated using crystalline residuum (data not shown). Further confirmation came from catalase. electron microscopic analysis ofthe material in peak 6, which showed predominantly 6D rather than 7D particles (i.e., Fig. RESULTS AND DISCUSSION le3). As in the case of the intact 7D subunit, we see here no consistent spatial arrangement of the domains in the isolated In previous studies, we examined the Mg2+-activated reas- subunits. That the single unit first removed by V8 protease is sociation of Octopus hemocyanin subunits into functional in fact domain g has been demonstrated in earlier studies (10). decamers, using light scattering, fluorescence, and sedimen- Purification of the 6D fragment allowed us to ask the tation techniques (12). The sedimentation studies showed following questions. (i) Is domain g required for reassociation that the process was slow, and indicated the major compo- into decamers? (ii) If 6D subunits can reassociate, can we nents present during reassociation to be completely assem- deduce from the structure of the reconstituted decamer the bled molecules and unreacted monomer. Light scattering and position of the g units in the molecule? fluorescence experiments showed that the rate-limiting step Reassociation experiments were performed by rapid dial- is second order in monomer.