PRINCIPLES IN THE ASSEMBLY OF ANNELID ERYTHROCRUORINS WAYNE A. HENDRICKSON AND WILLIAM E. ROYER, JR. Department ofBiochemistry and Molecular Biophysics. Columbia University. New York. New York 10032 ABSTRACT Erythrocruorins are giant extracellular respiratory proteins found freely dissolved in the blood ofannelids. We present here results from our ultracentrifugation, electron microscopy, spectroscopy, and diffraction experiments on these erythrocruorins. These data are rationalized in terms of a three-dimensional model of the quaternary structure. The proposed structure is arranged in a hierarchy of symmetry. The implications of this structure for the assembly process are considered with special attention to uniqueness and self-limitation. The hypothesis is consistent with observations not used in its construction and it serves as a working hypothesis to focus further experimentation. INTRODUCTION and many others. Some, such as the fibrin clot, occur in a 1 he extracellular respiratory proteins found in the bloods statistical distribution of sizes and configurations. Others, of many invertebrates comprise complex assemblages of such as microtubules or deoxyhemoglobin S fibers, are many subunits and have molecular weights in the millions well-defined in a repeating pattern but are not naturally (1). These giant molecules include the hemocyanins of limited to a specified length. However, the biological molluscs (up to 160 oxygen-binding domains in 20 poly­ activity of many macromolecular assemblages demands peptide chains for a total mass of 8-9 x 106d), the that they be specific, precisely defined entities. Mecha­ arthropod hemocyanins (up to 48 subunits for a molecular nisms must exist in the assembly process to avoid uncon­ mass of 3.6 x 106 d) and several types of hemoglobins, trolled or unlimited aggregation. This is not a trivial otherwise known as erythrocruorins. The erythrocruorins problem when the ensemble is a complex structure that of annelids are composed of some 200 subunits organized comprises dozens of subunits, many of which may be in a hierarchal symmetry that generates a characteristic identical or very similar. 3.9 x 106 d structure. There is, of course, a simple solution to the design of We have undertaken a structural study of these giant unique structures from multiple copies of identical sub­ respiratory proteins both to understand their cooperative units. This is by the use of one of the closed-point symme­ oxygen-binding properties and to examine principles try groups-----<:yclic, dihedral, tetrahedral, octahedral, or involved in the assembly process. Our immediate focus is icosahedral-as happens in many oligomeric proteins (2). on the annelid erythrocruorins and particularly on the Symmetry properties can have important functional conse­ earthworm protein. We report here on our initial experi­ quences as in the Monod, Wyman, and Changeaux inter­ mental observations on erythrocruorins and present a pretation of cooperative interactions of proteins with hypothesis of the structural organization in these mole­ ligands and substrates (3). Symmetry is also important in cules. Deductions about the assembly process are inti­ the economical packaging of genetic information in mately dependent on knowledge of the structure. Diffrac­ viruses. But, for most spherical viruses, simple point group tion studies that we have underway should provide defini­ symmetry does not suffice. More "identical" subunits exist tive structural information, but even now some aspects of than can be accommodated by the 60 equivalent positions the principles that govern assembly in these molecules can of icoshahedral point symmetry. Caspar and Klug intro­ be examined. We expect that important features of duced the principle of quasiequivalence to solve this prob­ assembly principles at work in the erythrocruorins will also lem in their seminal paper on virus construction (4). pertain to questions of assembly in other biological sys­ High-resolution crystallographic structures of RNA plant tems. viruses (5-7) have confirmed the spirit if not the literal predictions of this theory (8). The icosahedral viruses provide a fascinating and Questions of Assembly instructive arena for the study of macromolecular Large molecular aggregates play important roles in many assembly properties, especially in light of the theoretical biological structures. These include ribosomes, muscle foundation provided by Caspar and Klug. However, other filaments, viruses, multienzyme complexes, nuclear pores, important structures are very differently constructed and BIOPHYS. J. © Biophysical Society 0006-3495/86/01/177/13 $1.00 177 Volume49 January 1986 177-189 other rules ofassembly apply. These systems, often charac­ resolution by HoI and colleagues (17). Anthropodan hemo­ terized by quasisymmetry and hierarchies of symmetry, cyanins are composed of heterogeneous subunits and these also pose interesting assembly questions. are associated in multiples of 1, 2, 4, or 8 hexamers One such system is the acetylcholine receptor. Each depending on the species. Lamy and coworkers have found receptor unit comprises five polypeptide chains in the that the 24-mer of scorpion hemocyanin consists of eight stoichiometry of a.jJ-yo. The chains are homologous in electrophoretically and immunologically distinct subunits. sequence at the 40% level (9). Electron microscopy and These subunits occur in stoichiometric amounts and their labeling studies show that the protein subunits are positions in the molecule have been reproducibly located in arranged in a cylindrical wall about a central cavity in the electron micrographs of molecules labeled by specific Fab cyclic order of-{3-Ci-')'-Ci-O- (10). This poses very interest­ antibody fragments (18,19) as shown in Fig. 1. Amino­ ing questions of how the two identical a-chains assemble to acid sequences indicate that the subunits within one species adapt specifically to distinctive yet similar environments. and also between different species are similar (20). Another significant class of proteins that present puzzles Clearly, if all subunits were identical, a molecule con­ in assembly are the proton ATPases from mitrochondria structed in such a hierarchy of symmetries would not be and chloroplasts. The soluble catalytic F1 factor of these self-limited. Scorpion hemocyanin has evolved an elegant enzymes consists of five kinds of polypeptide chains in the solution to this problem. The distinctive roles ofquasisym­ stoichiometry a3{33-y0f:. Sequences of the large a and (3 metrically related subunits permit uniqueness through chains are found to be highly homologous, whereas the variations on a simple theme. minor chains are unique and smaller (11). Quite unex­ The erythrocruorins found freely dissolved in the bloods pectedly in light of the composition, F1 molecules lie on of annelids bind oxygen at the iron atoms of heme groups twofold axes in crystals (12). These results raise questions as in hemoglobin. However, unlike their mammalian coun­ about how identical chains achieve specialization and terparts, these molecules are very large and not all of the about how chemically distinct chains behave identically. protein subunits bear hemes. Functionally, they are highly Phycobilisomes provoke another set of assembly ques­ cooperative, with Hill coefficients as high as 5.5. There is tions. These light-harvesting pigments in cyanobacteria an abundant literature on erythrocruorins and many fea­ and red algae are large (6--7 x 106 d) complexes ofstacked tures are clear, but there is also considerable confusion. discs arrayed around a central core. The elements of the Details will be dealt with in the presentation of results. By disc are phycocyanins or phycoerythrins that have the way of introduction, let it suffice to give a brief overview. composition (a{3)6 and have D3 point symmetry (13, 14). These are giant molecules with a mass of nearly four Each disc has two or three such units in structurally million daltons each. They are arranged in two apposed distinct sites. These are connected by "linker" polypeptides layers of six-membered rings that can be dissociated into to the core allophycocyanins and to one another (15). The twelve major subunits. The 1/12th particles can in turn be role of linker proteins helps to explain the assembly further dissociated into a few kinds of 15-20,000 d chains. properties of phycobilisomes, but questions still remain Some of these chains are like the mammalian globins, but about how specificity is achieved in this complex hierarchy others appear to be nonhelical and without heme. It seems ofsymmetries and how the unique linkers interact with the clear that the annelid hemoglobins are organized in an symmetric phycoproteins. ' Many of the same assembly questions that are encoun­ tered in these and other systems also arise from work in the giant respiratory proteins of invertebrates. Giant Respiratory Proteins The molluscan hemocyanins appear to be organized with Cs or Ds symmetry, depending on the species, and have two polypeptide chains in the asymmetric unit. Each of these chains is a remarkable string ofseven or eight domains that can be cleaved into functional 50,OOO-d oxygen-binding units. The assembly properties of these exceedingly large and complex molecules is certainly of interest, but rela­ tively little is presently known about structural details (16). The state of knowledge about arthropodan hemocyanins FIGURE I A schematic representation of the quaternary structure of is more advanced.