View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Structure 14, 1167–1177, July 2006 ª2006 Elsevier Ltd All rights reserved DOI 10.1016/j.str.2006.05.011 Lumbricus Erythrocruorin at 3.5 A˚ Resolution: Architecture of a Megadalton Respiratory Complex William E. Royer, Jr.,1,2,* Hitesh Sharma,1,3 subunits occupy nearly equivalent positions with only Kristen Strand,1,4 James E. Knapp,1 slightly altered bonding patterns. Quasi-equivalence and Balaji Bhyravbhatla1 remains an important theoretical underpinning of our 1 Department of Biochemistry and Molecular understanding of spherical virus construction, although Pharmacology viral subunits have shown substantially greater bonding University of Massachusetts Medical School variations than originally envisioned (Harrison, 2001). Worcester, Massachusetts 01655 Symmetrical arrangements of multiple subunits are also observed in invertebrate giant extracellular respira- tory proteins. These include the copper-containing he- Summary mocyanins, from arthropods and mollusks (van Holde and Miller, 1995), and heme-containing respiratory pro- Annelid erythrocruorins are highly cooperative extra- teins, such as those found in annelid worms. The most cellular respiratory proteins with molecular masses prevalent of these annelid complexes are known as ei- on the order of 3.6 million Daltons. We report here ther erythrocruorins or hexagonal bilayer hemoglobins. the 3.5 A˚ crystal structure of erythrocruorin from the Our designation of these proteins as erythrocruorins earthworm Lumbricus terrestris. This structure re- emphasizes the requirement of nonhemoglobin linker veals details of symmetrical and quasi-symmetrical subunits for assembly (Kuchumov et al., 1999; Lamy interactions that dictate the self-limited assembly of et al., 2000; Zhu et al., 1996) and their similar overall ar- 144 hemoglobin and 36 linker subunits. The linker sub- chitectures to those of annelid chlorocruorins (de Haas units assemble into a core complex with D6 symmetry et al., 1996a, 1997; Schatz et al., 1995). Because of their onto which 12 hemoglobin dodecamers bind to form extracellular nature and giant size, erythrocruorins were the entire complex. Although the three unique linker important subjects for seminal biophysical investiga- subunits share structural similarity, their interactions tions. Lumbricus erythrocruorin was the first protein with each other and the hemoglobin subunits display ever reported to be crystallized, in 1840 by Hu¨ nefeld striking diversity. The observed diversity includes (McPherson, 1999), one of the first proteins investigated design features that have been incorporated into the in Svedberg’s initial ultracentrifugation experiments linker subunits and may be critical for efficient assem- (Svedberg and Ericksson-Quensel, 1933), and an early bly of large quantities of this complex respiratory molecular subject of electron microscopy (Roche protein. et al., 1960). Both erythrocruorins and chlorocruorins display overall D6 dihedral symmetry; similarly to icosa- hedral viruses, however, many more subunits are pres- Introduction ent than can be accommodated in the 12 equivalent D6 symmetry positions. Assembly of functional units into large macromolecular A number of biological advantages arise from the for- complexes permits greater coordination and regulation mation of the giant erythrocruorins. Such large com- in many biological processes. Design of large macromo- plexes can be readily retained as freely dissolved enti- lecular complexes often involves symmetrically arranged ties in the vascular system and each complex can be subunits. Symmetrical assemblages require surfaces endowed with a very large oxygen binding capacity. used for subunit interactions to be fully satisfied in order Moreover, subunits can be arranged in a manner to per- to prevent unwanted further assembly, thus ensuring mit intersubunit communication that can result in coop- such biological assemblies are limited to a discrete size. erative oxygen binding along with additional regulatory This can be achieved by the use of one of the closed features that promote efficient oxygen transport. In the point symmetry groups—cyclic, dihedral, tetrahedral, case of Lumbricus erythrocruorin, highly cooperative octahedral, or icosahedral. However, for a number of oxygen binding (Hill coefficient = 7.9, under conditions assemblages, the symmetry expressed in these point of maximum cooperativity) is coupled with the binding groups does not suffice for the quantity of subunits of cations and protons (Fushitani et al., 1986). Due to present. Noteworthy examples include spherical viruses their large size, extracellular nature, and resistance to that are commonly arranged with icosahedral symmetry, oxidation (Dorman et al., 2002; Harrington et al., 2000), but most often contain many more subunits than can erythrocruorins have been proposed as useful model be accommodated by the 60 equivalent icosahedral systems for developing therapeutic extracellular blood symmetry positions. Caspar and Klug (1962) proposed substitutes (Hirsch et al., 1997; Zal et al., 2002). an elegant solution to this problem with the principle Our previous 5.5 A˚ resolution crystal structure of Lum- of quasi-equivalence, in which chemically identical bricus erythrocruorin revealed an architecture of 144 he- moglobin subunits arranged into 12 dodecamers that assemble onto a central scaffold of 36 linker subunits *Correspondence: [email protected] (Royer et al., 2000). This has been complemented by 2 Lab address: http://www.umassmed.edu/bmp/faculty/royer.cfm a 2.6 A˚ resolution crystal structure of purified hemoglo- 3 Present address: Department of Molecular Biophysics and Bio- chemistry, Yale University, New Haven, Connecticut 06511. bin dodecamers (Strand et al., 2004). We report here the 4 Present address: Genzyme Corporation, Cambridge, Massachu- crystal structure of the entire Lumbricus erythrocruorin setts 02142. molecule at 3.5 A˚ resolution. Our results, which, to our Structure 1168 Figure 1. Stereodiagrams of 24-Fold Aver- aged Electron Density Maps for Lumbricus Erythrocruorin (A) The heme region for one hemoglobin sub- unit a is shown with gray contours at 1.5s and magenta surface at 12s around the heme iron position. Included with the density are atomic models for the heme group, proximal histi- dine (F8), distal histidine (E7), and B10 Phe. (B) An a-carbon trace (red) for the b barrel domain of L1 is shown with electron density at a contour level of 3s superimposed. (C) An a-carbon trace (red) for the linker triple- stranded coiled coil is shown with electron density contoured at 3s. The density for the coiled coil shows the course of the helices, but poorer density for the amino termini at the bottom and side chains (compare with middle panel) is consistent with high refined group B factors suggesting high mobility for this portion of the linker structures. knowledge, provide the first complete set of atomic we were able to obtain a readily interpretable map, por- models for an entire megadalton complex from this tions of which are shown in Figure 1. An atomic model, class of respiratory proteins, reveal the central role of in- including two whole molecules (7.2 million Daltons) per teractions among three distinct linker subunits to dictate asymmetric unit, has been refined to a conventional R the intricate hierarchy of symmetry of these complexes. factor of 0.288 and to a free R of 0.297. Crystallographic statistics are provided in Table 1. Results Overall Structure Growth of a new triclinic crystal form of Lumbricus Lumbricus erythrocruorin is assembled from 180 poly- erythrocruorin permitted the determination of this struc- peptide chains into an overall hexagonal bilayer shape ture to 3.5 A˚ resolution. As described in Experimental (Figure 2). The vertices of two hexagonal halves of the Procedures, the starting point for phasing the new dif- molecule are partially staggered, with one half rotated fraction data was the 5.5 A˚ electron density maps ob- about the 6-fold axis by about 16º from an eclipsed tained from orthorhombic crystals grown in 1.8 M phos- arrangement, as was evident from cryo-electron micro- phate. Using molecular averaging for phase extension, scopic investigations (de Haas et al., 1997; Schatz et al., Architecture of a Megadalton Respiratory Complex 1169 Table 1. Crystallographic Statistics quences. Chains a (B2), b (A1), and c (B1) are disulfide linked into a heterotrimer, whereas chain d (A2) is not Crystal Parameters disulfide linked with any other subunits. All hemoglobin Space group P1 subunits contain an intrasubunit disulfide bond. Each a(A˚ ) 176.08 dodecamer is arranged with three d chains clustering b(A˚ ) 257.96 near the local 3-fold axis and three disulfide-linked abc ˚ c(A) 436.53 trimers in an extended arrangement around the dodeca- Data Collection mer’s periphery (Figure 3). This gives rise to an overall Resolution range (A˚ ) 100–3.5 domed shape, with the d chains at the apex of the Highest shell (A˚ ) 3.63–3.5 dome forming a concave surface that interacts with Total observations 1,718,296 the linker subunits. All hemoglobin subunits are involved Unique reflections 890,862 in EF dimer pairing, in which an extensive dimeric inter- a Completeness (%) 89.3 (74.9) face forms from contacts involving the E and F helices <I/s(I)> 8.4 (2.0)a a and heme groups; EF dimer pairing has been found in Rmerge (%) 7.8 (35.4) all cooperative invertebrate hemoglobins