Domain Structure of the αβ′ε-COP Coatomer Complex
Coatomer purified from mammalian cytosol is a heptameric protein that can be disassembled using high salt concentrations into stable βδ/γζ-COP and αβ′ε-COP complexes (Lowe and Kreis, 1995
). To study the cage-forming αβ′ε-COP complex in isolation, we prepared the trimeric protein from Bos taurus by coexpression in baculovirus-infected insect cells. The purified αβ′ε-COP protein was soluble despite the absence of βδ/γζ-COP, and it migrated as a large (~600 kDa) particle on size-exclusion chromatography columns. To define domain boundaries and identify protein interactions in the αβ′ε-COP complex, we digested the protein with subtilisin and analyzed the proteolysis products by chromatography, SDS-PAGE and N-terminal sequencing (see Experimental Procedures). Three major protease-resistant products were obtained (see ): a subcomplex comprising almost full-length β′-COP and a central α-solenoid domain of α-COP; a C-terminal domain of α-COP bound to essentially full-length ε-COP (this interaction was identified previously by Eugster et al. 
); and an N-terminal region of α-COP that includes the β-propeller domain. (This final α-COP product was more difficult to define since a loop within the β-propeller domain is nicked by subtilisin in the vicinity of residue 191).
Domain Organization of the αβ′ε-COP Complex
The results of this preliminary analysis suggested that the αβ′-COP and αε-COP subcomplexes are amenable to structural analysis. Indeed, we were able to determine the crystal structures of both subcomplexes, as described below. Together they account for more than two thirds of the mass of the αβ′ε-COP complex.
Crystal Structure Determination
Crystals of the B. taurus αε-COP heterodimer grew in space group I422. The structure was determined by the multi-wavelength anomalous diffraction method using selenium as the anomalous scatterer ( and Experimental Procedures). The assignment of residues during model building was aided by the selenium atom positions, and the structure was refined with data to 2.6 Å resolution.
Data Collection and Refinement Statistics
It proved more difficult to determine the structure of the αβ′-COP subcomplex. Initially, we obtained crystals of αβ′-COP from B. taurus, but they were of low crystallographic quality. Instead, the equivalent αβ′-COP subcomplex from Saccharomyces cerevisiae (residues 1-814 of β′-COP and residues 642-818 of α-COP) crystallized in space group P3221, with three copies of αβ′-COP —a total of 330 kDa—in the asymmetric unit. The structure was determined by the multiple isomorphous replacement method using crystals derivatized with Ta6Br12 and mercury compounds (see Experimental Procedures). The structure of the yeast αβ′-COP subcomplex was refined to 2.5 Å resolution ().
Structure of the αε-COP Subcomplex
The αβ′-COP subcomplex is the more enlightening of the two crystal structures, so we discuss the αε-COP subcomplex only briefly here.
The molecular models of the two subcomplexes are presented in and . The αβ′-COP and αε-COP crystal structures do not share any overlapping elements. Rather the two subcomplexes are connected by a highly acidic ~80-residue linker region of α-COP (residues 818-900) that may be unstructured according to our proteolysis results (). This suggests that the 65 kDa αε-COP subcomplex is flexibly linked to a larger core region of αβ′ε-COP (). This is reminiscent of the C-terminal α-solenoid domain of Sec31 in the COPII cage, which is flexibly linked to the assembly unit core and probably projects from the cage in towards the membrane vesicle (Fath et al., 2007
Structural Analysis of the αβ′-COP Subcomplex
Architecture of the αβ′-COP and αε-COP Subcomplexes
The sequence of ε-COP contains tetratricopeptide repeats (TPR), and the αε-COP crystal structure reveals that ε-COP forms a characteristic TPR-protein super-helix (Das, et al., 1998
), which coils like a snake around one end of α-COP, to form tight dimer contacts (). Starting from its N-terminus, the TPR domain of ε-COP coils in a right-handed fashion along one end of the 80-Å long rod-shaped α-COP molecule. The TPR domain ends at residue 280, and the C-terminal twenty residues of ε-COP form a final α helix that makes additional contacts with α-COP, so that ε-COP envelops about one-third of the α-COP rod in total. The α-COP C-terminal domain has a mixed tertiary structure of five discrete elements: an N-terminal mostly helical region (residues 915-965); a β-hairpin “finger” (residues 967-983) that is encircled by ε-COP; a three-helix bundle (residues 1003-1074), followed by a short α-solenoid region (residues 1078-1151); and finally a nest of three orthogonally-oriented β-hairpins (residues 1165-1210) toward the C-terminus of the molecule. Overall, the 65 kDa αε-COP subcomplex is a compact rod, 115 Å long and ~35 Å diameter. Its possible location relative to other elements in the COPI lattice is discussed below.
Architectural Overview of the αβ′-COP Subcomplex
The αβ′-COP subcomplex forms a curved structure composed of the 90 kDa β′-COP molecule and 20 kDa α-COP α-solenoid domain ( and ). β′-COP has the domain arrangement β-propeller–β-propeller–α-solenoid, and three copies of the αβ′-COP subcomplex converge through the N-terminal β-propeller domains to form a triskelion ( and ). The α-solenoid domain of α-COP binds in an anti-parallel manner to the α-solenoid of β′-COP to extend the legs of the triskelion (), so that each of the legs is approximately 175 Å along the curved path and 120 Å measured radially (both distances are measured from the triskelion center). The αβ′-COP triskelion does not form around a crystallographic 3-fold axis; rather the three copies of αβ′-COP come together in the asymmetric unit of the crystal. Indeed, the interfacial contacts at the center of the triskelion are not exactly three-fold related, and the long α-solenoid legs deviate incrementally from the three-fold relation the farther they extend from the triskelion center, as might be expected for non-crystallographic symmetry.
Finally, the molecular model we have built for β′-COP most likely includes the entirety of the structured portion of the molecule. A C-terminal element (75-100 residues in yeast and mammalian sequences; see ) of the β′-COP polypeptide was omitted from the crystallographic study, but we conclude that this region is probably unstructured since it is highly acidic and its sequence is not conserved (indeed, this C-terminal element is absent in β′-COP from Schizosaccharomyces pombe).
Structure and Domain Organization of the αβ′-COP Subcomplex
The arrangement of protein domains in the β′-COP subunit is remarkably similar to that observed in the Sec13/31 complex of the COPII cage (which forms one half of the (Sec13/31)2
assembly unit [Fath et al., 2007
]). Sec13/31 adopts a β-propeller–β-propeller–α-solenoid arrangement in which the small Sec13 subunit forms the second β-propeller domain and is thus sandwiched between the N-terminal β-propeller and C-terminal α-solenoid domains of Sec31. The β-propeller domains of β′-COP, like Sec13/31, both comprise seven blades (Figure S1
and ). The N-terminal β-propeller (residues 1-301) is characterized by a regular and compact structure involving short connecting loops between the β-strands at both axial ends of the β-propeller. The effect is to create relatively flat axial ends, which seems to be important for interactions at the triskelion center ( and ). The C-terminal β-propeller (residues 304-600) interacts with the N-terminal β-propeller through a relatively small interaction area, whereby loops contributed from blades 1, 2 and 7 of the C-terminal β-propeller interact with residues on loops 5,6 and 7 of the N-terminal β-propeller (see Figure S1
for the numbering scheme). The small interaction area suggests that there might be some flexibility at this site connecting the two β-propellers. Sites of potential flexibility have been identified in the COPII (Sec13/31)2
assembly unit, and these are proposed to be important for lattice adaptability and the formation of different size cages (Lederkremer et al., 2001
; Fath et al., 2007
; Stagg et al., 2008
Contact surfaces of the αβ′-COP triskelion
In the COPII system, cage architecture is determined by the geometry of the protein-protein contacts at the dyad vertex center and by the spatial relationships of the β-propeller and α-solenoid domains of the assembly unit (Fath et al., 2007
; Stagg et al., 2007
; Stagg et al., 2008
). In particular, the axes of the β-propeller domains of Sec13 and Sec31 are inclined at a 50° angle and the domains are displaced ~15 Å from each other. The β-propeller domains of β′-COP are juxtaposed in a slightly different configuration. The axes of the two β-propellers are almost parallel (as can be seen most clearly in , which is viewed along the β-propeller axes and reveals the “pore” of each domain); and the axes are displaced ~25Å from each other. However, the prevailing observation in this context is that the polarity of the β-propeller domains is conserved in COPI and COPII (the arrows in are meant to convey this relationship). Finally, a characteristic feature of the αβ′-COP subcomplex is the ~90° angle between the axes of the α-solenoid and the C-terminal β-propeller domain, which helps to create curvature and yields the triskelion form of the trimer ( and ). The similarity of the curved aspect to clathrin heavy chain suggests that this is almost certainly an important feature of COPI cage design (Fotin et al., 2004
). The COPII assembly unit is different in this regard, as the axis of the Sec31 α-solenoid domain is roughly parallel with the Sec13 β-propeller axis (), the result of which is that the (Sec13/31)2
assembly unit is a relatively straight rod (Figure S2
, central panel).
Relationship among COPI, COPII and clathrin cages
The α-solenoid domain of β′-COP is ~90 Å long and is composed of sixteen α helices ( and S1
). The α-solenoid is a relatively straight rod, and the curvature present in the α-solenoid region arises not from β′-COP but from the 40° angled interaction with the α-COP α-solenoid domain (). The antiparallel interaction between the α-solenoids of α-COP and β′-COP is highly reminiscent of the homodimer interaction involving the α-solenoid region of Sec31 near the center of the COPII assembly unit (Fath et al., 2007
). Indeed, on closer inspection the α-COP and β′-COP α-solenoids are seen to interact around an approximate twofold symmetry axis; the axis runs between the centers of helix 11 of each α-solenoid domain, and is roughly in the plane of the triskelion. To highlight this relationship we numbered the α-COP α-helices to coincide with β′-COP (Figure S3
shows sequence and structural alignments of the α-COP and β′-COP α-solenoid domains). This relationship, together with their similar domain compositions, hints at a common evolutionary origin for the α-COP and β′-COP proteins ().
The functional relevance of the interaction we observe between α-COP and β′-COP is supported by the G688D mutation in β′-COP, which is present in the original sec27-1
yeast mutant (Duden et al., 1994
; Eugster et al., 2004
). The phenotype is a defect in Golgi-to-ER transport (of dilysine cargo) and, strikingly, a destabilization of α-COP in the cell (Eugster et al., 2004
). The G688D mutation maps to α helix 7 of β′-COP close to the interface with α-COP ( and ). The mutation may cause local instability of the α-solenoid structure in this region of β′-COP, and this would weaken the interactions with α-COP in the vicinity of α helices 13–15 (Figure S4
In summary, through this structural analysis of the αβ′-COP subcomplex, we can recognize differences between the assembly unit cores of COPI and COPII in terms of the juxtaposition of domains in the propeller–propeller–solenoid array. But the overriding conclusion is that the αβ′-COP subcomplex and the (Sec13/31)2 assembly unit of COPII are fundamentally related. The common features—the propeller–propeller–solenoid arrangement, the polarity of the β-propeller domains and their flat axial ends, the anti-parallel interactions of α-solenoid rods—imply an evolutionarily-conserved function for the propeller–propeller–solenoid array. And since these structural features are key to COPII vertex architecture, we propose that the αβ′-COP triskelion constitutes the vertex of the COPI cage ().
Model for the COPI Vertex and Common Architectural Principles of COPI, COPII and Clathrin Vertices
The COPI triskelion is intermediate in design between COPII and clathrin: the domain organization and vertex contacts are strikingly similar to COPII, but the triskelion form—curved legs radiating from a three-fold center—closely resembles the clathrin assembly unit ().
In the schematic diagram (), we have oriented the COPI and clathrin triskelions so that the α-solenoid legs adopt a clockwise curve. This corresponds to a view of the outer face of the triskelion in the clathrin cage (Fotin et al., 2004
). The COPII vertex is likewise a view of its outer face (Fath et al., 2007
). In the absence of an EM image of the cage, we cannot make this assignment for the COPI triskelion, but the difference (whether clockwise or anticlockwise) is a trivial one with respect to cage design, and the resemblance to clathrin in symmetry and form is evident.
The resemblance of the COPI vertex interactions to COPII is even more striking (). The COPI vertex has the simpler arrangement: three αβ′-COP molecules are situated around a threefold rotation axis, so all three N-terminal β-propellers of β′-COP interact in the same way (here we ignore deviations from threefold symmetry in the crystal asymmetric unit). In the COPII vertex, four copies of the assembly unit are situated around a twofold rotation axis, so all four cannot interact in the same way. A proximal pair of Sec31 β-propellers interacts in a somewhat different manner to the distal pair (Fath et al., 2007
). Nevertheless, the two types of Sec31 interactions and the β′-COP interaction are all variations on a geometric theme of β-propeller domains interacting via their flat axial ends ().
In the absence of an EM image of the COPI cage, we do not have direct evidence that the αβ′-COP triskelion constitutes the vertex. When we tested the αβ′-COP subcomplex for trimer formation in vitro we detected only monomeric αβ′-COP in gel filtration experiments using protein concentrations up to 30 mg/ml protein (data not shown). The same negative result was obtained in previous studies on the COPII vertex: a core Sec13/31 construct of two β-propellers remains monomeric (dimer and tetramer formation is undetectable) using as much as 30 mg/ml protein (Fath et al., 2007
). This was despite the fact that the role of Sec31 at the COPII cage vertex has been assigned definitively based on the concordance of the crystal structure and the EM density map (Fath et al., 2007
; Stagg et al., 2007
; Stagg et al., 2008
). The vertex interactions in COPI and COPII cages—both of which lack the α-solenoid interdigitation of the clathrin cage—seem to be exceptionally weak. This fits with the view that, in a protein polyhedron, very weak interactions between assembly units can yield a very stable cage, and even modest-strength interactions may severely compromise the cage disassembly reaction (Zlotnick, 1994
Indirect evidence for the role of the αβ′-COP triskelion as the vertex comes from the sec27-95
temperature-sensitive mutant of β′-COP, which harbors the mutation S114Y (Eugster et al., 2004
; Prinz et al., 2000
). Residue Ser114 is located on the N-terminal β-propeller of β′-COP close to the triskelion contact surface (). The mutation of Ser114 to tyrosine will affect key residues at the triskelion interface, in particular Pro97, Asp98, Tyr99, and possibly also Phe77 and Asp117 (described below). The sec27-95
mutant is defective in retrograde Golgi-to-ER transport (of the dilysine cargo molecule Emp47p) but, unlike the sec27-1
mutant, α-COP is not destabilized in mutant cells grown at the permissive temperature (Eugster et al., 2004
are the only β′-COP mutants to have been characterized to date, and the two mutations map to strategically important regions of the β′-COP molecule (). This concordance of structure and function lends support to our model for the coatomer vertex, but a direct test for a triskelion COPI vertex geometry probably will require an electron cryomicroscopy analysis of reconstituted cages.
At the triskelion center, the β′-COP subunits associate through pairwise interactions involving a small, circumscribed area of the axial end of one N-terminal β-propeller and the side of the adjacent β-propeller ( and ). (Two of the propeller–propeller interfaces form very similar interactions, whereas the third interface has rotated apart due to crystal packing distortions; hence this description applies to the two similar interfaces). The interface involves residues from loops 2B–2C, 2D–3A, 3B–3C and 3D-4A on the axial end of the N-terminal β-propeller with residues from loops 4B–4C and 4D–5A of the side of the adjacent β-propeller ( and S1
). Key contacts involve the side chains of residues Phe77, Asp98, Tyr99, Phe142, Arg163 and Glu184 (S. cerevisiae β′-COP numbering). The COPI interface cannot be compared in molecular detail with the corresponding COPII interfaces, since the COPII vertex is based on a model fit into relatively low-resolution EM maps. However, we note that the geometry of the β′-COP–β′-COP interaction seems most similar to the proximal–distal β-propeller contacts (defined as cII and cIII contacts) at the COPII vertex (Fath et al., 2007
; Stagg et al., 2008
). Finally, the contact interfaces at both the COPI and COPII vertices seem to involve small surface areas, consistent with the weak interactions required to facilitate cage disassembly.
Implications for the Architecture of the COPI Cage
In our schematic representation () we illustrate a possible arrangement of protein components in the COPI cage formed by αβ′ε-COP. The known elements of the cage design are the atomic structures of the αβ′-COP and αε-COP subcomplexes (which account for ~75% of the structured polypeptide in αβ′ε-COP) and the symmetry and form of the triskelion, which we infer is the vertex of the cage.
Model for the architecture of the COPI lattice
The unknown element of the design is the connection that joins adjacent triskelia. In the simplest model, two copies of αβ′ε-COP connect to form a (αβ′ε-COP)2 dimer as the assembly unit of the COPI cage. For simplicity in we have drawn this connection between the N-terminal regions of two copies of α-COP (this is entirely speculative). In this arrangement, the COPI cage assembles in a very similar manner to COPII. Thus, the assembly unit () is a dimeric molecule—rod-shaped (Sec13/31)2 or S-shaped (αβ′ε-COP)2—with terminal β-propeller domains that interact to drive self-assembly (). In the resultant lattice, , the edge is formed by the assembly unit (αβ′ε-COP)2, but the symmetry is governed by the three-fold centre of the triskelion to yield a clathrin-like array of hexagonal and pentagonal shapes. Finally, the compulsion to maximize the number of stable bonds between assembly units drives the formation of the spherical cage, with the same symmetry as a clathrin cage ().
According to our model, the architectural core of the COPI cage comprises the propeller–propeller–solenoid array of β-COP, the central α-solenoid domain of α-COP, and an additional N-terminal region (possibly the entirety) of α-COP to form the connection at the center of the assembly unit ( and ). We have as yet been unable to express soluble portions of the α-COP N terminus, but future biochemical and structural studies should address the role of this region in cage assembly. We infer that the αε-COP subcomplex is not part of the architectural core of the cage as it is flexibly linked to the αβ′-COP subcomplex via a highly acidic ~80-residue linker of α-COP (). Moreover, the ε-COP protein is not essential for yeast growth, although its absence does compromise the stability of α-COP (Duden, et al., 1998
), consistent with the intimate, coiled interaction observed in the crystal structure of the αε-COP subcomplex (). In the case of the COPII cage, the C-terminal α-solenoid domain of Sec31 is flexibly linked to the assembly unit core via a ~340-residue proline-rich linker; importantly, a 50-residue peptide at the center of this linker interacts with the cargo-binding Sec23/24•Sar1 complex (Bi, et al., 2007
). It will be interesting to test the role of the αε-COP subcomplex and acidic linker in the COPI coat, and specifically whether these regions project towards the membrane to interact with the cargo-binding βδ/γζ-COP complex.
In conclusion, this analysis of the αβ′ε-COP complex establishes architectural principles that are common to the three major classes of vesicular cages, and a simple transformation of design—from COPII to COPI to clathrin—is revealed (). The findings should provide a foundation for molecular-level studies of the dynamic processes of COPI coat assembly and disassembly.