Crystal structure of α-COP in complex with ϵ-COP provides ... · Crystal structure of α-COP in...

6
Crystal structure of α-COP in complex with ϵ-COP provides insight into the architecture of the COPI vesicular coat Kuo-Chiang Hsia and André Hoelz 1 Laboratory of Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 Communicated by Günter Blobel, The Rockefeller University, New York, NY, May 10, 2010 (received for review April 5, 2010) The heptameric coatomer complex forms the protein shell of mem- brane-bound vesicles that are involved in transport from the Golgi to the endoplasmatic reticulum and in intraGolgi trafficking. The heptamer can be dissected into a heterotetrameric F-subcomplex, which displays similarities to the adapter complex of the innercoat in clathrin-coated vesicles, and a heterotrimeric B-subcomplex, which is believed to form an outercoat with a morphology distinct from that of clathrin-coated vesicles. We have determined the crys- tal structure of the complex between the C-terminal domain (CTD) of α-COP and full-length ϵ-COP, two components of the B-subcom- plex, at a 2.9 Å resolution. The α-COP CTD ϵ-COP heterodimer forms a rod-shaped structure, in which ϵ-COP adopts a tetratricopeptide repeat (TPR) fold that deviates substantially from the canonical superhelical conformation. The α-COP CTD adopts a U-shaped archi- tecture that complements the TPR fold of ϵ-COP. The ϵ-COP TPRs form a circular bracelet that wraps around a protruding β-hairpin of the α-COP CTD, thus interlocking the two proteins. The α-COP CTD ϵ-COP complex forms heterodimers in solution, and we demonstrate biochemically that the heterodimer directly interacts with the Dsl1 tethering complex. These data suggest that the heterodimer is exposed on COPI vesicles, while the remaining part of the B-subcomplex oligomerizes underneath into a cage. coatomer interaction studies membrane coat U-shaped α-helical solenoid tetratricopeptide repeat (TPR) T he evolution of the eukaryotic cell entailed the formation of intricate intracellular membrane systems that divide the cell into approximately one dozen compartments. As a result of this compartmentalization, sophisticated transport systems have evolved which mediate and regulate macromolecular transport between different subcellular locations. In vesicular transport, cargo is ferried between organelles via spherical vesicles, which are stabilized by protein coats that are reminiscent of cages. Three types of coated vesicles have been characterized: (i) Cla- thrin-coated vesicles mediate the transport from the plasma membrane to the early endosome and from the Golgi to the endosome. (ii) Coat protein complex II (COPII)-coated vesicles bud from the endoplasmatic reticulum (ER) to export newly synthesized proteins to the Golgi. (iii) Coatomer (COPI)-coated vesicles are responsible for the transport from the cis-Golgi to the ER and also function in intraGolgi trafficking (1, 2). COPI vesicles were discovered as non-clathrin-coated vesicles that emanated from the Golgi membrane preparations of Chinese hamster ovary cells that were treated with ATP and a cytosolic protein fraction (3). The analysis by electron microscopy revealed that the vesicles lacked the characteristic hexagonal- pentagonal basketwork of clathrin-coated vesicles and had an average diameter of 100 nm (3). A further advance was the biochemical purification of COPI vesicles that were produced in a mammalian cell-free system in the presence of the nonhydro- lyzable GTP analog, GTPγS (4). The biochemical purification enabled the initial identification of four coat proteins (COPs): α-COP, β-COP, γ-COP, and δ-COP. Subsequently, three addi- tional members were identified, β-COP, ϵ-COP, and ζ-COP, that form an 800 kDa heptameric complex termed coatomer(59). Parallel to the discovery of the mammalian coatomer compo- nents, all seven members of the Saccharomyces cerevisiae coato- mer were identified in genetic screens, revealing mutants that displayed a temperature-sensitive secretion defect (1015). In addition, a heptameric coatomer homolog could be purified from yeast cells, demonstrating the evolutionary conservation of the secretory protein transport pathway (11). The next advance of the characterization of COPI vesicles was the demonstration of the mammalian heptameric coatomer form- ing the coated vesicles together with the ADP-ribosylation factor (ARF), a small GTPase that is required for the binding of the coatomer to the Golgi membranes, in stoichiometric amounts and as an intact unit (9, 16, 17). The dissection of the mammalian coatomer in high-salt conditions yielded two fractions, the B- and F-subcomplexes, which reassembled into the heptamer in low-salt conditions. The B- and F-subcomplexes contain α-COP, β-COP, and ϵ-COP and β-COP, γ-COP, δ-COP, and ζ-COP, respectively (18, 19). The four components of the COPI F-subcomplex display significant sequence homology and interact with each other in a fashion similar to the constituents of the heterotetrameric clathrin adapter protein complex, which recruits clathrin to the site of vesicle formation (1, 2022). This similarity has led to a model in which the F-subcomplex forms the membrane-adjacent layer, while the B-subcomplex is functionally related to clathrin and exposed to the outside (21). This hypothesis is supported by a phylogenetic analysis and the structural characterization of γ-COP and ζ-COP (2225). Moreover, components of the F-subcomplex cross link to membrane-bound photo-labeled ARFGTP, suggesting that this subcomplex is indeed localized close to the membrane (26). The interactions between the three components of the COPI B-subcomplex have been characterized by a yeast two-hybrid analysis and revealed associations between α-COP and ϵ-COP and α-COP and β-COP, respectively (20). While a plausible arrangement for the components of the F-subcomplex could be derived, an equivalent understanding of the architecture of the B-subcomplex remains elusive. In order to gain insight into the architecture of the outer layer of the COPI-coated vesicles, we have determined the crystal structure of the C-terminal domain (CTD) of α-COP in complex with ϵ-COP at a 2.9 Å resolution. Unexpectedly, ϵ-COP adopts a Author contributions: K.-C.H. and A.H. designed research; performed research; contributed new reagents/analytic tools; analyzed data; and wrote the paper. The authors declare no conflict of interest. Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3MV2 and 3MV3). 1 To whom correspondence should be addressed. E-mail: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/ doi:10.1073/pnas.1006297107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1006297107 PNAS June 22, 2010 vol. 107 no. 25 1127111276 BIOCHEMISTRY Downloaded by guest on March 22, 2020

Transcript of Crystal structure of α-COP in complex with ϵ-COP provides ... · Crystal structure of α-COP in...

Page 1: Crystal structure of α-COP in complex with ϵ-COP provides ... · Crystal structure of α-COP in complex with ϵ-COP provides insight into the architecture of the COPI vesicular

Crystal structure of α-COP in complex withϵ-COP provides insight into the architectureof the COPI vesicular coatKuo-Chiang Hsia and André Hoelz1

Laboratory of Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065

Communicated by Günter Blobel, The Rockefeller University, New York, NY, May 10, 2010 (received for review April 5, 2010)

The heptameric coatomer complex forms the protein shell of mem-brane-bound vesicles that are involved in transport from the Golgito the endoplasmatic reticulum and in intraGolgi trafficking. Theheptamer can be dissected into a heterotetrameric F-subcomplex,which displays similarities to the adapter complex of the “inner”coat in clathrin-coated vesicles, and a heterotrimeric B-subcomplex,which isbelievedto forman “outer” coatwithamorphologydistinctfrom that of clathrin-coated vesicles. We have determined the crys-tal structure of the complex between the C-terminal domain (CTD)of α-COP and full-length ϵ-COP, two components of the B-subcom-plex, at a 2.9 Å resolution. The α-COPCTD•ϵ-COP heterodimer forms arod-shaped structure, in which ϵ-COP adopts a tetratricopeptiderepeat (TPR) fold that deviates substantially from the canonicalsuperhelical conformation. The α-COP CTD adopts a U-shaped archi-tecture that complements theTPR foldof ϵ-COP. The ϵ-COPTPRs forma circular bracelet that wraps around a protruding β-hairpin of theα-COP CTD, thus interlocking the two proteins. The α-COPCTD•ϵ-COPcomplex forms heterodimers in solution, and we demonstratebiochemically that the heterodimer directly interacts with theDsl1 tethering complex. These data suggest that the heterodimeris exposed on COPI vesicles, while the remaining part of theB-subcomplex oligomerizes underneath into a cage.

coatomer ∣ interaction studies ∣ membrane coat ∣ U-shaped α-helicalsolenoid ∣ tetratricopeptide repeat (TPR)

The evolution of the eukaryotic cell entailed the formation ofintricate intracellular membrane systems that divide the cell

into approximately one dozen compartments. As a result of thiscompartmentalization, sophisticated transport systems haveevolved which mediate and regulate macromolecular transportbetween different subcellular locations. In vesicular transport,cargo is ferried between organelles via spherical vesicles, whichare stabilized by protein coats that are reminiscent of cages.Three types of coated vesicles have been characterized: (i) Cla-thrin-coated vesicles mediate the transport from the plasmamembrane to the early endosome and from the Golgi to theendosome. (ii) Coat protein complex II (COPII)-coated vesiclesbud from the endoplasmatic reticulum (ER) to export newlysynthesized proteins to the Golgi. (iii) Coatomer (COPI)-coatedvesicles are responsible for the transport from the cis-Golgi to theER and also function in intraGolgi trafficking (1, 2).

COPI vesicles were discovered as non-clathrin-coated vesiclesthat emanated from the Golgi membrane preparations ofChinese hamster ovary cells that were treated with ATP and acytosolic protein fraction (3). The analysis by electron microscopyrevealed that the vesicles lacked the characteristic hexagonal-pentagonal basketwork of clathrin-coated vesicles and had anaverage diameter of ≈100 nm (3). A further advance was thebiochemical purification of COPI vesicles that were producedin a mammalian cell-free system in the presence of the nonhydro-lyzable GTP analog, GTPγS (4). The biochemical purificationenabled the initial identification of four coat proteins (COPs):α-COP, β-COP, γ-COP, and δ-COP. Subsequently, three addi-

tional members were identified, β′-COP, ϵ-COP, and ζ-COP, thatform an ≈800 kDa heptameric complex termed “coatomer”(5–9).

Parallel to the discovery of the mammalian coatomer compo-nents, all seven members of the Saccharomyces cerevisiae coato-mer were identified in genetic screens, revealing mutants thatdisplayed a temperature-sensitive secretion defect (10–15). Inaddition, a heptameric coatomer homolog could be purified fromyeast cells, demonstrating the evolutionary conservation of thesecretory protein transport pathway (11).

The next advance of the characterization of COPI vesicles wasthe demonstration of the mammalian heptameric coatomer form-ing the coated vesicles together with the ADP-ribosylation factor(ARF), a small GTPase that is required for the binding of thecoatomer to the Golgi membranes, in stoichiometric amountsand as an intact unit (9, 16, 17). The dissection of the mammaliancoatomer in high-salt conditions yielded two fractions, the B- andF-subcomplexes, which reassembled into the heptamer in low-saltconditions. The B- and F-subcomplexes contain α-COP, β′-COP,and ϵ-COP and β-COP, γ-COP, δ-COP, and ζ-COP, respectively(18, 19).

The four components of the COPI F-subcomplex displaysignificant sequence homology and interact with each other in afashion similar to the constituents of the heterotetramericclathrin adapter protein complex, which recruits clathrin to the siteof vesicle formation (1, 20–22). This similarity has led to amodel inwhich the F-subcomplex forms the membrane-adjacent layer,while the B-subcomplex is functionally related to clathrin andexposed to the outside (21). This hypothesis is supported by aphylogenetic analysis and the structural characterization ofγ-COP and ζ-COP (22–25). Moreover, components of theF-subcomplex cross link to membrane-bound photo-labeledARF•GTP, suggesting that this subcomplex is indeed localizedclose to the membrane (26).

The interactions between the three components of the COPIB-subcomplex have been characterized by a yeast two-hybridanalysis and revealed associations between α-COP and ϵ-COPand α-COP and β′-COP, respectively (20). While a plausiblearrangement for the components of the F-subcomplex couldbe derived, an equivalent understanding of the architecture ofthe B-subcomplex remains elusive.

In order to gain insight into the architecture of the outer layerof the COPI-coated vesicles, we have determined the crystalstructure of the C-terminal domain (CTD) of α-COP in complexwith ϵ-COP at a 2.9 Å resolution. Unexpectedly, ϵ-COP adopts a

Author contributions: K.-C.H. andA.H. designed research; performed research; contributednew reagents/analytic tools; analyzed data; and wrote the paper.

The authors declare no conflict of interest.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,www.pdb.org (PDB ID codes 3MV2 and 3MV3).1To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1006297107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1006297107 PNAS ∣ June 22, 2010 ∣ vol. 107 ∣ no. 25 ∣ 11271–11276

BIOCH

EMISTR

Y

Dow

nloa

ded

by g

uest

on

Mar

ch 2

2, 2

020

Page 2: Crystal structure of α-COP in complex with ϵ-COP provides ... · Crystal structure of α-COP in complex with ϵ-COP provides insight into the architecture of the COPI vesicular

tetratricopeptide repeat fold that substantially differs from thecanonical superhelical topology. α-COP CTD has a unique foldwith a U-shaped architecture and contains a region at the top ofthe U that folds into a protruding β-hairpin structure. The twoproteins are tightly intertwined through the formation of anintermolecular tetratricopeptide repeat (TPR) and the encapsu-lation of the protruding α-COP CTD β-hairpin within the 8.5TPRs of ϵ-COP. We also demonstrate biochemically that theα-COPCTD

•ϵ-COP complex exists as a heterodimer in solution.Finally, we show that the heterodimer binds to a disorderedDsl1 surface loop of the heterotrimeric Dsl1 tethering complexthat targets COPI vesicles to the ER membrane.

ResultsStructure Determination. The polypeptide chain of the 1,201-resi-due α-COP can be divided into an ≈600-residue, N-terminal,all-β-strand region that is followed by an ≈200-residue, α-helicalregion, an ≈100-residue, unstructured region, and a C-terminal,≈300-residue, predominantly α-helical region. ϵ-COP is com-posed of 296 residues and is predicted to be entirely α-helical(Fig. 1A). A yeast two-hybrid analysis indicated an interactionbetween the C-terminal, α-helical domain of α-COP (residues686–1,201) and full-length ϵ-COP (residues 1–296) (21).

Based on this result, we designed a series of expression con-structs which encoded C-terminal fragments of α-COP. Whilefull-length ϵ-COP was soluble in the absence of α-COP, all ofthe tested α-COP constructs were insoluble. We identified anα-COP fragment (residues 900–1,201) that formed a stable com-plex with full-length ϵ-COP when coexpressed in Escherichia coli.In the following text, this complex is referred to as the α-COPCTD•ϵ-COP complex.

Crystals of the α-COPCTD•ϵ-COP heterodimer appeared

in the monoclinic space group C2, with three complexes in theasymmetric unit. The structure was solved by single-anomalousdispersion (SAD), using X-ray diffraction data obtained froma selenomethionine-labeled crystal. The structure of theα-COPCTD

•ϵ-COP complex was refined to a 2.9 Å resolution with

Rwork and Rfree values of 24.6% and 29.6%, respectively. No den-sity was observed for the final three residues of ϵ-COP; therefore,these residues have been omitted from the final model. Fordetails on the crystallographic statistics, see Table S1.

The α-COPCTD•ϵ-COP complex elutes as a single peak from a

gel filtration column, corresponding to a heterodimer. Analyticalultracentrifugation confirmed heterodimer formation in solutionwith a determined molecular weight of 69.2 kDa (theoreticalmolecular weight 71.9 kDa, Fig. S1). No higher-order oligomeri-zation states were detected in size-exclusion chromatography andanalytical ultracentrifugation. Hence, we focus on the descriptionof the α-COPCTD

•ϵ-COP heterodimer in the following text.

Structural Overview. The α-COPCTD•ϵ-COP heterodimer forms

a rod-like structure in which the ϵ-COP and α-COP CTD arearranged in a linear fashion (Fig. 1 B, C and Movies S1 and S2).Overall, the rod is ≈110 Å long and has a diameter of ≈40 Å.Unexpectedly, ϵ-COP forms an α-helical domain that is composedof 8.5 TPRs, which form a distorted right-handed superhelicalstructure. The α-COP CTD has an overall U-shaped architecturein which an α-helical domain forms the descending arm of the U.Contrary to the U-shaped solenoids found in other membranecoats, the ascending arm of the U of α-COP is not formed byan additional α-helical region, but by a small β-sheet domain thatis decorated with extensive loops. The α-COP CTD contains aβ-hairpin structure which protrudes from the top of the domain.This β-hairpin is encapsulated by the TPRs of ϵ-COP, therebyfacilitating the association of the two proteins via a large, evolu-tionarily conserved interface.

Structure of the α-COP CTD. The C-terminal domain of α-COPforms a U-shaped structure with an overall unique fold (Fig. 2).The descending arm of the U is formed by a 245-residue α-helicaldomain that is composed of 11 α-helices, αA–αK, which arearranged in a zigzag fashion and stacked on top of each other.A salient feature of the α-helical domain is a 37-residue insertionbetween helices αD and αE that forms a β-hairpin structure

Fig. 1. Overview of the structure of the α-COPCTD•ϵ-COP heterodimer. (A) Domain structures of yeast α-COP and ϵ-COP. ϵ-COP is an all-α-helical protein. Forα-COP, the two putative N-terminal β-propeller domains (light blue), the α-helical domain (dark blue), the unstructured region (U, gray), and the C-terminaldomain (steel blue and green) containing the DIM (magenta) are indicated. The numbering of α-COP and ϵ-COP is relative to the yeast proteins. The bars abovethe domain structures mark the crystallized fragments and are referred to as the α-COPCTD•ϵ-COP complex. (B) Ribbon representation of the α-COPCTD•ϵ-COPheterodimer colored according to A. The overall dimensions are indicated, and a 90° rotated view is shown on the right. (C) Schematic representation of theα-COPCTD•ϵ-COP heterodimer.

11272 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1006297107 Hsia and Hoelz

Dow

nloa

ded

by g

uest

on

Mar

ch 2

2, 2

020

Page 3: Crystal structure of α-COP in complex with ϵ-COP provides ... · Crystal structure of α-COP in complex with ϵ-COP provides insight into the architecture of the COPI vesicular

protruding with an ≈45° angle, approximately 25 Å away from thedomain. The base of the hairpin is formed by two antiparallelβ-strands, β1 and β2, that are connected by a nine-residue loopthat forms the outermost region of the domain. Together withhelix αD, this region forms the α-COP domain invasion motif(DIM). The ascending arm of the U is formed by a small β-sheetdomain that is composed of four β-strands, β3–β6, which bind tohelices αC, αF, αG, and αH, located approximately in the middleof the α-helical arm. This β-sheet domain is followed by a 27-re-sidue, extended segment that lies in a surface groove formed byhelices αB, αC, αF, and αG and extends to top of the U. The twoarms of the U are connected by an 18-residue linker region andare held together by an extensive hydrophobic core.

Structure of ϵ-COP. The polypeptide chain of ϵ-COP folds into adistorted, right-handed, superhelical structure that is composedof 8.5 degenerate TPRs, which were not predicted by primarysequence analysis (Fig. 3). The canonical TPR motif consistsof ≈34 residues that fold into a pair of antiparallel α-helices,A and B, which are connected by a short loop (27). Instead of

forming a canonical superhelical structure, the arrangement ofthe ϵ-COP TPRs is deformed by the intramolecular interactionbetween TPR1 and TPR6 that is caused by the noncanonicalangle between TPR3 and TPR4 and the binding of helix 1A tohelix 6A. This interaction results in the formation of a hollowcylinder that is closed on the top. The surface of the interiorof the cylinder is almost exclusively formed by the A-helicesand is lined with numerous aromatic side chains. This hydropho-bic chamber facilitates the binding to the α-COPCTD β-hairpin(see below). In addition to the unusual topology, the ϵ-COP TPRsdisplay three further differences to canonical TPR proteins:TPR2 lacks helix B and instead contains a long loop that connectshelices 2A with 3A, TPR7 contains an additional short helix 7Cthat is inserted into the 7B–8A interTPR connector loop, andTPR9 contains only helix 9A.

α-COPCTD-ϵ-COP Interface. Overall, the α-COPCTD•ϵ-COP hetero-

dimer forms an elongated structure in which the two proteins arearranged in a linear fashion (Fig. 1B). The open end of theU-shaped C-terminal domain of α-COP provides the binding site

Fig. 2. Structure of the α-COP CTD. A ribbon representation of the α-COP CTD structure is shown in rainbow colors along the polypeptide chain from the N tothe C terminus. The α-COP CTD has a unique fold with a U-shaped architecture. The α-helical descending arm of the “U”, containing the DIM at the top, thesmall β-sheet domain that forms the ascending arm of the “U”, as well as their secondary structure elements are indicated. The structure is shown from fourdifferent views that are related by 90° rotations.

Fig. 3. The Interaction of ϵ-COPwith the α-COP DIM. (A) ϵ-COP and the α-COP DIM are shown in yellow andmagenta, respectively. ϵ-COP adopts a TPR fold thatforms an intermolecular TPR with its C-terminal α-helix 9A and helix αD of the α-COP DIM. The β-hairpin of the α-COP DIM is encapsulated by the ϵ-COP TPRs. Asa reference, a cartoon representation of the α-COPCTD•ϵ-COP heterodimer is shown and corresponds to the orientation on the left. A 90° rotated view and aview from the top are shown on the right. (B) Schematic representation of the architecture of the ϵ-COP TPRs and their interaction with the α-COP DIM.

Hsia and Hoelz PNAS ∣ June 22, 2010 ∣ vol. 107 ∣ no. 25 ∣ 11273

BIOCH

EMISTR

Y

Dow

nloa

ded

by g

uest

on

Mar

ch 2

2, 2

020

Page 4: Crystal structure of α-COP in complex with ϵ-COP provides ... · Crystal structure of α-COP in complex with ϵ-COP provides insight into the architecture of the COPI vesicular

for ϵ-COP. The interaction between the two proteins is mediatedby helix αD and the protruding β-hairpin of the α-COP CTD andthe hydrophobic chamber and the C-terminal helix 9A of ϵ-COP.Strikingly, these regions facilitate the association of the twoproteins predominantly via two hydrophobic interactions thatresult in the complementation of the ϵ-COP fold: (i) Helix αDof α-COP mimics a TPR B-helix and binds to helix 9A ofϵ-COP, thereby forming an intermolecular TPR, and (ii) theβ-hairpin of α-COP is encapsulated by the hydrophobic chamberof ϵ-COP and completes the hydrophobic core (Fig. 3). Due tothe fold-complementing properties of helix αD and the followingβ-hairpin, we termed this region of α-COP the DIM (Fig. 3). Inaddition to these interactions, there are minor contacts betweenαA and 5B, αC and 9A, and αE∕αF and the 8AB loop of theα-COP CTD and ϵ-COP, respectively. The majority of α-COPand ϵ-COP residues that mediate the interaction between thetwo proteins are evolutionarily conserved (Figs. S2 and S3).Notably, the highest conservation is found in the surface patchesthat mediate the formation of the intramolecular TPR and formthe hydrophobic chamber of ϵ-COP, while the residues of theα-COP β-hairpin only display a conservation of their hydrophobiccharacter. Altogether, ≈4;500 Å2 of surface area are buriedbetween the two proteins, involving 47 and 63 residues of theα-COP CTD and ϵ-COP, respectively.

Structural Comparison. The formation of an intermolecular TPRhas been previously observed in the structure of the APC6TPR•CDC26N heterodimer (28). In this case, the APC6TPR domainfacilitates the binding to an N-terminal 29-residue peptideof CDC26 via the formation of an intermolecular TPR and byproviding a groove in the inner surface of the superhelix forthe binding of an N-terminal segment of this peptide (Fig. S4).However, the APC6TPR domain and ϵ-COP differ dramaticallyin their conformation, despite the fact that they are constructedof a comparable number of residues and an identical number ofTPRs.While the APC6TPR domain forms a canonical superhelicalstructure, the ϵ-COP conformation is distorted. The noncanonicalTPR sequence motifs of ϵ-COP may account for this difference;however, it could also be the result of the binding of theα-COPCTD DIM, which snugly fits into the hydrophobic chamberof ϵ-COP. The latter suggests that the conformation of ϵ-COP issubstantially different in the absence of α-COP.

The presence of DIMs is not limited to α-helical domains. Infact, the six-bladed β-propeller domains of Sec13 and Seh1 arecomplemented by an additional blade, which is provided bythe DIMs of their binding partners. These DIM-mediated inter-actions lead to the formation of heterodimeric complexes in theCOPII coat (Sec13•Sec31) and the coat for the nuclear poremembrane (Sec13•Nup145C and Seh1•Nup85) (29–32).

Surface Properties. Overall, the α-COPCTD•ϵ-COP heterodimer

displays two areas of distinct electrostatic surface potentials(Fig. S5 A, B). While the ϵ-COP end of the rod is negativelycharged, the opposite end of the rod is primarily hydrophobicand contains a basic surface patch (Fig. S5 A, B). An additionalhydrophobic surface is located on one side of the rod and is gen-erated by the ascending arm of the U-shaped α-COP CTD. Strik-ingly, with the exception of a small, invariant, negatively chargedpatch in the ϵ-COP surface, themapping of the conserved residuescoincides with the α-COP portion of the α-COPCTD

•ϵ-COPsurface (Fig. S5C). This result reflects the finding that while theprimary sequence of α-COP is evolutionarily well conserved fromyeast to human (43% identity), ϵ-COP displays a substantiallylesser degree of sequence conservation (22% identity) (Figs. S2and S3).

Biochemical Analysis. The α-COP CTD has been implicated inbinding to the Dsl1 subunit of the heterotrimeric Dsl1 tethering

complex that targets COPI vesicles to the ER membrane and isinvolved in the subsequent fusion of the two membranes (33, 34).The heterotrimeric Dsl1 complex is composed of Dsl1, Tip20,and Sec39 (35). While Tip20 and Sec39 facilitate the attachmentto the ER membrane via their binding to the membrane-embedded ER SNAREs (for soluble N-ethylmaleimide-sensitvefactor (NSF) attachment protein receptors), Dsl1 has been shownto directly interact with two subunits of the coatomer, α-COP andδ-COP (33). These interactions have been mapped to an unstruc-tured region of the Dsl1 structure using pullout experiments withglutathione S-transferase (GST)-fusions of Dsl1 peptides andyeast extracts (34, 36). Based on these observations, we testedwhether a GST-Dsl1 peptide comprised of residues 410–440directly interacts with the α-COPCTD

•ϵ-COP heterodimer orϵ-COP. The recombinant proteins were expressed, purified tohomogeneity, and monitored for complex formation throughsize-exclusion chromatography. While the α-COPCTD

•ϵ-COPheterodimer was capable of complex formation, ϵ-COP alonefailed to interact with the GST-Dsl1 peptide (Fig. 4). These dataare in line with the surface properties of the α-COPCTD

•ϵ-COPheterodimer, in which the majority of the conserved patches can

Fig. 4. The α-COPCTD•ϵ-COP heterodimer binds to a Dsl1 peptide. (A) Gel fil-trationprofiles of theGST-Dsl1410–440 fusionprotein (red), the α-COPCTD•ϵ-COPcomplex (green), and the coinjection of GST-Dsl1410–440 fusion proteinwith the α-COPCTD•ϵ-COP complex (blue). (B) Gel filtration profiles of theGST-Dsl1410–440 fusion protein (red), ϵ-COP (green), and the elution profile re-sulting from incubation of theGST-Dsl1410–440 fusion proteinwith ϵ-COP (blue)prior to injection are indicated. All proteins were injected at approximatelythe same concentrations.

11274 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1006297107 Hsia and Hoelz

Dow

nloa

ded

by g

uest

on

Mar

ch 2

2, 2

020

Page 5: Crystal structure of α-COP in complex with ϵ-COP provides ... · Crystal structure of α-COP in complex with ϵ-COP provides insight into the architecture of the COPI vesicular

be mapped within the surface areas that are contributed by theα-COP CTD (Fig. S5). Because the α-COP CTD is insoluble inour bacterial expression system in the absence of ϵ-COP, we wereunable to test whether the α-COP CTD interacts with the Dsl1peptide in the absence of ϵ-COP. These data demonstrate a directinteraction between Dsl1 and the α-COPCTD

•ϵ-COP complex.

DiscussionThe heterotrimeric B-subcomplex of the COPI vesicular coat hasbeen implicated in the formation of an “outer” layer that is mor-phologically distinct from clathrin cages. We have determined thecrystal structure of the complex between the C-terminal domain ofα-COP and ϵ-COP, two components of the B-subcomplex.Unexpectedly, ϵ-COP adopts a superhelical TPR fold that facili-tates complex formation by encapsulating a protruding β-hairpinelement of the U-shaped structure of the α-COP CTD. We showthat the α-COPCTD

•ϵ-COP complex forms heterodimers in solu-tion and directly interacts with Dsl1, a component of the Dsl1tethering complex. Strikingly, the remaining components of theB-subcomplex, β′-COP and the N-terminal part of α-COP (α-COPN), share a common domain organization with the proteinsthat form the outer layer of COPII cages (Fig. S6). Together, thesedata suggest that the α-COPCTD

•ϵ-COP complex is exposed onCOPI vesicles in order to facilitate targeting, while the remainingpart of the B-subcomplex would form a cage with a COPII-likearchitecture (Fig. 5).

Intracellular membrane systems have been proposed to be theresult of the internalization of the plasma membrane, which isfacilitated by protein complexes that have evolved to inducemembrane invaginations and fission (37). The finding that mostmembrane coat proteins share a common fold composition led tothe proposal of a progenitor “protocoatomer” (38). To date, fourmembrane-coating complexes have been well characterized: thecage-forming vesicular coats, clathrin, COPI, and COPII, as wellas the cylindrical coat for the nuclear pore membrane, the NPCcoat (1, 2, 30).

The clathrin coat is formed by the oligomerization of clathrintriskelia that are composed of two proteins, termed light chainand heavy chain, into vesicular coats with a hexagonal morphol-ogy. The heterotetrameric adapter complex recruits clathrin tothe membrane and thereby facilitates the formation of clathrinvesicles (1).

The COPII vesicular coat is composed of an inner and an outerlayer that are composed of Sec23•Sec24 and Sec13•Sec31,respectively. The outer layer forms cage-like structures of varioussizes and geometries, which result from the oligomerization of theSec13•Sec31 complex. The polypeptide chain of Sec31 encodesan N-terminal, seven-bladed β-propeller, which is followed bya DIM, a central α-helical domain, and a C-terminal α-helicaldomain that is separated by a proline-rich linker which is believedto be unstructured (Fig. S6A). The Sec31 DIM complements thesix blades of Sec13 with an additional blade, forming a seven-bladed β-propeller, resulting in the tight association betweenSec31 and Sec13 (29). The formation of the COPII cage is theresult of homodimerization of the central α-helical domainand of the asymmetric homotetramerization of the N-terminalβ-propeller of Sec31, which form the edge and vertex elementsof the cage, respectively. The proline-rich linker and the C-term-inal α-helical domain of Sec31 are dispensable for cage formation(29). While the function of the C-terminal α-helical domain ispoorly understood, the proline-rich linker has been implicatedin the linkage of the inner and outer coat (39).

The heterotetrameric F-subcomplex of the COPI coatomercontains four proteins that share remarkable sequence similaritywith the heterotetrameric adapter complex of clathrin coats (22).This finding has led to the proposal that the F-subcomplex is anadapter complex homolog (1, 20–25). It has been suggested thatthe remaining three proteins of the B-subcomplex are function-

ally equivalent to the cage-forming proteins of other membranecoats, but no homology at the primary sequence level could bedetected. However, α-COP and the COPII outer coat componentSec31 share a common domain architecture (Fig. S6A). In addi-tion, α-COPN and β′-COP share an ≈40% sequence homology,and fragments of the two proteins that lack the N-terminal β-pro-peller domains have been shown to interact with each other in ayeast two-hybrid analysis (20). Finally, we have shown that theα-COPCTD

•ϵ-COP heterodimer fails to oligomerize beyond theheterodimer in solution, and that it facilitates the binding tothe Dsl1 tethering complex.

Together, these data are consistent with a model in which theα-COPCTD

•ϵ-COP heterodimer protrudes from the COPI coat tobe accessible for targeting by the Dsl1 complex (Fig. 5). The si-milar domain structure of α-COPN and β′-COP may allow theseproteins to form an α-COPN

•β′-COP heterotetramer that oligo-merizes in a fashion similar to Sec13•Sec31 heterotetramersto form COPII-like cages (Fig. S6). In this scenario, the COPIvesicular coat would be an “evolutionary hybrid” between theclathrin and COPII coats; it would be formed by an inner layercomposed of a heterotetrameric adapter-like complex and an

Fig. 5. Model for the architecture and ER-targeting of the COPI vesicularcoat. Schematic diagram of the outer coat of the COPI complex. The hetero-trimeric B-subcomplex of the coatomer that is composed of α-COP, β′-COP,and ϵ-COP is believed to form a coat that is distinct from the hexagonalmorphology of clathrin cages. The α-COPCTD•ϵ-COP heterodimer binds toDsl1, a component of the heterotrimeric Dsl1 tethering complex that targetsCOPI vesicles to the ER membrane. We propose that the α-COPCTD•ϵ-COPheterodimer is exposed on the COPI coat, while the remaining part of theB-subcomplex, the N-terminal region of α-COP (α-COPN) and β′-COP, oligo-merize into a cage with a COPII-like architecture, as discussed in the text.The α-COPCTD•ϵ-COP heterodimer is attached to the cage via an ≈100 residueregion that is predicted to be unstructured. For clarity, the heterotetramericF-subcomplex of the coatomer that shares a similar architecture with theadapter complex and facilitates the oligomerization of clathrin has beenomitted. The arrangement of the Dsl1 tethering complex and its interactionwith ER-SNARES are according to Ren et al. (36). The interaction of theα-COPCTD•ϵ-COP heterodimer with the exposed Dsl1 loop is shown schema-tically. Notably, the illustration only reflects one of several possibilities for theplacement of α-COPN and β′-COP in a putative COPII-like cage, and, thus, isnot meant to reflect the precise position of these proteins.

Hsia and Hoelz PNAS ∣ June 22, 2010 ∣ vol. 107 ∣ no. 25 ∣ 11275

BIOCH

EMISTR

Y

Dow

nloa

ded

by g

uest

on

Mar

ch 2

2, 2

020

Page 6: Crystal structure of α-COP in complex with ϵ-COP provides ... · Crystal structure of α-COP in complex with ϵ-COP provides insight into the architecture of the COPI vesicular

outer layer that would be architecturally similar to the COPIIcage (Fig. 5). Future biochemical and structural analyses areexpected to illuminate the precise architecture of the outer coatof COPI.

MethodsThe details of protein expression, purification, crystallization, structure deter-mination, protein interaction analysis, and analytical ultracentrifugation aredescribed in the SI Text published online. In short, the α-COP CTD and ϵ-COPwere cloned into a pET28a vector modified to contain a PreScission protease-cleavable N-terminal hexa-histidine tag (40) and into pET-Duet1 (Novagen),respectively. The GST-Dsl1 expression clone was the kind gift of Hans Schmitt(41). All proteins were expressed in E. coli using the appropriate expressionconstructs and purified using several chromatographic techniques. X-raydiffraction data were collected at the General Medicine and Cancer Institutes

Collaborative Access Team (GM/CA-CAT) beamline at the Advanced PhotonSource, Argonne National Laboratory. The structure was solved by SAD, usingdata obtained from selenomethionine-labeled crystals. Data collection andrefinement statistics are summarized in Table S1.

ACKNOWLEDGMENTS. We thank Andrew Davenport, Erik Debler, JanaMitchell, Johanna Napetschnig, Alina Patke, Pete Stavropoulos, andHyuk-Soo Seo for critical reading of the manuscript, Stephanie Ethertonfor help with editing the manuscript, Hans Schmitt for providing theDsl1 peptide expression construct, and Günter Blobel for continuing support.In addition, we thank Erik Debler for help and Nagarajan Venugopalan(GM/CA-CAT) for support during data collection. Analytical ultracentri-fugation was carried out by the Wadsworth Center Biochemistry CoreFacility. A.H. was supported by a grant from the Leukemia and LymphomaSociety.

1. Kirchhausen T (2000) Three ways to make a vesicle. Nat Rev Mol Cell Biol 1:187–198.2. Springer S, Spang A, Schekman R (1999) A primer on vesicle budding. Cell 97:145–148.3. Orci L, Glick BS, Rothman JE (1986) A new type of coated vesicular carrier that appears

not to contain clathrin: its possible role in protein transport within the Golgi stack.Cell 46:171–184.

4. Malhotra V, Serafini T, Orci L, Shepherd JC, Rothman JE (1989) Purification of a novelclass of coated vesicles mediating biosynthetic protein transport through the Golgistack. Cell 58:329–336.

5. Serafini T, et al. (1991) A coat subunit of Golgi-derived non-clathrin-coatedvesicles with homology to the clathrin-coated vesicle coat protein beta-adaptin.Nature 349:215–220.

6. Waters MG, Serafini T, Rothman JE (1991) “Coatomer”: a cytosolic proteincomplex containing subunits of non-clathrin-coated Golgi transport vesicles. Nature349:248–251.

7. Hara-Kuge S, et al. (1994) En bloc incorporation of coatomer subunits during theassembly of COP-coated vesicles. J Cell Biol 124:883–892.

8. Kuge O, et al. (1993) zeta-COP, a subunit of coatomer, is required for COP-coatedvesicle assembly. J Cell Biol 123:1727–1734.

9. Stenbeck G, et al. (1993) beta′-COP, a novel subunit of coatomer. EMBO J12:2841–2845.

10. Stenbeck G, et al. (1992) Gamma-COP, a coat subunit of non-clathrin-coated vesicleswith homology to Sec21p. FEBS Lett 314:195–198.

11. Hosobuchi M, Kreis T, Schekman R (1992) SEC21 is a gene required for ER to Golgiprotein transport that encodes a subunit of a yeast coatomer. Nature 360:603–605.

12. Duden R, et al. (1994) Yeast beta-and beta'-coat proteins (COP). Two coatomersubunits essential for endoplasmic reticulum-to-Golgi protein traffic. J Biol Chem269:24486–24495.

13. Letourneur F, et al. (1994) Coatomer is essential for retrieval of dilysine-taggedproteins to the endoplasmic reticulum. Cell 79:1199–1207.

14. Cosson P, Démollière C, Hennecke S, Duden R, Letourneur F (1996) Delta- andzeta-COP, two coatomer subunits homologous to clathrin-associated proteins, areinvolved in ER retrieval. EMBO J 15:1792–1798.

15. Duden R, Kajikawa L, Wuestehube L, Schekman R (1998) epsilon-COP is a structuralcomponent of coatomer that functions to stabilize alpha-COP. EMBO J 17:985–995.

16. Serafini T, et al. (1991) ADP-ribosylation factor is a subunit of the coat of Golgi-derivedCOP-coated vesicles: a novel role for a GTP-binding protein. Cell 67:239–253.

17. Gaynor EC, Graham TR, Emr SD (1998) COPI in ER/Golgi and intra-Golgi transport: doyeast COPI mutants point the way? Biochim Biophys Acta 1404:33–51.

18. Fiedler K, Veit M, Stamnes MA, Rothman JE (1996) Bimodal interaction of coatomerwith the p24 family of putative cargo receptors. Science 273:1396–1399.

19. Lowe M, Kreis TE (1996) in Vivo assembly of coatomer, the COP-I coat precursor. J BiolChem 271:30725–30730.

20. Faulstich D, et al. (1996) Architecture of coatomer: molecular characterization ofdelta-COP and protein interactions within the complex. J Cell Biol 135:53–61.

21. Eugster A, Frigerio G, Dale M, Duden R (2000) COP I domains required for coatomerintegrity, and novel interactions with ARF and ARF-GAP. EMBO J 19:3905–3917.

22. Schledzewski K, Brinkmann H, Mendel RR (1999) Phylogenetic analysis of componentsof the eukaryotic vesicle transport system reveals a common origin of adaptor protein

complexes 1, 2, and 3 and the F subcomplex of the coatomer COPI. J Mol Evol48:770–778.

23. Hoffman GR, Rahl PB, Collins RN, Cerione RA (2003) Conserved structural motifs inintracellular trafficking pathways: structure of the gammaCOP appendage domain.Mol Cell 12:615–625.

24. Watson PJ, Frigerio G, Collins BM, Duden R, Owen DJ (2004) Gamma-COP appendagedomain—structure and function. Traffic 5:79–88.

25. Yu W, Lin J, Jin C, Xia B (2009) Solution structure of human zeta-COP: directevidences for structural similarity between COP I and clathrin-adaptor coats. J Mol Biol386:903–912.

26. Sun Z, et al. (2007) Multiple and stepwise interactions between coatomer andADP-ribosylation factor-1(Arf1)-GTP. Traffic 8:582–593.

27. Blatch GL, Laessle M (1999) The tetratricopeptide repeat: a structural motif mediatingprotein-protein interactions. Bioessays 21:932–939.

28. Wang J, Dye BT, Rajashankar KR, Kurinov I, Schulman BA (2009) Insights into anaphasepromoting complex TPR subdomain assembly from a CDC26-APC6 structure.Nat StructMol Biol 16:987–989.

29. Fath S, Mancias JD, Bi X, Goldberg J (2007) Structure and organization of coat proteinsin the COPII cage. Cell 129:1325–1336.

30. Hsia KC, Stavropoulos P, Blobel G, Hoelz A (2007) Architecture of a coat for the nuclearpore membrane. Cell 131:1313–1326.

31. Debler EW, et al. (2008) A fence-like coat for the nuclear pore membrane. Mol Cell32:815–826.

32. Brohawn SG, Leksa NC, Spear ED, Rajashankar KR, Schwartz TU (2008) Structuralevidence for common ancestry of the nuclear pore complex and vesicle coats. Science322:1369–1373.

33. Andag U, Schmitt HD (2003) Dsl1p, an essential component of the Golgi-endoplasmicreticulum retrieval system in yeast, uses the same sequence motif to interact withdifferent subunits of the COPI vesicle coat. J Biol Chem 278:51722–51734.

34. Zink S, Wenzel D, Wurm CA, Schmitt HD (2009) A link between ER tethering andCOP-I vesicle uncoating. Dev Cell 17:403–416.

35. Kraynack BA, et al. (2005) Dsl1p, Tip20p, and the novel Dsl3(Sec39) protein arerequired for the stability of the Q/t-SNARE complex at the endoplasmic reticulumin yeast. Mol Biol Cell 16:3963–3977.

36. Ren Y, et al. (2009) A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1. Cell 139:1119–1129.

37. Blobel G (1980) Intracellular protein topogenesis. Proc Natl Acad Sci USA77:1496–1500.

38. Devos D, et al. (2004) Components of coated vesicles and nuclear pore complexes sharea common molecular architecture. PLoS Biol 2:e380.

39. Bi X, Mancias JD, Goldberg J (2007) Insights into COPII coat nucleation from the struc-ture of Sec23. Sar1 complexedwith the active fragment of Sec 31.Dev Cell 13:635–645.

40. Hoelz A, Nairn AC, Kuriyan J (2003) Crystal structure of a tetradecameric assemblyof the association domain of Ca2+/calmodulin-dependent kinase II. Mol Cell11:1241–1251.

41. Zink S, Wenzel D, Wurm CA, Schmitt HD (2009) A link between ER tethering andCOP-I vesicle uncoating. Dev Cell 17:403–416.

11276 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1006297107 Hsia and Hoelz

Dow

nloa

ded

by g

uest

on

Mar

ch 2

2, 2

020