Architecture of Sec23•Sar1 Bound to the Active Fragment of Sec31
The overall conformations of Sec23 and Sar1 observed in the crystal structure of the ternary complex ( and ) are essentially the same as in the Sec23•Sar1 binary complex (Bi et al., 2002
), with only subtle alterations observed in the vicinity of the Sar1 active site. Sar1 is stabilized in the GTP conformation by the non-hydrolyzable analog, such that its β2–β3 switching element has shifted to eliminate the binding site for the Sar1 N-terminal “membrane anchor” (Bi et al., 2002
Layered Appearance of COPII Coat Proteins in a Model of Sec23/24•Sar1 Bound to Sec31
Thirty-six residues of Sec31, residues 907–942, are included in the molecular model, as no electron density is observed for the eight N-terminal and five C-terminal positions. This is consistent with the results of the biochemical dissection (see ), and suggests that as few as 35–40 residues of Sec31 are required for GAP stimulation.
The Sec31 active fragment binds as an extended polypeptide across a composite surface of the Sec23 and Sar1 molecules. It does not adopt a folded tertiary structure; moreover, it has hardly any secondary structure other than a single turn of α helix, residues 915–919, that binds to the switch 2 element of Sar1 (). The active fragment is oriented with its N terminus bound to Sar1 and C terminus bound to Sec23. A 16-residue segment (907–922) that includes the turn of α helix, extends ~15 Å across the Sar1 surface, interacting with switch 2 and the adjacent α3 helix (a common binding site for GAPs on G proteins [Vetter and Wittinghofer, 2001
]). The 20 C-terminal residues 923–942 bind in a highly extended conformation, 47 Å long, across Sec23, and contact the gelsolin, trunk and β-barrel domains of the Sec23 molecule ((Bi et al., 2002
The observation that the Sec31 active fragment binds to a composite surface of Sec23 and Sar1 provides a molecular explanation for the ordered recruitment of the Sec23/24 and Sec13/31 complexes to ER membranes. Only upon GTP-dependent formation of Sec23/24•Sar1 is the receptor site for Sec13/31 formed on the membrane (Barlowe et al., 1994
; Matsuoka et al., 1998
). A structural corollary of this is the layered arrangement of the membrane and proteins in the coat, as highlighted in . Thus, the Sec31 active fragment binds to the membrane-distal surface of Sec23•Sar1. All of its residues reside a uniform distance from the membrane surface, which we estimate to be ~45 Å (this assumes that the membrane-proximal surface of Sec23/24•Sar1 closely apposes the phospholipid membrane).
The sequence composition of the active fragment () strongly suggests that the isolated peptide will adopt an unstructured conformation in solution. Only residue Trp922 of the ~40 amino acids is conserved as a large hydrophobic side chain. Moreover, a limited proteolysis analysis of Sec13/31 did not identify a stably-folded domain in this region or in the proline-rich region of Sec31 (residues 770–1110) as a whole. Whether the Sec31 active fragment is thoroughly unstructured in the context of full-length Sec13/31 is unclear. The 3-fold differences in catalytic rate and affinity on Sec23/24•Sar1 that we reported above may hint at regulatory conformational constraints imposed on the active fragment by adjacent domains of Sec13/31, but the development of this speculative idea must await further experimental work.
Sec31 Interactions at the Sec23•Sar1 Interface and the GTPase Active Site
In , the Sec31 active fragment is colored to delineate five segments. The N-terminal residues, 907–919 (colored purple) interact solely with Sar1. These residues are not conserved in Sec31 sequences (), but we observe close interactions between this region and the switch 2 and α3 elements of Sar1. Next, residue Asp920 and the highly conserved residues Gly921 and Trp922 of the active fragment (colored white in ) contact both Sec23 and Sar1 to form a tripartite protein interface near the Sar1 active site (). The peptide geometry around residue Gly921 guides the insertion of the Trp922 side chain close to the seat of reaction (), and these interactions are key to the stimulation of GAP activity, as described in more detail below.
The remainder of the active fragment, residues 923–942, interacts solely with Sec23. A central stretch, residues 928–934 (yellow in ), appears to interact loosely, whereas the regions on either side form more intimate interactions with Sec23. Residues 923–927 (blue in ) are well conserved among Sec31 sequences (), and form a series of interactions with Sec23 that appear to be important for affinity and for buttressing Trp922 at the Sar1 active site (). Finally, the C-terminal stretch comprising residues 935–942 (green in ) is not well conserved, but several side chains—in particular Ala936, Val939 and Val941—form intimate contacts with residues of the trunk and β-barrel domains of Sec23 ().
In summary, the binding site for the Sec31 active fragment extends ~60 Å across the surface of Sec23 and Sar1, and involves three quasi-independent binding regions (colored purple, white/blue and green in ). Most residue positions in the Sec31 active fragment are not well conserved, in particular the N-terminal sequence that interacts with Sar1 and the C-terminal portion that interacts with Sec23. Nevertheless, these two terminal interaction regions are important for binding and stimulation of GAP activity, according to the results of the dissection experiment (). Sequence conservation is restricted to a central set of ~6 residues of the active fragment that is clustered around the tripartite interface near to the Sar1 active site.
Sec31 Residues Complete the Sar1 Active Site for Rapid GTP Hydrolysis
Sar1 has a very slow intrinsic rate of GTP hydrolysis because, like other Ras proteins, it lacks key catalytic residues (Bi et al., 2002
; Vetter and Wittinghofer, 2001
). The mechanism by which Sec23 acts as a GAP to accelerate the reaction involves the insertion of an arginine side chain, Arg722, into the active site to form bonds to the phosphates via its guanidinum group (Bi et al., 2002
) (). This type of mechanism, involving an “arginine-finger” residue that neutralizes negative charge in the GTPase transition state is likewise a common feature of Ras proteins (Vetter and Wittinghofer, 2001
The crystal structure of the Sec23•Sar1 binary complex revealed two catalytic features in addition to the identity of the arginine-finger residue. Firstly, an extensive interface between Sec23 and the switch 1 and 2 elements of Sar1 stabilizes these regions close to the active site. Secondly, a water molecule bridges the imidazole side chain of His77 and the γ-phosphate group of GppNHp, and is suitably located for nucleophilic attack (Bi et al., 2002
, and ). Taken together, the catalytic features of the Sec23•Sar1 active site are very similar to those seen in other GAP•G-protein complexes, so it is not immediately obvious from the Sec23•Sar1 crystal structure how Sec31 might stimulate GAP activity (discussed in Bi et al., 2002
Inspection of the Sar1 active site and the tripartite interface in the ternary complex now reveals how Sec31 stimulates GAP activity of Sec23 (). The active fragment inserts two residues, Trp922 and Asn923, close to the active site, with the plane of the indole ring of Trp922 oriented almost parallel with the imidazole ring of His77 (). We propose that this interaction optimizes the geometry of the key histidine side chain for bonding to the nucleophilic water molecule (His77 is equivalent to Gln61 of H-Ras). The imidazole ring of His77 has rotated ~15° upon interaction with Trp922, to align with the plane of the tryptophan indole ring (the χ2 torsion angle of His 77 is −60° in the binary complex and −75° in the ternary complex). Other changes at the active site induced by Sec31 binding appear to support the role of Trp922. In particular, Sec23 residue Gln720, which is oriented away from the active site in the binary complex, is turned toward the active site and forms hydrogen bonds to residues Trp922 and Asn923 of the active fragment.
Thus, Sec31 side chains do not have a chemical catalytic role at the Sar1 active site like the arginine finger residue of Sec23—indeed, only residue Pro926 is invariant in the subset of sequences shown in . Rather, GAP stimulation is likely caused by Trp922 of Sec31 interacting with His77 to orient its imidazole ring at the Sar1 active site. In so doing, Trp922 and Asn923 plug a solvent-filled cavity that extends from bulk solvent to the vicinity of the active site, an unfavorable arrangement for catalysis in the binary complex that leaves one surface of the indole ring of His77 exposed to solvent ().
Structure-based mutagenesis of the Sec31 peptide confirmed the importance of these key residues. Mutation of residue Trp922 and Asn923 to alanine caused complete loss of GAP stimulatory activity (). Likewise, changes to Leu925 and residue Val939 in the C-terminal region were highly disruptive, even though these are not highly conserved positions (). Two very conservative residue changes in the N-terminal portion of the active fragment, Q910A and N915A, were tolerated with only modest loss of stimulatory activity; the asparagine residue does not in fact contact Sec23 or Sar1 directly.
In summary, the active-site configuration of the ternary complex explains how Sec31 synergizes with Sec23 to accelerate GTP hydrolysis. Sec31 cannot act alone on Sar1 because it only binds to the Sec23•Sar1 complex. In this way, the sequential binding reaction confers a two-gear mechanism for GTP hydrolysis on Sar1 (Antonny et al., 2001
), whereby hydrolysis is initiated upon Sec23/24 binding and is accelerated further upon recruitment of Sec13/31. Thus GTP hydrolysis is programmed into the COPII system upon assembly—the slow rate of reaction on Sec23/24•Sar1 may provide the pre-budding complex the opportunity to gather cargo and SNARE molecules prior to Sec13/31 binding. Upon Sec13/31 recruitment, the rapid rate of hydrolysis—which we estimate could be as high as 0.5 sec−1
—may compromise the stable attachment of ternary complexes to the membrane. But in the late stages of budding the dependence on Sar1–GTP for stabilization of COPII proteins on the ER probably diminishes as Sec13/31 polymerizes and as Sec23/24 collects cargo to provide additional bonds linking coat proteins laterally and to the membrane (discussed in Antonny et al., 2001
The F382L Disease Mutation in Human Sec23A Maps Close to the Interface of Sec23 and Sec31
A substitution in human Sec23A, F382L, causes cranio-lenticulo-sutural dysplasia, a craniofacial and skeletal dysmorphic syndrome (Boyadjiev et al., 2006
). Fibroblasts homozygous for the mutation have a disorganized endoplasmic reticulum. Molecular analysis of F382L Sec23A reveals that although it can combine with Sec24 and is recruited efficiently to membranes by Sar1, the mutant protein is deficient in vesicle formation. Moreover, Sec13/31 is mislocalized to the cytoplasm in the mutant cells, suggesting that the mutation affects the interaction of Sec23/24•Sar1 with Sec13/31 (Boyadjiev et al., 2006
, and see accompanying manuscript by Fromme et al., 2007
The corresponding phenylalanine residue in S. cerevisiae Sec23, Phe380, is located on helix αI of the trunk domain, which makes key interactions with the Sec31 active fragment (). The phenylalanine residue is highly conserved (Boyadjiev et al., 2006
), and its side chain resides on the internal face of helix αI, contributing to the hydrophobic core via contacts to adjacent hydrophobic side chains of Sec23—including Phe346, Phe375 and Tyr384. Although Phe380 does not contact Sec31 directly, residues 924–931 of the active fragment make contacts along the length of helix αI, the most notable of which is the electrostatic interaction between the side chain of Asp924 and the positive charge of the N terminus of the α helix (). As noted above, this set of residues of the active fragment—in particular 923–927—are important for buttressing Trp922 at the Sar1 active site. The disease mutation is a subtle change to leucine, but we predict that this will perturb the local structure of helix αI so as to weaken contacts to the active fragment. We infer that the Sec23 disease mutation impairs the recruitment and nucleation of Sec13/31 at sites of COPII budding.
Connection of Inner and Outer Shell Complexes in the COPII Coat
presents a model for the arrangement of Sec23/24 complexes in the COPII coat, and is based on recent structural data on the 60 nm cuboctahedron cage built from 24 assembly units (Fath et al., 2007
; Stagg et al., 2006
). Since each assembly unit is a Sec13/Sec31•Sec31/Sec13 heterotetramer, there are 48 copies of Sec31 per cage, and binding sites for 48 copies of Sec23/24•Sar1. Put differently, the cage has twelve vertices, each vertex formed from the convergence of four Sec13/31 rods, so there are binding sites for four Sec23/24•Sar1 molecules to nestle on the membrane underneath each vertex.
A Model for the Interactions and Organization of Proteins in the COPII Coat
With respect to the present study, there are three salient features of the model. Firstly, the membrane vesicle is drawn as a 40 nm sphere inside the cage, to allow an ~5 nm space for Sec23/24•Sar1 (see Barlowe et al., 1994
, and Fath et al., 2007
). Secondly, the polypeptide linker (zigzag line) that connects the Sec31 active fragment to the upstream α-solenoid domain is a 130-residue portion of the proline-rich region, and is very likely unstructured. Thirdly, the C-terminus of the Sec31 α-solenoid domain, from where the linker projects down toward the membrane, is roughly equidistant (~70 Å) from the vertex dyad and the center of the assembly unit.
The flexible connection between the cage and Sec23/24•Sar1, via the 130-residue linker, suggests that Sec23/24•Sar1 complexes may be somewhat mobile on the membrane, such that the 432 symmetry of the cage is not imposed strictly on the inner shell proteins. Nevertheless, the mobility of Sec23/24•Sar1 is probably restricted by its dense packing on the membrane surface—we previously estimated that 48 copies of Sec23/24•Sar1 would cover as much as 80% of the surface area of a 40 nm membrane vesicle (Fath et al., 2007
We have depicted this situation as four Sec23/24•Sar1 complexes under a vertex but not conforming closely to the cage symmetry (). The complexes are drawn nestled close to the vertex center, and paired in an approximate fashion with the Sec31 α-solenoid domain to which each is attached, rather than in a jumbled arrangement. But we predict that this order arises as much from the dense surface packing of Sec23/24•Sar1 as it does from the arrangement and symmetry of the outer shell assembly units.
A flexible connection between the inner and outer shells of the vesicular coat is highly reminiscent of the situation in clathrin cages, where the N-terminal β-propeller domain of the clathrin heavy chain interacts with adaptors via short peptide elements that are nested in flexible regions of polypeptide (ter Haar et al., 2000
). Thus, a flexible connection between the architectural outer shell and the cargo-gathering inner shell complex may be a common feature of vesicle coat organization that facilitates the packaging of a range of cargo molecules of different shapes and sizes.