Sec23/24:Sar1
Created by Zenad Jarrar
“A distinctive feature of eukaryotic cells is the presence of an endomembrane system enclosing various intracellular compartments. Transport between these compartments is mediated by coat protein complexes such as COPII” (2). Coat protein complex II is responsible for the formation of vesicles leaving the endoplasmic reticulum and targeting the cis-Golgi (5). The COPII coat forms through the sequential recruitment of three cytoplasmic proteins—Sar1, Sec23/24, and Sec13/31—to the ER membrane. Budding is initiated by the activation of the G-protein Sar1 to its GTP-bound form, causing it to embed an N-terminal α helix in the bilayer. Sar1-GTP recruits the Sec23/24 heterodimer to form the inner shell or “pre-budding” complex that binds to SNARE and cargo molecules. Finally, the pre-budding complex recruits Sec13/31 to induce coat polymerization and membrane deformation (3).
Sar1 (PDB ID = 1M2O, chain B), from Saccharomyces cerivisiae, is a GTPase in the Ras superfamily of GTPases and functions as a molecular switch controlled by the exchange of GDP and GTP (2). Its primary structure consists of 190 amino acids, and its secondary structure is 26% helical and 21% beta sheet. The structural core of Sar1 has six central β-strands (5 parallel, 1 antiparallel [β2]) in a relatively flat β-sheet that is surrounded by five α-helices (6). The antiparallel strand is not shown and the N-terminal helix is truncated from the structure. Sar1 has four distinct domains: a guanine nucleotide binding pocket, switch I, switch II, and an N-terminal amphipathic α-helix (truncated from structure). The structural core composed of the six β-strands forms the nucleotide binding pocket with a highly conserved nucleotide binding motif GxxxxGKT37 (P-loop), where x can be any amino acid. The switch I domain mediates the interactions between the guanine nucleotide and Mg2+. Switch I covers residues 44-57, which are located close to the guanine nucleotide (2). Residue Thr54 in the switch 1 region of Sar1-GppNHp (non-hydrolysable form of GTP) forms bonds to the γ-phosphate and Mg2+, and residue Gly76 in switch II coordinates the γ-phosphate (1). Consequently, switch II (residues 73-89) plays an important role in the hydrolysis of GTP (2). In Sar1 proteins, an N-terminal extension of about ten residues replaces the myristoyl or prenyl group found in most other Ras GTPases and precedes the α-helix (1). The N-terminal extension contains a conserved cluster of bulky hydrophobic amino acids, referred to as the Sar1-NH2-terminal activation recruitment (STAR) motif (6). The molecular weight of Sar1 is 21450.66 Da, and its isoelectric point (pI) is 5.21.
In Sar1-GDP, the N-terminal extension and α-helix are retracted into a surface pocket formed in part by the linker region of the β2-β3 hairpin (1). The placement of the NH2 terminus of Sar1 adjacent to switch I facilitates the recognition of Sec12 through the STAR motif (6). Sec12, the GTP exchange factor (GEF) for Sar1, is a membrane protein that is localized to the ER. The localization and activity of Sec12 promotes the localization of Sar1 to the ER (2). GEFs induce the GDP/GTP exchange through the transient disruption of the Mg2+ coordination in the nucleotide binding pocket while stabilizing a nucleotide-free and cation-free conformation. The absence of Mg2+ causes an average displacement of 8.8 Å of switch I, exposing the guanine nucleotide binding pocket. This is utilized by Sec12 to promote the exchange of GDP for GTP. After the dissociation of Mg2+, and in the absence of GTP, a water molecule stabilizes GDP through hydrogen bonds. Sec12 inserts one or more than one residue from the seven-bladed β-propeller type WD40 motif as a “finger” to disrupt the GDP binding site (4). GTP binding to Sar1causes an approximately 7Å displacement of the β2-β3 hairpin—involving a change in register of the hydrogen bonding between strands β3 and β1—to eliminate the pocket for the anchor (refer to Figure A). The anchor comprises an N-terminal amphipathic α-helix that forms tight interactions with the ER membrane by inserting hydrophobic side chains into the bilayer. The newly formed Sar1-GTP (bound to ER membrane) displays an increased affinity for the Sec23/24 heterodimer. This initiates membrane recruitment and the formation of the pre-budding complex, which comprises Sar1 and the Sec23/24 heterodimer (1).
Crystal structures of the Sec23/24 heterodimer revealed it to have a general bow-tie shape (2). Sec23/24-Sar1 is relatively thin and extends about 40Å in the direction away from the ER membrane, except for two protrusions formed by a gelsolin-like domain on each subunit that extend about 60Å. An important feature of the pre-budding complex is its concave inner surface (refer to Figure B), which matches the size of a 60 nm vesicle. Although the binding of Sec13/31 is thought to be the driving force behind vesiculation, the inherent curvature of Sec23/24-Sar1 suggests that it will favor membrane deformation. Sec23/24-Sar1 is complementary to the vesicle surface in chemistry as well as shape. The concave, inner surface of the complex is generally positively charged, which explains the results of in vitro budding experiments that demonstrated a requirement for acidic (negatively charged) phospholipids for the Sar1-dependent binding of Sec23/24 to synthetic lysosomes. The extensive interaction between Sar1-GppNHp and Sec23 causes one complex to cover ~8200 Å2 of membrane, roughly 0.7% of the surface area of a 60 nm vesicle (1).
Sec23 (PDB ID = 1M2O, chain A), from Saccharomyces cerivisiae, is classified as a transport protein. Its primary structure consists of 768 amino acids, and its secondary structure is 32% helical and 21% beta sheet. The Sec23 polypeptide folds into five distinct domains: a β-barrel, a zinc finger, an α/β vWA or “trunk” domain, an all-helical region, and a carboxy-terminal domain that closely resembles a gelsolin module. The β-barrel is formed from ~180 residues contributed by three polypeptide segments. The strands of the barrel lie almost parallel to the membrane such that one end of the barrel forms part of the concave inner surface of the coat and the other end part of the membrane-distal surface. The barrel is constructed from two apposed sheets: the six-stranded sheet β4-10-24-23-19-21 faces partly toward the zinc finger and partly toward the solvent; sheet β1-3-22-20 faces the helical domain. The ~55 residue zinc-finger domain lies at the periphery of the complex. Zinc bound to Cys56-61-80-83 is situated toward the membrane-proximal surface and, along with several neighboring arginine and lysine side chains, contributes to the basic (positively charged) inner surface. The trunk domain is formed from a single ~270 residue (118-395) segment plugged into the barrel between strands β1 and β19. The trunk has an α/β fold and it forms the Sec23/24 dimer interface, primarily involving strand β14. This domain also contacts Sar1. The α-helical domain covers residues 525-626 and 731-768. Helices αK-L-M form a three-helix bundle with αL facing the concave inner surface. Helices αN-O-S complete the domain, with the αM-αN linker contacting Sar1. Finally, the gelsolin-like domain is formed by a stretch of 105 residues. This is the only domain that does not contribute to the inner surface; however, it contains the catalytically important Arg722 residue that accelerates Sar1-GTP GTPase activity (1). The molecular weight of Sec23 is 85384.69 Da, and its isoelectric point (pI) is 5.39.
The highly conserved residuesVFR (181-183) of Sec23 are located in the β14 strand and the β14-15 loop. They are responsible for recognizing and forming van der Waals’ interactions with residues Phe 385 and Pro 387 of the highly conserved FLP (positions 385-377) region located in the β14-β15 loop of Sec24. This forms the Sec23/24 heterodimer interface, which buries ~900 Å2 of Sec24 surface area (1).
Interaction of Sec23 with Sar1-GTP forms an interface that encompasses roughly 20% of Sar1 surface area. Compared to uncomplexed Sec23, Sec23 recruited to Sar1 shows a 1.5° increase in curvature. This results from the rotation of the barrel, zinc finger, helical and gelsolin-like domains together as a rigid body with respect to the trunk domain. The increase in curvature provides more evidence for the possible involvement of Sec23/24 in the deformation of the ER membrane (1+2).
Sec23 uses residues from loops rather than secondary structure elements to contact switch 1, switch II, and the β2-β3 hairpin regions of GTP activated Sar1. Switch I is contacted by the helical domain, and switch II and the hairpin by loops from the trunk domain. The Sar1 binding site is the most conserved region of extended surface on Sec23. Twelve of the 24 residues contacting Sar1-GTP are invariant among most species. Interaction patch I involves the invariant residues FNNS (599-602) and Glu 605 found in the αM-αN loops of the helical domain. These are the most critical for recognition of Sar1-GTP. First, residue Phe 599 enters a hydrophobic pocket created by switch I, and Asn 600 accepts a hydrogen bond from the side chain of Thr 54, which also engages Mg2+ in the GTP state. Then, Ser 602 and Glu 605 form interactions with switch I residues WHP (55-57). These interactions are reinforced by trunk-domain residues from loops β17-αH and β18-α1. The second set of interactions occurs between residues LTD (746-748), found in the αR-αS loop of the helical domain of Sec23, and switch 1 of Sar1-GTP. This second interaction patch is notable owing to van der Waals interaction between Leu 746 and the guanine base. The final set of interactions, involving patch III, is formed between the gelsolin-like domain and the nucleotide binding site of Sar1. Rather than functioning to recruit Sec23/24 in response to GTP binding, it controls GTP hydrolysis and uncoating (1).
Sar1-GTP GTPase activity is very low because it lacks critical catalytic residues. Sec23, possessing these residues, acts as the Sar1-GTP GAP (GTPase Activating Protein). Sec23 inserts an arginine side chain, Arg 722, into the Sar1 active site. Through its guanidinium group, arginine bonds to the phosphates and neutralizes their negative charges. Arg 722 and adjoining residues form helix αR on one face of the gelsolin-like domain, and side chain atoms of Ser 719, Arg 722, Phe 723, and Ser 726 directly contact Sar1. The OH group of Ser719 bonds to Asp 32 of the P-loop, and its C'O group forms a hydrogen bond to the catalytic Arg 722 to assist in its positioning. The productive configuration at the active site is stabilized by hydrophobic interactions between Phe 723 of Sec23 and Pro 53 of switch I. This creates a sandwich of van der Waals contacts, the layers of which comprise Phe723-Pro53-Arg722-P loop. This rigid arrangement forms a productive complex with the phosphates even in this ground-state structure (1).
In addition to the insertion of a catalytic arginine residue into the active sites of G-proteins, GAPs also act by stabilizing switch II primarily to position a catalytic glutamine side chain and its associated nucleophilic water molecule. In Sar1 proteins, His 77 replaces the glutamine residue, and in electron density maps, His 77 bonds to an appropriately positioned water molecule. But, reconfiguration of switch II is not a principal feature of Sec23 action. Sar1 proteins avoid alterations to switch II by bonding the nucleophilic water in a new way. The change from QXXI to HXXA (77-80) in Sar1 allows His 77 to lie against Ala 80 and bond the nucleophilic water molecule in a manner not seen in other G-protein structures, through a configuration not possible with the bulkier isoleucine and longer glutamine side chains (1).
Sec24 (PDB ID = 1M2V, chain B), has an approximate 17% sequence similarity to Sec23 and an almost identical folding pattern. The results of DALI (Z = 30.9, rmsd = 2.7) and protein BLAST (E = 5e-8) searches show that Sec24 has both primary and tertiary structure similarities (Z>2 and rmsd<5). Like Sec23, Sec24 folds into the five distinct domains: a β-barrel, a zinc finger, a trunk domain, an all-helical region, and a gelsolin-like domain. Sec24 lacks the Sar1 recognition residues found in Sec23, which include FNNS (599-602), Glu 605, and Leu 746. Sec24 also lacks the catalytic arginine residue in the orientation needed to accelerate the GTPase activity of Sar1. Therefore, Sec24 is not a Sar1 GAP and it does not associate with Sar1 directly (1).
The Sec31 (PDB ID = 2QTV, chain C) active fragment (residues 907-942) binds as an extended polypeptide across a composite surface of the Sec23 and Sar1 molecules. This active fragment has no tertiary structure, and its secondary structure consists of a single turn of α-helix (residues 915-919) that binds to switch II of Sar1. A 16 residue segment (907-922), which includes the turn of α-helix, interacts with switch II and the adjacent α3 helix of Sar1. The 20 residue long C-terminus (923-942) binds in a highly extended conformation across Sec23 and contacts its gelsolin-like, trunk, and β-barrel domains. The fact that the Sec31 active fragment only binds to a composite surface of Sec23 and Sar1 explains the sequential recruitment of the Sec23/24 and Sec13/31 complexes to the ER membrane. The active fragment inserts two residues, Trp922 and Asn923, close to the active site. The plane of the indole ring of Trp 922 orients almost parallel with the imidazole ring of His77. This interaction optimizes the geometry of the key histidine side chain for bonding to the nucleophilic water molecule (3). This interaction causes a tenfold increase in the rate of GTP hydrolysis by Sec23. The COPII triggers its own disassembly, but the lifetime of GTP on Sar1 may be matched to the rate of budding to allow pre-budding complexes time to gather SNARE and cargo molecules (1). Several mechanisms slow down the turnover of Sec23/24 even after GTP hydrolysis to make sure the coated vesicle reaches its target (cis-Golgi). The interactions between Sec24 and the cytosolic tails of cargo are strong enough for this purpose. Binding of dynactin at ER exit sites slows down the rate of Sec23/24 turnover as well. In metazoans, ER-to-Golgi vesicles are transported along microtubules in a dynein motor-dependent manner. A human Sec23A paralog interacts with the p150 subunit of the dynactin complex, which is essential for dynein function. Disruption of the Sec23-dynactin interaction leads to an increase in the turnover rate of COPII components (5).
Lastly, after leaving the ER and just before reaching the cis-Golgi, Sec23 binds the Bet3 subunit of TRAPPI, a multi-subunit complex responsible for specifically tethering vesicles to target membranes (5).
The effect of a single amino acid substitution in human Sec23A demonstrates the importance of a normal functioning ER-cis Golgi transport system. Cranio-lenticulo-sutural dysplasia results from the substitution of Phe382 with Leu. The mutant form can still interact with Sec24 and is recruited efficiently to membranes by Sar1; however, the mutation affects the interaction of Sec23/24-Sar1 with Sec13/31. The corresponding phenylalanine residue in S. cerevisiae, Phe380, is located on helix α1 of the trunk domain which makes key interactions with the Sec31 active fragment. A mutation to a leucine residue perturbs the local structure of helix α1 and weakens contacts to the active fragment. This impairs the recruitment and nucleation of Sec13/31 at sites of COPII budding. The ERs of affected individuals are disorganized with long tubule-like protrusions, indicating a failure of the COPII vesicle to bud off the membrane. Symptoms include late-closing fontanels, sutural cataracts, facial dysmorphisms and skeletal defects (7).