Structure of the SARlb protein

Although the SAR1 protein is included in the GTPase superfamily (and, in particular, the RAS superfamily) members of which are present in most living cells, from bacteria to vertebrates, it is only slightly related to other RAS or ARF proteins and is distant from the RAB/YPT1/SEC4 subclass [50, 51]. SAR1 is conserved from an evolutionary standpoint and appears to present in all eukaryotes. However, whereas yeast and insects have a single SAR1 protein, higher organisms express two forms, SAR1b and SAR1a (both with 198 amino acids), which differ by 20 amino-acid residues [52] .The function of SAR1a has not been elucidated yet and, to date, no variant in the SAR1A gene has been described. The sequence alignment of SAR1b as compared to SAR1p (Figure 3) illustrates the different regions that are highly conserved across species and shows the different functional motifs in SAR1b that participate in vesicle budding, in GDP/GTP binding and hydrolysis and in interactions with other COP proteins.

Five X-ray crystallographic-derived structures for SAR1b bound to GDP or GTP, alone or complexed with other COPII components, have been deposited in the Protein Data Bank. Three of these structures are derived from S. cerevisiae (yeast) recombinant protein and two from Cricetulus griseus (hamster) recombinant protein. These structures provide insights into the structural changes that SAR1b may undergo upon GDP/GTP binding as well as demonstrating which parts of the protein constitute interfaces with other COPII components. No X-ray derived structures of SAR1b complexed with components of the PCTV are available to our knowledge. There is also one X-ray derived structure for SAR1a using human recombinant protein.

Using the 1F6B model Cricetulus griseus SAR1b [53]

(which lacks the first twelve AA)

and Swiss pdb Viewer:

two residues were modified (I80V, V163I)

in order to produce a structural module having a sequence identical to that of human SAR1b.

In yellow: p strands

In blue: a helixes

In white: loops

In green: GDP

Figure 4. Three dimensional structure of SAR1B protein

The X-ray structures show that SAR1b has six central |3 strands (5 parallel, |32 antiparallel) that are sandwiched between three a helixes on each side (Figure 4). In SAR1-GDP (the inactive form), the a1helix is retracted into a pocket formed by the |2- |3 hairpin. The | strands 1-2-3 are approximately parallel to the membrane allowing their juxtaposition with the membrane (the N and C terminus and | 2- | 3 hairpin would participate in this membrane interaction) [36]. The Mg2+ ion is coordinated by an oxygen atom of the phosphate of the GDP and the hydroxyl oxygen of Threonine residue 39 (in SAR1-GDP) [37]. Many H bonds stabilize the structure and could be altered by mutations (see discussion below), for example Ser 179 with Asp 137 and Leu 181 [2].

The X-ray data also provide insights into the roles played by the different parts of the structure in SAR1b functions (see the protein alignment Figure 3). The amino- (N) terminal part of SAR1b contains the STAR (SAR1 NH2 Terminal Activation Recruitment) motif, a hydrophobic sequence of amino acids (AA) (1-9), a structure different from other ARF superfamily GTPases, which recruits SEC12, and the a1 amphipathic helix (AA 15-19, residues VLNFL). The role of the a1 amphipathic helix is fundamental as demonstrated by the loss of all export activity of SAR1B following the substitution of the 4 hydrophobic AA by 4 Alanine [53]. Between the STAR motif and the a1 helix, a short domain (AA 9-14, YSGFS) participates in deforming the ER membrane [38]. Three other regions contact the membrane, one each in the N- (AA 1-25) and carboxyl- (C) (AA 195-198) terminii and a central motif in the p2- p3 strand (AA 65-70) [36, 38]. There is one motif that recognizes the guanine base (AA 134-137, NKXD) and two active sites for GTP hydrolysis (AA 32-38, motif GXXXXGK and AA 75-78, motif DXXG) [54]. Close to the GTP hydrolysis site, Threonine 39 is a highly conserved residue and the substitution T39N inhibits SAR1 function by interfering with activation by SEC12 [53].

The two switch regions (AA 48-59 and AA 78-94) contain two very important residues, the Threonine at position 56 and the Glycine at position 78, respectively [53]. A second unique structural region of SAR1, not observed in the ARF GTPases, is a long surface-exposed loop (AA 156-171) which connects the a4 helix and the p6 strand and which regulates the function of SAR1b. The substitution Thr158Ala abolishes the activity of SAR1 [53]. A specific C-terminal motif (AA 171-181, PXEVFMC/VSV/L), present in the p6 strand, targets SAR1b to the ER [55].

The three-dimensional structure was obtained by crystallography [36, 53] and then by a computational approach. By crystallography (without the nine first and the 48-55 residues ), SAR1-GDP appeared as a dimer [37, 53]. Nothing is available about an in vivo GTPase activity with this dimer structure. Moreover, Long and coll (2010) showed that SAR1b may function as a monomer [56], so we will only consider the monomer form.

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