The Sac3-Thp1-Sem1 Complex

The main function of the TREX-2 transcription export complex is to link transcription and processing of the mRNA transcript with its export out of the nucleus and into the cytoplasm where it can undergo translation. The Sac3-Thp1-Sem1 complex has been shown to be critical for this functioning.

Figure 1. The Sac3-Thp1-Sem1 Complex. Sac3, shown in red, and Thp1, shown in green, are both nuclear mRNA export proteins, while Sem1, shown in blue, is a 26S proteasome complex subunit.

Ellisdon et al. (2012) managed to successfully generate a 2.9-Å-resolution crystal structure of the Sac3_250-563-Thp1-Sem1 complex of the Saccharomyces cerevisiae TREX-2. This complex, shown above and found on the distal region of TREX-2, was shown to mediate TREX-2’s interaction with the mRNA transcript.

The crystal structure was achieved through vapor diffusion, hanging drop method, at 292K, and was refined using synchrotron radiation. The structure of Sac3-Thp1-Sem1 and the electron density within the crystal was determined using selenomethionine single isomorphous replacement phasing. This method is used to determine the phase angles of reflection through insertion of the heavy atom selenomethionine; this insertion leads to intensity changes, which reveals the positions of the heavy atoms within the crystal and hence helps determine the overall positioning of the other atoms and the 3D structure.

The Interaction Between Thp1 and Sem1

Figure 2. SDS-PAGE pull-down assays, stained with Coomassie blue, determine the interactions between the three proteins within the complex and the overall assembly of the complex. The resin used for the His-tagged proteins was nickel-nitrilotriacetic acid while for the GST-tagged proteins, the agarose beads were coated with GST's substrate, forming a glutathione-Sepharose 4B resin.

Sem1 is a small negatively charged cofactor. It is made evident, in lanes 1 and 2 of Figure 2, that the solubility of Thp1 is dependent on co-expression of this Sem1, since when it is expressed on its own in lane 1, it is insoluble, while in lane 2 its band is visible, confirming solubility. The solubility of Sac3, on the other hand, as seen in lanes 3 and 4, does not depend on Sem1 co-expression, since Sem1 is not pulled down. In effect, lanes 3-5 reveal that Sem1 is not pulled down unless Thp1 is present. Hence, Thp1 and Sem1 must be associated to enable the formation of the Sac3-Thp1-Sem1 complex, since Sem1 is required for the stabilization of Thp1. Western blots were performed to confirm all the proteins were expressed.

This interaction can be observed in Figure 1, where a single Sem1 chain is seen to associate closely with Thp1, while making little contact with Sac3. In fact, the residues of Sem1 that make contact with Sac3 are not very conserved and hence do not supply much to the overall binding. The ones that bind to Thp1, however, are highly conserved and are necessary for the stabilization of Thp1 and consequently the uniting of the whole complex.

Figures 3 and 4 provide a much clearer look at these interactions:



Figure 3

The Sem1 C-terminal conserved hydrophobic residues: Phe73, Leu77 and Leu81, bind to the Thp1 winged helix domain, locking the Sem1 helix in place. Winged helix domains are nucleic acid binding motifs, which bind to double- and single-stranded DNA, and RNA.

Figure 4

The Interaction Between Thp1 and Sac3

Sac3 and Thp1 both have PCI folds, which are stretches of around 200 residues in purely alpha-helical conformations. They have an N-terminal right-handed superhelical domain and a C-terminal winged helix domain.

Thp1’s superhelical domain is composed of 15 right-handed alpha helices; its winged helix domain is made up of helices α16-18 and β-strands 1-3. As illustrated in figure 5, α1-4 form a 4-helix bundle that covers the N-terminal, while α5-9 adopt an antiparallel helix-turn-helix motif. Similarly, α10-15 also have a helix-turn-helix motif, and interact strongly with α16 in Thp1's winged helix domain, visible in figure 6. This α16 helix is very important, as it provides the main interaction interface between the superhelical and winged helix domains of Thp1. It is therefore highly conserved between yeasts and humans.

Figure 5. Thp1 Superhelical Domain

Figure 6. Thp1 Winged Helix Domain

Sac3_250-563’s superhelical domain is composed of right-handed helices α1-9. As depicted in figure 7, it is shorter than the Thp1 superhelical domain. Helices α1-4 follow an antiparallel helix-turn-helix motif, while α5-9 form a helical bundle, which interfaces with helix α10 of Sac3's winged helix domain. This winged helix domain, illustrated in figure 8, is made up of α10-12 and β-strands 1-3. Its α10 helix is analogous in function to Thp1's α16: creating a bridge between the two domains.

Figure 7. Sac3 Superhelical Domain

Figure 8. Sac3 Winged Helix Domain

Both Thp1 and Sac3’s winged helix domains share a homologous structure with a αβααββ motif, and they come together at the surface of the complex to form a six-stranded antiparallel β-sheet winged helix dimer. This interface helps recruit Thp1-Sem1 to Sac3, uniting the complex.

Figure 9. The juxtaposed Thp1 winged helix, shown in green, and the Sac3 winged helix, shown in red.



Figure 10. The Thp1 residues are colored cyan; the Sac3
residues are colored orange. They are all essential for proper
binding to mRNA, and consequently its export into the cytoplasm.

This juxtaposition also creates a positively charged stripe, which contains Thp1 conserved basic residues Arg414, Lys427 and Lys428, and Sac3 less conserved basic residues Lys467, 468 and 509, which are all solvent exposed. When these basic residues were mutated to aspartate (R414D, K427D-K428D, K467D-K468D, K509D), via PCR-based site directed mutagenesis, the affinity of the complex for nucleic acids decreased. This is illustrated in the Electrophoretic mobility shift assay (EMSA) in Figure 11 below. Only the wild-type complex showed binding to the poly(U)₂₅ RNA.

Figure 11. (j) SDS-PAGE of the wild-type Sac3-Thp1-Sem1 complex, the single mutants: R414D and K509D and the double mutants: K427D K428D and K467D K468D. (k) EMSA of the poly(U)₂₅ RNA incubated with the wild-type and specific mutants of the complex.

Hence, this interface and each of these residues are necessary for proper nucleic acid binding, as they provide a platform. Furthermore, they bind with a specificity for poly(U)₂₅ RNA (Fig. 12).

Figure 12. (c) EMSA of poly(A)₂₅ RNA incubated with increasing concentrations of Sac3-Thp1-Sem1 complex. (d) EMSA of poly(U)₂₅ RNA incubated with increasing concentrations of Sac3-Thp1-Sem1 complex. (e, f) Graphs detailing the EMSA binding of Sac3-Thp1-Sem1 complex to the following nucleic acids and to varying lengths of RNA.

The Sac3-Thp1-Sem1 complex shows a higher affinity for poly(U). As seen in figure 12c-d, the complex binds to poly(U)₂₅ RNA at a lower concentration, starting at around 0.5μM, than to poly(A)₂₅ RNA, which needs to be present at at least 2μM concentration for binding to occur; hence, less poly(U)₂₅ RNA needs to be present for binding, highlighting a stronger specificity and affinity for it. Figure 12e-f show that the complex binds preferentially to RNA lengths of 25 bases and specifically to uracil bases. In effect, a significant decrease is visible for RNA lengths of 10 bases or less.

Figure 13. Highlights the positions of the basic residues on the surface of the complex.

The predominant interaction interface between Thp1 and Sac3, is formed by the α17-18 loop within Thp1’s winged helix domain that binds to a cleft generated by helix α9 of Sac’3s superhelical domain and helix α10 of Sac3’s winged helix domain. This interface and the side chains of the interacting residues involved are shown and labelled in the video below:

When two bulky side chains, tyrosine and tryptophan, were inserted in the place of Val405 and Thr406 respectively, forming V405Y and T406W mutants, it inhibited the close associations between the Thp1 and Sac3 helices, leading to a reduced binding between Thp1 and Sac3. Sem1 binding to Thp1 was retained, as seen by comparison to wild-type in figure 14, indicating that the structure of Thp1 was not completely altered by the mutations. Hence, this confirms that these side chains are necessary for the binding of Sem1-Thp1 to Sac3 and the resulting formation of the nucleic acid binding platform.

Figure 14. 6Histidine-tag pulldown of His-Thp1 co-expressed with Sem1.

Overall, the interaction between the winged helix domains of Thp1 and Sac3 is highly important for the proper functioning of the complex, and reveals why the Sac3-Thp1-Sem1 complex is necessary for TREX-2 binding to mRNA.

- Written by Tamara Casteels