# Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2
## 1 Introduction
For successful translation initiation and recovery, IF2 interacts with many biological partners and undergoes functional cycling, including GTP hydrolysis, GDP/GTP exchange, initiator tRNA binding, and binding to the 30S ribosome subunit. While these activities are facilitated by different domains of IF2, they all act in an orchestrated, sequential manner that is indicative of allosteric-like domain-domain communication at individual interaction stages. Our understanding of the function-structure relationship has been largely improved by structural data. First, the full-length three-dimensional structure of IF2 was resolved for IF2/eIF5B from _M. thermoautotrophicum_[18]. This remarkable structure displays a novel and unusual chalice-like protein fold with two domains separated by a 4 nm long \(\alpha\)-helix. This fold possesses some common properties with the domain architectures of EF-Tu and EF-G, suggesting that all three proteins may interact similarly. Interestingly, the structure of IF2 does not explain how the signal of GTP hydrolysis in the G-domain is propagated to the distal C2 domain. In 2005, a cryo-EM model of ribosome-bound IF2 from _E. coli_ was published [19], in which IF2 undergoes conformational rearrangement upon binding to the ribosome. In particular, the authors observed that the portion attributable to the GTP-binding domain (G domain) of IF2 was significantly larger than the G domain of the reoriented crystal structure [19]. Hence, IF2 undergoes large conformational changes during translation initiation. In 2016, 11 cryo-EM structures of the _Thermus thermophilus_ translation preinitiation complex were published [12]. The complex contains 30S-IF1-IF2-IF3-mRNA-tRNA. Again, the authors observed that the conformation of ribosome-bound IF2 differs from the crystal structure of _Thermus_ IF2 [20]. Similar conformational rearrangements of IF2 were also reported in recent cryo-EM structures of the 70S initiation complex from _Pseudomonas aeruginosa_, with a bound compact form of IF2-GDP and initiator tRNA [21]. NMR and circular dichroism of _E. coli_ IF2 revealed a flexible linker connecting the N-terminal and central segments of the protein with the C-terminal domain [22]. Thus, multidomain IF2 may be considered flexible, with the capacity to undergo dynamic conformational transitions. Interestingly, the structure of release factor 2, another translation factor, also exhibits differences between its free form in solution compared with the conformation of its ribosome-bound form [23; 24; 25; 26]. The X-ray crystal structures of full-length _Thermus thermophilus_ apo IF2 and its complex with GDP have been obtained at 3.1 A resolution [20; 27]. The structure of IF2 bears a resemblance to beads on a string, rather than exhibiting a chalice shape similar to eIF5B. IF2 adopts a stable and ordered conformation when interacting with the ribosome, which is facilitated by the predetermined inherent flexibility and expandability of the protein (Figure 1). Possible elements of flexibility are long helices. The overall dimensions of IF2 depend on the nucleotide status of IF2. The apoform of _T. thermophilus_ IF2 is more extended, while the GDP-bound IF2 appears more compact. However, IF2 is significantly larger when in a functionally active state, bound to the 30S ribosomal subunit. Unlike all other translational GTPases, IF2 does not have an effect dom  
Figure 1: The division (upper part) of translation initiation factor 2 from _Thermus thermophilus_ into the G fragment (red), consisting of G1 and G2/3 domains, and the C fragment (blue) consisting of C1 and C2; cryo-EM model (lower part) of ribosome-bound IF2 prepared based on the structure in PDB [5 LMV], with binding partners (IF1, tRNA, mRNA, and IF3) shown as colored molecular surfaces. with the switch II region of the GTPase domain.