PROTEOSYNTHETIC ELONGATION FACTOR EF-Tu DOMAINS: THERMAL STABILITY, ADAPTATION AND FUNCTIONS

Jiří Jonák^1,2,3, Hana Šanderová^1,2, Marta Hůlková¹, Petr Maloň⁴, Libor Krásný^1,2, Markéta Kepková¹

¹Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic

²Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic

³Institute of Medical Biochemistry, First Medical Faculty, Charles University, Kateřinská 32, 12108 Prague 2, Czech Republic

⁴Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nám. 2, 16637 Prague 6, Czech Republic

Elongation factors EF-Tu are essential components of the bacterial protein synthesis machinery. They are members of the family of GTP-binding proteins, their activity is regulated by guanine nucleotides GDP and GTP. Binding with GTP induces an active-GTP conformation rendering EF-Tu capable of binding aminoacyl-tRNA (aa-tRNA) and transferring it to the ribosomal A-site during mRNA decoding. Hydrolysis of the bound GTP to GDP by the EF-Tu intrinsic GTPase center, activated by the codon-anticodon contact, induces a great conformational change in EF-Tu resulting in an “inactive”-GDP conformation. EF-Tu.GDP leaves the ribosome because its affinity for aa-tRNA is lost, thus allowing a proper accommodation of aa-tRNA at the ribosomal A-site. All elongation factors EF-Tu are composed of a ~400 amino acid residue long polypeptide chain folded into three clearly distinct domains. The N-terminal domain 1 (~200 residues) has an alpha/beta structure. It is called a catalytic domain as it is the site of GDP/GTP binding and GTPase activity. The middle domain 2 and the C-terminal domain 3 (each composed of ~100 residues) are beta barrels. They have no known binding or catalytic activity.

The two guanine nucleotide-induced EF-Tu conformations that determine EF-Tu functional state (as described above) are the results of both interdomain and intradomain rearrangements. To better understand how individual domains are involved in the building of conformational stability and functions of the EF-Tu we compared, in several assays, the properties of isolated domains 1 and full length EF-Tus from mesophilic, Gram- Escherichia coli (Ec), growing at 37oC, and thermophilic, Gram+ Bacillus stearothermophilus (Bst), growing at 60 oC and six chimeric forms of EF-Tu constructed by combination of domains of Ec and Bst EF-Tus. We found that 1) thermal stability of both EF-Tu proteins in both conformations resulted from domain 1 and domains 2+3 positive cooperation. Domain 1 set up a “basal” level (~20 oC higher with Bst domain 1 than with Ec domain 1) of the thermostability of either EF-Tu and domains 2+3 (mainly domain 3) finally raised the thermostability to the level consistent with the optimal growth temperature of either organism by stabilization of alpha-helical regions of domain 1 (as revealed by CD spectroscopy); 2) non catalytic domains 2+3 of Ec and Bst origin principally differed in their contributions to GDP and GTP binding and GTPase hydrolyzing activities of their respective EF-Tus: (i) Whereas in EcEF-Tu, the physiological GTP/GDP binding activity was attainable only in the presence of all three domains, in BstEF-Tu this activity appeared to be intrinsic to the G-domain alone. Here, the affinity for GTP/GDP was not affected by removal of domains 2+3. (ii) Domains 2+3 (mainly domain 2) of EcEF-Tu suppressed whereas domains 2+3 (mainly domain 2) of BstEF-Tu stimulated the GTPase activity of their respective G-domains. These results suggest that despite a high degree of structural homology (~ 80%) EF-Tu proteins developed, in various bacteria, a different inter-domain mechanics to fulfil the same functions. The non-catalytic domains of EF-Tu proteins might represent new potential tools for selective manipulation of G+ and G- bacteria.

Acknowledgements:

This work was supported by grant No NR9138-3 from the Ministry of Health of the Czech Republic and by grant No 2B06065 from the Ministry of Education, Youth and Sports of the Czech Republic