Thermodynamics of the GTP-GDP-operated Conformational Switch of Selenocysteine-specific Translation Factor SelB. Alena Paleskava, Andrey L. Konevega and Marina V. Rodnina . The Journal of Biological Chemistry, 287, 27906-27912 (2012). doi: 10.1074/jbc.M112.366120 .
Background from article:
Selenocysteine (Sec) is the 21st genetically encoded amino acid identified in selected proteins in all three kingdoms of life. In bacteria, Sec is encoded by the UGA stop codon in combination with a specific hairpin structure on the mRNA, the Sec insertion sequence. Sec-tRNASec is delivered to the ribosome by a specialized translation factor, SelB, a 69-kDa GTP-binding protein that consists of four domains and recognizes both Sec-tRNASec and the Sec insertion sequence. SelB shares significant sequence similarity with other translation factors that deliver aminoacyl-tRNA to the ribosome, such as EF-Tu and eIF2/eIF5B. Domains I, II, and III of SelB have the same secondary elements as the respective domains of EF-Tu and are highly conserved among all organisms. Domain I binds guanine nucleotides, and domains I, II, and III together provide most of the contact surface for Sec-tRNASec binding. Domain IV of SelB, which consists of four winged-helix motifs and recognizes the Sec insertion sequence element, has no analogs in other translational GTPases and differs in bacteria, archaea, and eukarya.
Despite the structural similarities of the nucleotide-binding domains of SelB and EF-Tu, their nucleotide-binding properties are markedly different. SelB from Escherichia coli binds GTP with higher affinity than GDP. In contrast, EF-Tu binds GTP much more weakly than GDP (values of Kd for GTP and GDP are 60 and 1 nm, respectively). Furthermore, the arrangement of domains I–III in EF-Tu changes dramatically upon GTP hydrolysis, which is used to control aminoacyl-tRNA binding and delivery to the ribosome. In contrast, crystal structures suggested that domains I–III in SelB from the archaeon Methanococcus maripaludis adopt similar, GTP-like conformations in the presence of the GTP analog GDPNP or GDP or in the absence of guanine nucleotides. This finding is difficult to reconcile with the 6 orders of magnitude differences in the binding affinity of Sec-tRNASec to SelB·GTP and SelB·GDP, which prompted us to probe the conformational changes of SelB upon binding of different guanine nucleotides in solution.
In principle, a number of techniques can be used to probe conformational rearrangements of proteins upon ligand binding. Among them, isothermal titration calorimetry (ITC) is an underexploited method to estimate structural changes by measuring the heat released or absorbed during complex formation. The figure below illustrates the components of an ITC system.
This enthalpy change determined at different temperatures allows calculating the heat capacity change, which, in turn, provides information about conformational changes of macromolecules upon binding their ligands. Thus, ITC provides a sensitive method to draw the link between the thermodynamic data and structural rearrangement of the molecules. The investigators utilized ITC to estimate the magnitude of conformational changes in SelB upon binding of different guanine nucleotides
Background/Review Questions:
a. Looking at the calorimetry cells above, what components would most likely be placed in the reference cell, sample cells, and syringe?
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