Abstract
SelB is a specialized translation factor that binds GTP and GDP and delivers selenocysteyl-tRNA (Sec-tRNA(Sec)) to the ribosome. By analogy to elongation factor Tu (EF-Tu), SelB is expected to control the delivery and release of Sec-tRNA(Sec) to the ribosome by the structural switch between GTP- and GDP-bound conformations. However, crystal structures of SelB suggested a similar domain arrangement in the apo form and GDP- and GTP-bound forms of the factor, raising the question of how SelB can fulfill its delivery function. Here, we studied the thermodynamics of guanine nucleotide binding to SelB by isothermal titration calorimetry in the temperature range between 10 and 25 °C using GTP, GDP, and two nonhydrolyzable GTP analogs, guanosine 5'-O-(γ-thio)triphosphate (GTPγS) and guanosine 5'-(β,γ-imido)-triphosphate (GDPNP). The binding of SelB to either guanine nucleotide is characterized by a large heat capacity change (-621, -467, -235, and -275 cal × mol(-1) × K(-1), with GTP, GTPγS, GDPNP, and GDP, respectively), associated with compensatory changes in binding entropy and enthalpy. Changes in heat capacity indicate a large decrease of the solvent-accessible surface area in SelB, amounting to 43 or 32 amino acids buried upon binding of GTP or GTPγS, respectively, and 15-19 amino acids upon binding GDP or GDPNP. The similarity of the GTP and GDP forms in the crystal structures can be attributed to the use of GDPNP, which appears to induce a structure of SelB that is more similar to the GDP than to the GTP-bound form.
Highlights
Translation factor SelB was reported to have similar structure in the GTP, GDP, and apo forms
isothermal titration calorimetry (ITC) Measurements with SelB—The thermodynamic parameters of SelB binding to guanine nucleotides were measured for GTP, GDP, guanosine 5-O-(␥-thio)triphosphate (GTP␥S), and GDPNP (Table 1)
GTP binding to SelB caused a large change in heat capacity, Ϫ621 cal ϫ molϪ1 ϫ KϪ1, with an estimated alteration in the accessible surface area ranging from 1380 to 2388 Å2, corresponding to 41– 43 amino acids
Summary
Translation factor SelB was reported to have similar structure in the GTP, GDP, and apo forms. 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 [13]. This finding is difficult to reconcile with the 6 orders of magnitude differences in the binding affinity of SectRNASec to SelB1⁄7GTP and SelB1⁄7GDP [14], which prompted us to probe the conformational changes of SelB upon binding of different guanine nucleotides in solution. We utilized ITC to estimate the magnitude of conformational changes in SelB upon binding of different guanine nucleotides
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