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Free keywords:
ABC transporters; Nucleotide binding; Differential scanning calorimetry; Protein unfolding; Dimerization
Abstract:
GlcV is the nucleotide binding domain of the ABC-type glucose transporter of the hyperthermoacidophile Sulfolobus solfataricus. GlcV
consists of two domains, an N-terminal domain containing the typical nucleotide binding-fold and a C-terminal β-barrel domain with unknown
function. The unfolding and structural stability of the wild-type (wt) protein and three mutants that are blocked at different steps in the ATP
hydrolytic cycle were studied. The G144A mutant is unable to dimerize, while the E166A and E166Q mutants are defective in ATP hydrolysis and
dimer dissociation. Unfolding of the wt GlcV and G144A GlcVoccurred with a single transition, whereas the E166A and E166Q mutants showed
a second transition at a higher melting temperature indicating an increased stability of the ABCα/β subdomain. The structural stability of GlcV
was increased in the presence of nucleotides suggesting that the transition corresponds to the unfolding of the NBD domain. Unfolding of the Cterminal
domain appears to occur at temperatures above the unfolding of the NBD which coincides with the aggregation of the protein. Analysis of
the domain organization of GlcV by trypsin digestion demonstrates cleavage of the NBD domain into three fragments, while nucleotides protect
against proteolysis. The cleaved GlcV protein retained the ability to bind nucleotides and to dimerize. These data indicate that the wt GlcV NBD
domain unfolds as a single domain protein, and that its stability is modified by mutations in the glutamate after the Walker B motif and by
nucleotide binding.
