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An enzyme's inherent structural plasticity is frequently associated with substrate binding, yet detailed structural characterization of flexible proteins remains challenging. This study employs complementary biophysical methods to characterize the partially unfolded structure of substrate-free AAC(6′)-Ii, an N-acetyltransferase of the GCN5-related N-acetyltransferase ( GNAT) superfamily implicated in conferring broad-spectrum aminoglycoside resistance on Enterococcus faecium. The X-ray crystal structure of AAC(6′)-Ii is analyzed to identify relative motions of the structural elements that constitute the dimeric enzyme. Comparison with the previously elucidated crystal structure of AAC(6′)-Ii with acetyl coenzyme A (AcCoA) reveals conformational changes that occur upon substrate binding. Our understanding of the enzyme's structural plasticity is further refined with small-angle X-ray scattering and circular dichroism analyses, which together reveal how flexible structural elements impact dimerization and substrate binding. These results clarify the extent of unfolding that AAC(6′)-Ii undergoes in the absence of AcCoA and provide a structural connection to previously observed allosteric cooperativity of this enzyme. Database Structural data are available in the PDB database under the accession number . [ABSTRACT FROM AUTHOR]

Allostery has been studied for many decades, yet it remains challenging to determine experimentally how it occurs at a molecular level. We have developed an approach combining isothermal titration calorimetry, circular dichroism and nuclear magnetic resonance spectroscopy to quantify allostery in terms of protein thermodynamics, structure and dynamics. This strategy was applied to study the interaction between aminoglycoside N-(6′)-acetyltransferase-Ii and one of its substrates, acetyl coenzyme A. It was found that homotropic allostery between the two active sites of the homodimeric enzyme is modulated by opposing mechanisms. One follows a classical Koshland-Némethy-Filmer (KNF) paradigm, whereas the other follows a recently proposed mechanism in which partial unfolding of the subunits is coupled to ligand binding. Competition between folding, binding and conformational changes represents a new way to govern energetic communication between binding sites. [ABSTRACT FROM AUTHOR]