The biochemical impact of extracting an embedded adenylate kinase domain using circular permutation

Adenylate kinases (AKs) are phosphotransferases that are frequently employed as models to investigate protein structure-function relationships. Prior studies have shown that AK homologs of different stabilities retain cellular activity in cells following circular permutation that split the AMP binding domain into fragments coded at different ends of the primary structure, such that this domain was no longer embedded as a continuous polypeptide within the core domain. Herein, we show mesophilic and thermophilic AKs having this topological restructuring retain activity and substrate-binding characteristics of the parental AK. While permutation decreased the activity of both AK homologs at physiological temperatures, the catalytic activity of the thermophilic AK increased upon permutation when assayed >30{degrees}C below the melting temperature of the native AK. The thermostabilities of the permuted AKs were uniformly lower than native AKs, and they exhibited multi-phasic unfolding transitions, unlike the native AKs, which presented cooperative thermal unfolding. In addition, proteolytic digestion revealed that permutation destabilized each AK, and mass spectrometry suggested that the new termini within the AMP binding domain were responsible for the increased proteolysis sensitivity. These findings illustrate how changes in contact order can be used to tune enzyme activity and alter folding dynamics in multidomain enzymes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/564053v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1ee9ecdorg.highwire.dtl.DTLVardef@fbb415org.highwire.dtl.DTLVardef@ebdd90org.highwire.dtl.DTLVardef@11f4271_HPS_FORMAT_FIGEXP M_FIG C_FIG