Anion Activation Site of Insulin-degrading Enzyme

Abstract

Insulin-degrading enzyme (IDE) (insulysin) is a zinc metallopeptidase that metabolizes several bioactive peptides, including insulin and the amyloid ␤ peptide. IDE is an unusual metallopeptidase in that it is allosterically activated by both small peptides and anions, such as ATP. Here, we report that the ATPbinding site is located on a portion of the substrate binding chamber wall arising largely from domain 4 of the four-domain IDE. Two variants having residues in this site mutated, IDE K898A,K899A,S901A and IDE R429S , both show greatly decreased activation by the polyphosphate anions ATP and PPP i . IDE K898A,K899A,S901A is also deficient in activation by small peptides, suggesting a possible mechanistic link between the two types of allosteric activation. Sodium chloride at high concentrations can also activate IDE. There are no observable differences in average conformation between the IDE-ATP complex and unliganded IDE, but regions of the active site and C-terminal domain do show increased crystallographic thermal factors in the complex, suggesting an effect on dynamics. Activation by ATP is shown to be independent of the ATP hydrolysis activity reported for the enzyme. We also report that IDE K898A,K899A,S901A has reduced intracellular function relative to unmodified IDE, consistent with a possible role for anion activation of IDE activity in vivo. Together, the data suggest a model in which the binding of anions activates by reducing the electrostatic attraction between the two halves of the enzyme, shifting the partitioning between open and closed conformations of IDE toward the open form.

Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme

“…Crystal structures of human and rat IDE have only revealed the IDE dimer that has the fully enclosed catalytic chamber (7,21). We suspect that the inability to capture another conformational state of IDE might be due to extensive intermolecular contacts conducive to crystal formation that preferentially stabilizes the closed state (Fig.…”

“…Catalytic activity and substrate selectivity of IDE is allosterically regulated by its oligomerization (dimer/tetramer), binding to substrates, ATP, and cellular partners (3,9,21,(28)(29)(30)(31). For example, the mixed oligomer of WT and mutant IDE can exhibit enzyme kinetics distinctly different from the homogeneous counterparts, suggesting cross-talk between subunits (31).…”

“…For example, the mixed oligomer of WT and mutant IDE can exhibit enzyme kinetics distinctly different from the homogeneous counterparts, suggesting cross-talk between subunits (31). ATP binds the catalytic chamber of IDE-C to selectively accelerate the degradation of the short peptides (e.g., substrate V) (21,30). Nestin, an intermediate filament protein, can enhance the degradation of substrate V by IDE while suppressing degradation of insulin (29).…”

Inositol phosphates and phosphoinositides activate insulin-degrading enzyme, while phosphoinositides also mediate binding to endosomes

“…In addition, IDE has been reported to have noncatalytic functions, such as acting as a receptor for varicella-zoster virus (11) and serving as a heat shock protein in stressed cells (12). IDE also modulates the activity of the proteasome (13), reportedly in conjunction with the retinoblastoma tumor-suppressor protein (14).We previously established that polyanions, such as free ATP and triphosphate, increase IDE activity by up to 100-fold toward a synthetic peptide substrate (15) and that ATP binds to a strongly electropositive inner surface of IDE that forms one-half of the substrate-binding chamber (16,17). Significantly, mutations in IDE that reduce its activation by polyanions also decrease its ability to rescue production of a mature yeast mating factor, indicating that activation by polyanions plays an important physiological role in cells (16).…”

“…We previously established that polyanions, such as free ATP and triphosphate, increase IDE activity by up to 100-fold toward a synthetic peptide substrate (15) and that ATP binds to a strongly electropositive inner surface of IDE that forms one-half of the substrate-binding chamber (16,17). Significantly, mutations in IDE that reduce its activation by polyanions also decrease its ability to rescue production of a mature yeast mating factor, indicating that activation by polyanions plays an important physiological role in cells (16).…”