Molecular Basis of Catalytic Chamber-assisted Unfolding and Cleavage of Human Insulin by Human Insulin-degrading Enzyme
Insulin is a hormone vital for glucose homeostasis, and insulin-degrading enzyme (IDE) plays a key role in its clearance. IDE exhibits a remarkable specificity to degrade insulin without breaking the disulfide bonds that hold the insulin A and B chains together. Using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to obtain high mass accuracy, and electron capture dissociation (ECD) to selectively break the disulfide bonds in gas phase fragmentation, we determined the cleavage sites and composition of human insulin fragments generated by human IDE. Our time-dependent analysis of IDE-digested insulin fragments reveals that IDE is highly processive in its initial cleavage at the middle of both the insulin A and B chains. This ensures that IDE effectively splits insulin into inactive N- and C-terminal halves without breaking the disulfide bonds. To understand the molecular basis of the recognition and unfolding of insulin by IDE, we determined a 2.6-A resolution insulin-bound IDE structure. Our structure reveals that IDE forms an enclosed catalytic chamber that completely engulfs and intimately interacts with a partially unfolded insulin molecule. This structure also highlights how the unique size, shape, charge distribution, and exosite of the IDE catalytic chamber contribute to its high affinity ( approximately 100 nm) for insulin. In addition, this structure shows how IDE utilizes the interaction of its exosite with the N terminus of the insulin A chain as well as other properties of the catalytic chamber to guide the unfolding of insulin and allowing for the processive cleavages.
- Research Organization:
- Argonne National Laboratory (ANL)
- Sponsoring Organization:
- USDOE
- OSTI ID:
- 1005646
- Journal Information:
- J. Biol. Chem., Journal Name: J. Biol. Chem. Journal Issue: (21) ; 05, 2009 Vol. 284; ISSN JBCHA3; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- ENGLISH
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Related Subjects
99 GENERAL AND MISCELLANEOUS
ACCURACY
AFFINITY
CHARGE DISTRIBUTION
CLEARANCE
CLEAVAGE
DISSOCIATION
DISULFIDES
ELECTRON CAPTURE
ENZYMES
FRAGMENTATION
GLUCOSE
HOMEOSTASIS
HORMONES
HUMAN POPULATIONS
INSULIN
INTERACTIONS
ION CYCLOTRON-RESONANCE
MASS SPECTROSCOPY
RESOLUTION
SHAPE
SIZE
SPECIFICITY