PDBsum entry 2wc0

Hydrolase/hormone

PDB id: 2wc0

Contents

Protein chains

953 a.a. *

21 a.a. *

20 a.a. *

Ligands

DIO ×8

Metals

_ZN ×2

Waters ×360

* Residue conservation analysis

PDB id:

2wc0

Name:

Hydrolase/hormone

Title:

Crystal structure of human insulin degrading enzyme in complex with iodinated insulin

Structure:

Insulin-degrading enzyme. Chain: a, b. Fragment: residues 42-1019. Synonym: insulin protease, insulysin, insulinase. Engineered: yes. Mutation: yes. Insulin a chain. Chain: c, e. Fragment: residues 90-110.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Organism_taxid: 9606

Resolution:

2.80Å R-factor: 0.172 R-free: 0.220

Authors:

M.Manolopoulou,Q.Guo,E.Malito,A.B.Schilling,W.J.Tang

Key ref:

M.Manolopoulou et al. (2009). Molecular Basis of Catalytic Chamber-assisted Unfolding and Cleavage of Human Insulin by Human Insulin-degrading Enzyme. J Biol Chem, 284, 14177-14188. PubMed id: 19321446 DOI: 10.1074/jbc.M900068200

Date:

06-Mar-09 Release date: 24-Mar-09

Protein chains

P14735  (IDE_HUMAN) -  Insulin-degrading enzyme from Homo sapiens

Seq: 1019 a.a.

Struc: 953 a.a.*

Protein chains

P01308  (INS_HUMAN) -  Insulin from Homo sapiens

Seq: 110 a.a.

Struc: 21 a.a.

Protein chains

P01308  (INS_HUMAN) -  Insulin from Homo sapiens

Seq: 110 a.a.

Struc: 20 a.a.

* PDB and UniProt seqs differ at 12 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains A, B: E.C.3.4.24.56 - insulysin.
• Reaction: Degradation of insulin, glucagon and other polypeptides. No action on proteins.
• Cofactor: Zn(2+)

DOI no: 10.1074/jbc.M900068200

J Biol Chem 284:14177-14188 (2009)

PubMed id: 19321446

Molecular Basis of Catalytic Chamber-assisted Unfolding and Cleavage of Human Insulin by Human Insulin-degrading Enzyme.

M.Manolopoulou, Q.Guo, E.Malito, A.B.Schilling, W.J.Tang.

ABSTRACT

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.

Selected figure(s)

Figure 3.
Structure of insulin-bound IDE. A, global view of the structure of insulin-bound IDE-CF-E111Q monomer. IDE domains 1–4 (IDE-D1 to IDE-D4) are colored green, blue, yellow, and red, respectively. Insulin A and B chains are colored magenta and cyan, respectively. The zinc ion is colored gray. B, composite omit map of insulin contoured at 1.5σ. C, electrostatic surface representation of insulin and IDE. The molecular surface is color-coded as calculated by Adaptive Poisson-Boltzmann Solver. The molecular surface is colored as calculated by Adaptive Poisson-Boltzmann Solver (51) (<–6 kT in red, 0 kT in white, and >+6 kT in blue). The interaction surface between the N- and C-terminal domains of IDE and insulin is marked by yellow dashed lines based on the contact residues displayed using CCP4 molecular graphics (30).

Figure 4.
Characterization of the IDE-insulin interaction. A, details of the interaction between insulin and IDE. Additional hydrogen bond networks are shown in the three right panels for clarity. IDE and insulin are colored as in Fig. 3. B, comparison of insulin in its free T-state form (PDB code 1G7A) and IDE-bound form. C, comparison of surface charge distribution of the partially unfolded insulin in insulin-bound IDE with T-state insulin modeled into the catalytic chamber of IDE. Surface representation of the substrate binding chamber of IDE was generated by the software Voidoo (31). The outer surface of IDE and the substrate chamber are colored pale green and dark green. The electrostatic surface representations of the IDE-bound insulin and T-state insulin models are calculated by Adaptive Poisson-Boltzmann Solver. D, comparison of IDE-bound insulin structure with the solution structures of insulin Ala^A2-DKP. Insulin Ala^A2-DKP has an Ile to Ala mutation in A2 residue of insulin-DKP, which is an engineered monomeric insulin. Left shows the comparison of IDE-bound insulin (colored in red and salmon for insulin A and B chain, respectively) with an exemplary NMR structure of insulin Ala^A2-DKP (PDB code 1K3M; colored in green and lime green for insulin A and B chain, respectively), and an exemplary NMR structure of insulin-DKP (PDB code 2JMN; colored in blue and light blue for insulin A and B chain, respectively). For a fair comparison, solution structures of insulin Ala^A2-DKP and insulin-DKP are also shown in the middle and right, respectively.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 14177-14188) copyright 2009.

Figures were selected by an automated process.