PDBsum entry 2h67

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Hormone/growth factor PDB id
Jmol PyMol
Protein chains
21 a.a.
30 a.a. *
* Residue conservation analysis
PDB id:
Name: Hormone/growth factor
Title: Nmr structure of human insulin mutant his-b5-ala, his-b10- asp pro-b28-lys, lys-b29-pro, 20 structures
Structure: Insulin a chain. Chain: a. Engineered: yes. Insulin b chain. Chain: b. Engineered: yes. Mutation: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Gene: ins. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_taxid: 562
NMR struc: 20 models
Authors: Q.X.Hua,M.Liu,S.Q.Hu,W.Jia,P.Arvan,M.A.Weiss
Key ref:
Q.X.Hua et al. (2006). A conserved histidine in insulin is required for the foldability of human proinsulin: structure and function of an ALAB5 analog. J Biol Chem, 281, 24889-24899. PubMed id: 16728398 DOI: 10.1074/jbc.M602617200
30-May-06     Release date:   18-Jul-06    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P01308  (INS_HUMAN) -  Insulin
110 a.a.
21 a.a.
Protein chain
Pfam   ArchSchema ?
P01308  (INS_HUMAN) -  Insulin
110 a.a.
30 a.a.*
Key:    PfamA domain  Secondary structure
* PDB and UniProt seqs differ at 4 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     extracellular region   1 term 
  Biochemical function     hormone activity     1 term  


DOI no: 10.1074/jbc.M602617200 J Biol Chem 281:24889-24899 (2006)
PubMed id: 16728398  
A conserved histidine in insulin is required for the foldability of human proinsulin: structure and function of an ALAB5 analog.
Q.X.Hua, M.Liu, S.Q.Hu, W.Jia, P.Arvan, M.A.Weiss.
The insulins of eutherian mammals contain histidines at positions B5 and B10. The role of His(B10) is well defined: although not required in the mature hormone for receptor binding, in the islet beta cell this side chain functions in targeting proinsulin to glucose-regulated secretory granules and provides axial zincbinding sites in storage hexamers. In contrast, the role of His(B5) is less well understood. Here, we demonstrate that its substitution with Ala markedly impairs insulin chain combination in vitro and blocks the folding and secretion of human proinsulin in a transfected mammalian cell line. The structure and stability of an Ala(B5)-insulin analog were investigated in an engineered monomer (DKP-insulin). Despite its impaired foldability, the structure of the Ala(B5) analog retains a native-like T-state conformation. At the site of substitution, interchain nuclear Overhauser effects are observed between the methyl resonance of Ala(B5) and side chains in the A chain; these nuclear Overhauser effects resemble those characteristic of His(B5) in native insulin. Substantial receptor binding activity is retained (80 +/- 10% relative to the parent monomer). Although the thermodynamic stability of the Ala(B5) analog is decreased (DeltaDeltaG(u) = 1.7 +/- 0.1 kcal/mol), consistent with loss of His(B5)-related interchain packing and hydrogen bonds, control studies suggest that this decrement cannot account for its impaired foldability. We propose that nascent long-range interactions by His(B5) facilitate alignment of Cys(A7) and Cys(B7) in protein-folding intermediates; its conservation thus reflects mechanisms of oxidative folding rather than structure-function relationships in the native state.
  Selected figure(s)  
Figure 1.
FIGURE 1. Structure of proinsulin and the B5-related interchain crevice. A, sequence of human proinsulin. The insulin moiety is shown in red (A chain) and blue (B chain). The connecting region is shown in black: flanking dibasic cleavage sites (filled circles) and the C-peptide (open circles). B, structural model of the insulin-like moiety and disordered connecting peptide (dashed black line). The A and B domains are shown in red and blue, respectively. Cystines (orange) are labeled in yellow boxes. C, structure of the B5-related crevice in multiple high resolution crystal structures. His^B5 and surrounding residues A6-A11, B4, B6, and B7 are shown in 15 independent T-state protomers. Structural variability reflects differences among crystal structures; two families of His^B5 side chain orientations are observed. Residues in the A chain are shown in red; residues in the B chain are shown in blue; and disulfide bridges are shown in orange. Structural coordinates were obtained from Protein Data Bank codes 1APH, 1BPH, 1CPH, 1DPH, 1G7A, 1LPH, 1MSO, 1TRZ, 1TYL, 1TYM, 1ZNI, 2INS, and 4INS. Structures were aligned with respect to the main chain atoms of residues A6-A11 and B4-B7.
Figure 5.
FIGURE 5. Pathway of insulin biosynthesis and transient transfection assay for expression and secretion. A, nascent proinsulin folds as a monomer in the rough ER (rER), wherein the zinc ion concentration is low. In the Golgi apparatus, the zinc-stabilized proinsulin hexamer assembles and is processed by cleavage of the connecting peptide to yield mature insulin. Zinc insulin crystals are observed in secretory granules. B, insulin hexamers dissociate in bloodstream to yield active monomers. C, SDS-PAGE assay of proinsulin expression in the ER (lanes 1, 3, 5, and 7) and secretion into the medium (lanes 2, 4, 6, and 8). The transfection assay was carried out in 293T cells: human wild-type proinsulin and variants Ala^B5 and [Ser^A6, Ser^A11]. The empty vector control is shown in lanes 1 and 2. Cells were pulse-labeled with ^35S-labeled amino acids for 1 h and chased for 1 h. The cells were lysed, and both lysates (C) and chase media (M) were immunoprecipitated with anti-insulin antiserum. The most rapidly migrating species contains the native disulfide-pairing scheme; less rapidly migrating species represent disulfide isomers.
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 24889-24899) copyright 2006.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20069126 M.C.Beauchamp, A.Yasmeen, A.Knafo, and W.H.Gotlieb (2010).
Targeting insulin and insulin-like growth factor pathways in epithelial ovarian cancer.
  J Oncol, 2010, 257058.  
20724178 M.Liu, I.Hodish, L.Haataja, R.Lara-Lemus, G.Rajpal, J.Wright, and P.Arvan (2010).
Proinsulin misfolding and diabetes: mutant INS gene-induced diabetes of youth.
  Trends Endocrinol Metab, 21, 652-659.  
20948967 M.Liu, L.Haataja, J.Wright, N.P.Wickramasinghe, Q.X.Hua, N.F.Phillips, F.Barbetti, M.A.Weiss, and P.Arvan (2010).
Mutant INS-gene induced diabetes of youth: proinsulin cysteine residues impose dominant-negative inhibition on wild-type proinsulin transport.
  PLoS One, 5, e13333.  
20540164 S.Chen, L.Adijanto, and N.H.Wang (2010).
In vitro folding of methionine-arginine human lyspro-proinsulin S-sulfonate-disulfide formation pathways and factors controlling yield.
  Biotechnol Prog, 26, 1332-1343.  
20034470 S.Y.Park, H.Ye, D.F.Steiner, and G.I.Bell (2010).
Mutant proinsulin proteins associated with neonatal diabetes are retained in the endoplasmic reticulum and not efficiently secreted.
  Biochem Biophys Res Commun, 391, 1449-1454.  
19321435 B.Xu, K.Huang, Y.C.Chu, S.Q.Hu, S.Nakagawa, S.Wang, R.Y.Wang, J.Whittaker, P.G.Katsoyannis, and M.A.Weiss (2009).
Decoding the cryptic active conformation of a protein by synthetic photoscanning: insulin inserts a detachable arm between receptor domains.
  J Biol Chem, 284, 14597-14608.  
19618407 G.Le Flem, J.Pecher, V.Le Flem-Bonhomme, A.Withdrawn, J.Rochette, J.P.Pujol, and P.Bogdanowicz (2009).
Human insulin A-chain peptide analog(s) with in vitro biological activity.
  Cell Biochem Funct, 27, 370-377.  
19395706 M.A.Weiss (2009).
Proinsulin and the genetics of diabetes mellitus.
  J Biol Chem, 284, 19159-19163.  
19850922 M.Liu, Z.L.Wan, Y.C.Chu, H.Aladdin, B.Klaproth, M.Choquette, Q.X.Hua, R.B.Mackin, J.S.Rao, P.De Meyts, P.G.Katsoyannis, P.Arvan, and M.A.Weiss (2009).
Crystal structure of a "nonfoldable" insulin: impaired folding efficiency despite native activity.
  J Biol Chem, 284, 35259-35272.
PDB code: 3gky
19321436 Q.X.Hua, B.Xu, K.Huang, S.Q.Hu, S.Nakagawa, W.Jia, S.Wang, J.Whittaker, P.G.Katsoyannis, and M.A.Weiss (2009).
Enhancing the activity of a protein by stereospecific unfolding: conformational life cycle of insulin and its evolutionary origins.
  J Biol Chem, 284, 14586-14596.
PDB codes: 2k91 2k9r
18772127 J.I.Park, J.Semyonov, W.Yi, C.L.Chang, and S.Y.Hsu (2008).
Regulation of receptor signaling by relaxin A chain motifs: derivation of pan-specific and LGR7-specific human relaxin analogs.
  J Biol Chem, 283, 32099-32109.  
18492668 Z.L.Wan, K.Huang, S.Q.Hu, J.Whittaker, and M.A.Weiss (2008).
The structure of a mutant insulin uncouples receptor binding from protein allostery. An electrostatic block to the TR transition.
  J Biol Chem, 283, 21198-21210.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.