Enhancing the Activity of Insulin at the Receptor Interface:  Crystal Structure and Photo-Cross-Linking of A8 Analogues

Wan, Zi Yi; Xu, Bin; Huang, Kun; Chen, Ying; Li, Biaoru; Nakagawa, Satoe H.; Qu, Yan; Hu, Shiyan; Katsoyannis, Panayotis G.; Weiss, Michael A.

doi:10.1021/bi048223f

Cited by 39 publications

(44 citation statements)

References 80 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…5E) in a semiexposed position but well away from F25. This finding supports the suggestion (36,47) that the A-chain residues V3 and T8 may contact a region of the insert domain that is peripheral to the critical site involved in contacting the B-chain residue F25. Such disparate contact points by the IR insert domain are possible given that it appears to be intrinsically disordered based on predictions with the DisProt web server (48).…”

Section: Docking Of Insulin On the L1 Domainsupporting

confidence: 85%

The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity

Lou¹,

Garrett²,

McKern³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

117

129

View full text Add to dashboard Cite

The insulin receptor (IR) and the type-1 insulin-like growth factor receptor (IGF1R) are homologous multidomain proteins that bind insulin and IGF with differing specificity. Here we report the crystal structure of the first three domains (L1-CR-L2) of human IR at 2.3 Å resolution and compare it with the previously determined structure of the corresponding fragment of IGF1R. The most important differences seen between the two receptors are in the two regions governing ligand specificity. The first is at the corner of the ligand-binding surface of the L1 domain, where the side chain of F39 in IR forms part of the ligand binding surface involving the second (central) ␤-sheet. This is very different to the location of its counterpart in IGF1R, S35, which is not involved in ligand binding. The second major difference is in the sixth module of the CR domain, where IR contains a larger loop that protrudes further into the ligand-binding pocket. This module, which governs IGF1-binding specificity, shows negligible sequence identity, significantly more ␣-helix, an additional disulfide bond, and opposite electrostatic potential compared to that of the IGF1R.crystal structure ͉ ectodomain ͉ insulin-binding site T he insulin receptor (IR), like the type-1 insulin-like growth factor receptor (IGF1R), is a member of the receptor tyrosine kinase family, and is a large, transmembrane, glycoprotein dimer consisting of several structural domains (1, 2). The N-terminal half of the ectodomain contains two leucine-rich repeat domains (L1 and L2) separated by a cys-rich region (CR) (1, 3). The C-terminal half of the IR ectodomain consists of three fibronectin type III domains, the second of which contains an insert region of Ϸ120 residues (1, 2).Although there is no high-resolution structural information available for the IR ectodomain, the three-dimensional structure is known for the first three domains (L1-CR-L2) of the closely related IGF1R (4). This structure has provided a framework to interpret previous studies on receptor chimeras, site-specific mutants, and mutants from patients with defective receptors (see refs. 1 and 2) and has guided subsequent studies on the insulin-binding site using mutational analysis (5, 6). Three regions of the ectodomain are known to be involved in low-affinity binding by the soluble IR ectodomain. These are the L1 domain, the CR region and the last 16 residues of the ␣-chain (see refs. 1 and 7). Of these, only the first two (L1 and the CR) are important determinants of ligand specificity, because IR͞IGF1R chimeras of whole receptors (8) or minireceptors (9) are little affected by swapping the regions that contained the last 16 residues of the ␣-chain.The major determinants in L1 for insulin binding specificity lie in the first 68 residues of this domain (10, 11), based on the analysis of receptor chimeras. Twelve residues in this N-terminal segment have been further confirmed as part of the ligand-binding region by site-specific mutagenesis (see Table 1). Surprisingly, nine of these 12 residues a...

show abstract

Section: Docking Of Insulin On the L1 Domainsupporting

confidence: 85%

The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity

Lou¹,

Garrett²,

McKern³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

117

129

View full text Add to dashboard Cite

show abstract

“…It should be noted that of the six substitutions studied in this work, only Phe 49 has been shown to play a role in binding to the 49 to alanine decreased IGF-I binding to IGFBP-1 80,000-fold and to IGFBP-3 15-fold. It is not known what effect the mutation to threonine reported here has on IGFBP binding.…”

Section: A17mentioning

confidence: 68%

“…2). This position is believed to introduce favorable and unfavorable interactions at the edge of the IR and has recently been analyzed in regard to receptor binding (49). Analysis of a photoactive A8 analogue showed that it exhibited efficient cross-linking to the insert in the second fibronectin type III domain of the IR that cooperates (probably in trans (50)) with the L1 domain to create binding site 1.…”

Section: A17mentioning

confidence: 99%

Structural Basis for the Lower Affinity of the Insulin-like Growth Factors for the Insulin Receptor

Gauguin¹,

Klaproth²,

Sajid³

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

Insulin and the insulin-like growth factors (IGFs) bind with high affinity to their cognate receptor and with lower affinity to the noncognate receptor. The major structural difference between insulin and the IGFs is that the IGFs are single chain polypeptides containing A-, B-, C-, and D-domains, whereas the insulin molecule contains separate A-and B-chains. The C-domain of IGF-I is critical for high affinity binding to the insulin-like growth factor I receptor, and lack of a C-domain largely explains the low affinity of insulin for the insulin-like growth factor I receptor. It is less clear why the IGFs have lower affinity for the insulin receptor. In this study, 24 insulin analogues and four IGF analogues were expressed and analyzed to explore the role of amino acid differences in the Aand B-domains between insulin and the IGFs in binding affinity for the insulin receptor. showed affinities similar to that of insulin for the insulin receptor. The mitogenic potency of these analogues correlated well with the binding properties. Thus, a small number of A-and B-domain substitutions that map to the IGF surface equivalent to the classical binding surface of insulin weaken two hotspots that bind to the insulin receptor site 1.

show abstract

“…At position B8 (occupied in native insulin by an invariant Gly with a positive angle) (33), only the D-isomer was prepared. The synthesis and characterization of protoprobes at positions A1, A3, A21, B0, B16, B24, and B25 have been described previously (6,17,25,26,34,35).…”

Section: Methodsmentioning

confidence: 99%

Decoding the Cryptic Active Conformation of a Protein by Synthetic Photoscanning

Huang

Chen

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Proteins evolve in a fitness landscape encompassing a complex network of biological constraints. Because of the interrelation of folding, function, and regulation, the ground-state structure of a protein may be inactive. A model is provided by insulin, a vertebrate hormone central to the control of metabolism. Whereas native assembly mediates storage within pancreatic ␤-cells, the active conformation of insulin and its mode of receptor binding remain elusive. Here, functional surfaces of insulin were probed by photocross-linking of an extensive set of azido derivatives constructed by chemical synthesis. Contacts are circumferential, suggesting that insulin is encaged within its receptor. Mapping of photoproducts to the hormone-binding domains of the insulin receptor demonstrated alternating contacts by the B-chain ␤-strand (residues B24 -B28). Whereas even-numbered probes (at positions B24 and B26) contact the N-terminal L1 domain of the ␣-subunit, odd-numbered probes (at positions B25 and B27) contact its C-terminal insert domain. This alternation corresponds to the canonical structure of a ␤-strand (wherein successive residues project in opposite directions) and so suggests that the B-chain inserts between receptor domains. Detachment of a receptor-binding arm enables photo engagement of surfaces otherwise hidden in the free hormone. The arm and associated surfaces contain sites also required for nascent folding and self-assembly of storage hexamers. The marked compression of structural information within a short polypeptide sequence rationalizes the diversity of diabetes-associated mutations in the insulin gene. Our studies demonstrate that photoscanningmutagenesiscandecodetheactiveconformationofaprotein and so illuminate cryptic constraints underlying its evolution.

show abstract

Enhancing the Activity of Insulin at the Receptor Interface: Crystal Structure and Photo-Cross-Linking of A8 Analogues

Cited by 39 publications

References 80 publications

The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity

The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity

Structural Basis for the Lower Affinity of the Insulin-like Growth Factors for the Insulin Receptor

Decoding the Cryptic Active Conformation of a Protein by Synthetic Photoscanning

Contact Info

Product

Resources

About