Diabetes-Associated Mutations in Insulin: Consecutive Residues in the B Chain Contact Distinct Domains of the Insulin Receptor ²,‡ Bin Xu, § Shi-Quan Hu, | Ying-Chi Chu, | Kun Huang, § Satoe H. Nakagawa, ⊥ Jonathan Whittaker, §,∇ Panayotis G. Katsoyannis, | and Michael A. Weiss* ,§ Department of Biochemistry, School of Medicine, Case Western ReserVe UniVersity, CleVeland, Ohio 44106-4935, Department of Pharmacology and Biological Chemistry, Mt. Sinai School of Medicine of New York UniVersity, New York, New York 10029, Department of Biochemistry and Molecular Biology, The UniVersity of Chicago, Chicago, Illinois 60637, and Department of Nutrition, School of Medicine, Case Western ReserVe UniVersity, CleVeland, Ohio 44106-4906 ReceiVed January 30, 2004; ReVised Manuscript ReceiVed April 8, 2004 ABSTRACT: How insulin binds to and activates the insulin receptor has long been the subject of speculation. Of particular interest are invariant phenylalanine residues at consecutive positions in the B chain (residues B24 and B25). Sites of mutation causing diabetes mellitus, these residues occupy opposite structural environments: Phe B25 projects from the surface of insulin, whereas Phe B24 packs against the core. Despite these differences, site-specific cross-linking suggests that each contacts the insulin receptor. Photoactivatable derivatives of insulin containing respective p-azidophenylalanine substitutions at positions B24 and B25 were synthesized in an engineered monomer (DKP-insulin). On ultraviolet irradiation each derivative cross-links efficiently to the receptor. Packing of Phe B24 at the receptor interface (rather than against the core of the hormone) may require a conformational change in the B chain. Sites of cross-linking in the receptor were mapped to domains by Western blot. Remarkably, whereas B25 cross-links to the C-terminal domain of the R subunit in accord with previous studies (Kurose, T., et al. (1994) J. Biol. Chem. 269, 29190-29197), the probe at B24 cross-links to its N-terminal domain (the L1 -helix). Our results demonstrate that consecutive residues in insulin contact widely separated sequences in the receptor and in turn suggest a revised interpretation of electron-microscopic images of the complex. By tethering the N- and C-terminal domains of the extracellular R subunit, insulin is proposed to stabilize an active conformation of the disulfide-linked transmembrane tyrosine kinase. The biological activities of insulin are mediated by binding of the hormone to receptors on target cells (1). Insulin is a globular protein containing two chains, A (21 residues) and B (30 residues). Stored in the pancreatic  cell as a zinc- stabilized hexamer, insulin functions in the blood stream as a zinc-free monomer. The structure of the free hormone has been extensively characterized as a dimer and hexamer by X-ray crystallography (Figure 1A; 2-5). 1 Complementary NMR studies of engineered monomers have demonstrated that major features of such structures are retained in solution (Figure 2A; 6-8). The insulin receptor (IR) comprises two extracellular R subunits and two transmembrane  subunits (Figure 3A,B; for a review, see ref 9). Binding of insulin to the R subunits activates the tyrosine kinase activity of the cytoplasmic domains of the  subunits, which leads in turn to a cascade of signal-transduction events. Here, we dem- onstrate by residue-specific photo-cross-linking (10) that consecutive residues in the B chainseach a site of mutation causing diabetes mellitus (11)scontact distinct domains of the IR. Implications for the mechanism of transmembrane signaling are discussed. Despite decades of investigation, how insulin binds to and activates the IR is not well understood. The present study is motivated by anomalous structure-activity relationships in the B chain (12-14). Our approach highlights similarities and differences between Phe B24 and its neighboring residue Phe B25 . Invariant among vertebrate insulin sequences (Figure ² This work was supported in part by the Diabetes Research & Training Center at the University of Chicago (S.H.N.) and by grants from the National Institutes of Health to P.G.K. (DK56673) and M.A.W. (DK40949). ‡ This paper is dedicated to the memory of the late Howard S. Tager. * To whom correspondence should be addressed. E-mail: michael.weiss@case.edu. Phone: (216) 368-5991. Fax: (216) 368- 3419. § Department of Biochemistry, School of Medicine, Case Western Reserve University. | Mt. Sinai School of Medicine of New York University. ⊥ The University of Chicago. ∇ Department of Nutrition, School of Medicine, Case Western Reserve University. 1 Crystal structures of zinc-insulin hexamers define three alterative conformations: T6,T3R f 3, and R6 hexamers (2, 4, 49). The solution structure of an engineered insulin monomer (Figure 2A) resembles the crystallographic T-state protomer (7, 8). 2 Abbreviations: Aib, R-aminoisobutyric acid; CR, cysteine-rich domain of the receptor R subunit; DTT, dithiothreitol; EM, electron microscopy; IGF-I, insulin-like growth factor I; IR, insulin receptor; IRR-N, polyclonal antibody that recognizes 20 amino-terminal residues of the R subunit; kDa, kilodalton (unit of mass); NAv, NeutrAvidin; Pap, p-azido-Phe; Pmp, p-amino-Phe; UV, ultraviolet; WGA, wheat- germ agglutinin. Amino acids are designated by standard three- and one-letter codes. “Native” elements of structure designate features of crystal structures (3) and may not correspond to the functional conformation in a receptor complex (22). 8356 Biochemistry 2004, 43, 8356-8372 10.1021/bi0497796 CCC: $27.50 © 2004 American Chemical Society Published on Web 06/08/2004