Crystal structure of allo-Ile(A2)-insulin, an inactive chiral analogue: implications for the mechanism of receptor binding.
The crystal structure of an inactive chiral analogue of insulin containing nonstandard substitution allo-Ile(A2) is described at 2.0 A resolution. In native insulin, the invariant Ile(A2) side chain anchors the N-terminal alpha-helix of the A-chain to the hydrophobic core. The structure of the variant protein was determined by molecular replacement as a T(3)R(3) zinc hexamer. Whereas respective T- and R-state main-chain structures are similar to those of native insulin (main-chain root-mean-square deviations (RMSD) of 0.45 and 0.54 A, respectively), differences in core packing are observed near the variant side chain. The R-state core resembles that of the native R-state with a local inversion of A2 orientation (core side chain RMSD 0.75 A excluding A2); in the T-state, allo-Ile(A2) exhibits an altered conformation in association with the reorganization of the surrounding side chains (RMSD 0.98 A). Surprisingly, the core of the R-state is similar to that observed in solution nuclear magnetic resonance (NMR) studies of an engineered T-like monomer containing the same chiral substitution (allo-Ile(A2)-DKP-insulin; Xu, B., Hua, Q. X., Nakagawa, S. H., Jia, W., Chu, Y. C., Katsoyannis, P. G., and Weiss, M. A. (2002) J. Mol. Biol. 316, 435-441). Simulation of NOESY spectra based on crystallographic protomers enables the analysis of similarities and differences in solution. The different responses of the T- and R-state cores to chiral perturbation illustrates both their intrinsic plasticity and constraints imposed by hexamer assembly. Although variant T- and R-protomers retain nativelike protein surfaces, the receptor-binding activity of allo-Ile(A2)-insulin is low (2% relative to native insulin). This seeming paradox suggests that insulin undergoes a change in conformation to expose Ile(A2) at the hormone-receptor interface.
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Zhu-li
ZL
Wan
Bin
B
Xu
Ying-Chi
YC
Chu
Panayotis G
PG
Katsoyannis
Michael A
MA
Weiss
D001665:Binding Sites; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry*; D007532:Isoleucine / Q000737:chemistry*; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular; D011972:Receptor, Insulin / Q000737:chemistry*; D012996:Solutions; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D007328:Insulin; D008024:Ligands; D012996:Solutions; D007532:Isoleucine; D011972:Receptor, Insulin