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J. Biol. Chem., Vol. 283, Issue 21, 14703-14716, May 23, 2008

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Design of an Active Ultrastable Single-chain Insulin Analog

SYNTHESIS, STRUCTURE, AND THERAPEUTIC IMPLICATIONS^*diamf

Qing-xin Hua¹, Satoe H. Nakagawa¹², Wenhua Jia, Kun Huang, Nelson B. Phillips³, Shi-quan Hu, and Michael A. Weiss⁴

From the Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 and the Department of Medicine, University of Chicago, Chicago, Illinois 60637

Single-chain insulin (SCI) analogs provide insight into the inter-relation of hormone structure, function, and dynamics. Although compatible with wild-type structure, short connecting segments (<3 residues) prevent induced fit upon receptor binding and so are essentially without biological activity. Substantial but incomplete activity can be regained with increasing linker length. Here, we describe the design, structure, and function of a single-chain insulin analog (SCI-57) containing a 6-residue linker (GGGPRR). Native receptor-binding affinity (130 ± 8% relative to the wild type) is achieved as hindrance by the linker is offset by favorable substitutions in the insulin moiety. The thermodynamic stability of SCI-57 is markedly increased (G_u = 0.7 ± 0.1 kcal/mol relative to the corresponding two-chain analog and 1.9 ± 0.1 kcal/mol relative to wild-type insulin). Analysis of inter-residue nuclear Overhauser effects demonstrates that a native-like fold is maintained in solution. Surprisingly, the glycine-rich connecting segment folds against the insulin moiety: its central Pro contacts Val^A3 at the edge of the hydrophobic core, whereas the final Arg extends the A1-A8 -helix. Comparison between SCI-57 and its parent two-chain analog reveals striking enhancement of multiple native-like nuclear Overhauser effects within the tethered protein. These contacts are consistent with wild-type crystal structures but are ordinarily attenuated in NMR spectra of two-chain analogs, presumably due to conformational fluctuations. Linker-specific damping of fluctuations provides evidence for the intrinsic flexibility of an insulin monomer. In addition to their biophysical interest, ultrastable SCIs may enhance the safety and efficacy of insulin replacement therapy in the developing world.

Received for publication, January 11, 2008 , and in revised form, February 20, 2008.

The atomic coordinates and structure factors (code 2jzq) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported, in whole or in part, by National Institutes of Health Grant DK040949 (to M. A. W.). Native fragment ligation of SCI-57 was supported by United States Department of Energy Grant DE-FG02-07ER64501 (to S. B. Kent). This article is a contribution from the Cleveland Center for Structural Biology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental "Experimental Procedures," Figs. S1-S5, Tables S1-S6, and additional references.

^diamf This article was selected as a Paper of the Week.

¹ Both authors contributed equally to this work.

² Supported by the Diabetes Research and Training Center at the University of Chicago.

³ Supported in part by a pilot grant from the Dietrich Diabetes Research Institute of the Diabetes Association of Greater Cleveland.

⁴ To whom correspondence should be addressed. E-mail: michael.weiss{at}case.edu.

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