Modularity of Serpins: A BIFUNCTIONAL CHIMERA POSSESSING α1-PROTEINASE INHIBITOR AND THYROXINE-BINDING GLOBULIN PROPERTIES

Helmut Grasberger,Christoph Buettner,Onno E Janssen

doi:10.1074/jbc.274.21.15046

Helmut Grasberger, Christoph Buettner + Show 1 more

Open Access

https://doi.org/10.1074/jbc.274.21.15046

Copy DOI

Abstract

An exciting application of protein engineering is the creation of proteins with novel functions by the retrofitting of native proteins. Such attempts might be facilitated by the idea of a mosaic architecture of proteins out of structural units. Even though numerous theoretical concepts deal with the delineation of structural "modules," their potential in the design of proteins has not yet been sufficiently exploited. To address this question we used a gain of function approach by designing modular chimeric molecules out of two structurally homologous but functionally diverse members of the superfamily of serine-proteinase inhibitors, alpha1-proteinase inhibitor and thyroxine-binding globulin. Substitution of two of four alpha1-proteinase inhibitor modules (Lys222 to Leu288 and Pro362 to Lys394, respectively), identified by alpha-backbone distance analysis, with their thyroxine-binding globulin homologues resulted in a bifunctional chimera with inhibition of human leukocyte elastase and high affinity thyroxine binding. To our knowledge, this is the first report on a bifunctional chimera engineered from modules of homologous globular proteins. Our results demonstrate how a modular concept can facilitate the design of new functional proteins by swapping structural units chosen from members of a protein superfamily.

Highlights

In all but the smallest proteins, crystallography has revealed that polypeptide chains form several more or less compact units
To address this question we used a gain of function approach by designing modular chimeric molecules out of two structurally homologous but functionally diverse members of the superfamily of serine-proteinase inhibitors, ␣1-proteinase inhibitor and thyroxine-binding globulin
Our results demonstrate how a modular concept can facilitate the design of new functional proteins by swapping structural units chosen from members of a protein superfamily

Summary

Modularity of Serpins

A BIFUNCTIONAL CHIMERA POSSESSING ␣1-PROTEINASE INHIBITOR AND THYROXINE-BINDING GLOBULIN PROPERTIES*. Even though numerous theoretical concepts deal with the delineation of structural “modules,” their potential in the design of proteins has not yet been sufficiently exploited To address this question we used a gain of function approach by designing modular chimeric molecules out of two structurally homologous but functionally diverse members of the superfamily of serine-proteinase inhibitors, ␣1-proteinase inhibitor and thyroxine-binding globulin. We present a novel concept of a modular architecture of the serpin structure and construction of an ␣1PI-TBG chimera with both inhibitory activity and high affinity T4 binding. N or C denote N- or C-terminal external primers, specific for the TBG or ␣1PI coding sequences or their reverse complements, respectively (bold letters)

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION