Journal Article

Engineering allosteric regulation into the hinge region of a circularly permuted TEM-1 β-lactamase

Valéry Mathieu, Jacques Fastrez and Patrice Soumillion

in Protein Engineering, Design and Selection

Volume 23, issue 9, pages 699-709
Published in print September 2010 | ISSN: 1741-0126
Published online June 2010 | e-ISSN: 1741-0134 | DOI: http://dx.doi.org/10.1093/protein/gzq041
Engineering allosteric regulation into the hinge region of a circularly permuted TEM-1 β-lactamase

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In nature, the activity of many enzymes involved in important biochemical pathways is controlled by binding a ligand in a site remote from the active site. The allosteric sites are frequently located in hinge regulatory subunits, in which a conformational change can occur and propagate to the active site. The enzymatic activity is then enhanced or decreased depending on the type of effectors. Many artificial binding sites have been created to engineer an allosteric regulation. Generally, these sites were engineered near the active site in loops or at the surface of contiguous helices or strands but rarely in hinge regions. This work aims at exploring the possibility of regulating a monomeric enzyme whose active site is located at the interface between two domains. We anticipated that binding of a ligand in the hinge region linking the domains would modify their positioning and, consequently, modulate the activity. Here, we describe the design of two mutants in a circularly permuted TEM-1 (cpTEM-1) β-lactamase. The first one, cpTEM-1-His3 was created by a rational design. It shows little regulation upon metal ion binding except for a weak activation with Zn2+. The second one, cpTEM-1-3M-His2, was selected by a directed evolution strategy. It is allosterically down-regulated by Zn2+, Ni2+ and Co2+ with binding affinities around 300 µM.

Keywords: allosteric regulation; hinge region; metallic ions; permuted TEM-1 β-lactamase; phage-display

Journal Article.  8221 words.  Illustrated.

Subjects: Proteins

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