Abstract
Engineering novel allostery into existing proteins is a challenging endeavor to obtain novel sensors, therapeutic proteins, or modulate metabolic and cellular processes. The RG13 protein achieves such allostery by inserting a circularly permuted TEM-1 β-lactamase gene into the maltose binding protein (MBP). RG13 is positively regulated by maltose yet is, serendipitously, inhibited by Zn2+ at low µM concentration. To probe the structure and allostery of RG13, we crystallized RG13 in the presence of mM Zn2+ concentration and determined its structure. The structure reveals that the MBP and TEM-1 domains are in close proximity connected via two linkers and a zinc ion bridging both domains. By bridging both TEM-1 and MBP, Zn2+ acts to “twist tie” the linkers thereby partially dislodging a linker between the two domains from its original catalytically productive position in TEM-1. This linker 1 contains residues normally part of the TEM-1 active site including the critical β3 and β4 strands important for activity. Mutagenesis of residues comprising the crystallographically observed Zn2+ site only slightly affected Zn2+ inhibition 2- to 4-fold. Combined with previous mutagenesis results we therefore hypothesize the presence of two or more inter-domain mutually exclusive inhibitory Zn2+ sites. Mutagenesis and molecular modeling of an intact TEM-1 domain near MBP within the RG13 framework indicated a close surface proximity of the two domains with maltose switching being critically dependent on MBP linker anchoring residues and linker length. Structural analysis indicated that the linker attachment sites on MBP are at a site that, upon maltose binding, harbors both the largest local Cα distance changes and displays surface curvature changes, from concave to relatively flat becoming thus less sterically intrusive. Maltose activation and zinc inhibition of RG13 are hypothesized to have opposite effects on productive relaxation of the TEM-1 β3 linker region via steric and/or linker juxtapositioning mechanisms.
Highlights
Allosteric regulation is a common mechanism cells utilize to regulate protein activity
The structure reveals that despite the domain shuffling, circular permutation, and different connections, RG13 contains two folded domains comprised of the regulatory maltose binding protein (MBP) and the catalytic TEM-1 (Figure 1B)
The overall dimensions of each RG13 molecule are roughly 50670670Awith the TEM-1 and MBP domains lying alongside each other connected by two linkers
Summary
Allosteric regulation is a common mechanism cells utilize to regulate protein activity. One approach to engineering allosteric proteins is to mimic nonhomologous recombination via domain/module combinations and rearrangements. This approach can develop complex new functions involving molecular recognition and regulation [3] and can lead to development of analytical molecular sensors [4]. A successful example of this approach is the allosteric protein RG13 which is a molecular switch created by recombining the nonhomologous genes encoding MBP and TEM-1 b-lactamase and subjecting the resulting library to evolutionary pressure [5]. The constructs were subjected to an in vivo selection for high ampicillin hydrolysis activity in the presence of maltose, followed by an in vitro screen for variants with maltose-dependent rate of b-lactam hydrolysis. RG13 activity was serendipitously found to be negatively regulated by Zn2+ at low mM concentrations in a noncompetitive and reversible mode, a characteristic that neither parent protein MBP nor TEM-1 possesses [6]
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