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Structure of an engineered ?-lactamase maltose binding protein fusion protein: insights into heterotropic allosteric regulation.

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 ?-lactamase gene into the maltose binding protein (MBP). RG13 is positively regulated by maltose yet is, serendipitously, inhibited by Zn(2+) at low µM concentration. To probe the structure and allostery of RG13, we crystallized RG13 in the presence of mM Zn(2+) 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, Zn(2+) 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 ?3 and ?4 strands important for activity. Mutagenesis of residues comprising the crystallographically observed Zn(2+) site only slightly affected Zn(2+) inhibition 2- to 4-fold. Combined with previous mutagenesis results we therefore hypothesize the presence of two or more inter-domain mutually exclusive inhibitory Zn(2+) 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 C? 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 ?3 linker region via steric and/or linker juxtapositioning mechanisms.

SUBMITTER: Ke W

PROVIDER: S-EPMC3375305 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Structure of an engineered β-lactamase maltose binding protein fusion protein: insights into heterotropic allosteric regulation.

Ke Wei W Laurent Abigail H AH Armstrong Morgan D MD Chen Yuchao Y Smith William E WE Liang Jing J Wright Chapman M CM Ostermeier Marc M van den Akker Focco F

PloS one 20120614 6

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 Zn(2+) at low µM concentration. To probe the structure and allostery of RG13, we crystallized RG13 in the presenc ...[more]

PMID: 22720063

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Project description:Beta-lactamases are enzymes that catalyze the hydrolysis of beta-lactam antibiotics. beta-lactamase/beta-lactamase inhibitor protein (BLIP) complexes are emerging as a well characterized experimental model system for studying protein-protein interactions. BLIP is a 165 amino acid protein that inhibits several class A beta-lactamases with a wide range of affinities: picomolar affinity for K1; nanomolar affinity for TEM-1, SME-1, and BlaI; but only micromolar affinity for SHV-1 beta-lactamase. The large differences in affinity coupled with the availability of extensive mutagenesis data and high-resolution crystal structures for the TEM-1/BLIP and SHV-1/BLIP complexes make them attractive systems for the further development of computational design methodology. We used EGAD, a physics-based computational design program, to redesign BLIP in an attempt to increase affinity for SHV-1. Characterization of several of designs and point mutants revealed that in all cases, the mutations stabilize the interface by 10- to 1000-fold relative to wild type BLIP. The calculated changes in binding affinity for the mutants were within a mean absolute error of 0.87 kcal/mol from the experimental values, and comparison of the calculated and experimental values for a set of 30 SHV-1/BLIP complexes yielded a correlation coefficient of 0.77. Structures of the two complexes with the highest affinity, SHV-1/BLIP (E73M) and SHV-1/BLIP (E73M, S130K, S146M), are presented at 1.7 A resolution. While the predicted structures have much in common with the experimentally determined structures, they do not coincide perfectly; in particular a salt bridge between SHV-1 D104 and BLIP K74 is observed in the experimental structures, but not in the predicted design conformations. This discrepancy highlights the difficulty of modeling salt bridge interactions with a protein design algorithm that approximates side chains as discrete rotamers. Nevertheless, while local structural features of the interface were sometimes miscalculated, EGAD is globally successful in designing complexes with increased affinity.

Dataset Information

Structure of an engineered ?-lactamase maltose binding protein fusion protein: insights into heterotropic allosteric regulation.

Publications

Structure of an engineered β-lactamase maltose binding protein fusion protein: insights into heterotropic allosteric regulation.

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