Structure of an Engineered β-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 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.

Protein Switch Engineering by Domain Insertion

“…The MBP–BLA switch genes function in E. coli cells and confer a new phenotype: maltose-dependent resistance to β-lactam antibiotics (Guntas et al, 2005, 2004). Many of the MBP–BLA switches function as heterotropic allosteric enzymes, and NMR and crystallographic studies of one switch are consistent with the expectation that the individual domain structures of RG13 are substantially conserved from MBP and BLA (Ke et al, 2012; Wright, Majumdar, Tolman, & Ostermeier, 2010). More recently, we have identified MBP–BLA switch genes that do not encode allosteric proteins but rather encode a protein whose cellular accumulation increases in the presence of the effector, thereby conferring an effector-dependent switching phenotype to cells (Heins, Choi, Sohka, & Ostermeier, 2011; Sohka et al, 2009).…”

“…Finally, our MBP–BLA switch work has shown how domain fusion can result in emergent properties. One of our MBP–BLA switches is negatively, allosterically regulated by Zn 2+ , which is unexpected since neither MBP nor BLA has significant affinity for Zn 2+ (Ke et al, 2012; Liang, Kim, Boock, Mansell, & Ostermeier, 2007). …”

Engineering allosteric control to an unregulated enzyme by transfer of a regulatory domain

“…For example, random domain insertion of the β-lactamase gene into maltose-binding protein created enzymes with activity both up-and down-regulated by maltose, and inhibited, in one chimera, by Zn 2+ that binds at an interface between the two domains (30,33,35). The full structural details of this system have only recently been reported (36,37). Light-dependent catalytic activity has been observed when dihydrofolate reductase was linked to a light-sensing signaling domain (32).…”

Full and Partial Agonism of a Designed Enzyme Switch

“…These systems have typically been designed through an engineering-centric “bottom-up” approach 5 : functional modules from different biological systems are mixed and matched to obtain the desired function. In another example, maltose-binding protein (input domain) was fused to β-lactamase (output domain) such that sugar binding regulated β-lactamase activity 11 . However, engineering these switches is still far from rational; it entails extensive trial-and-error and/or directed evolution, particularly with regards to the linkers used for tethering together the functional modules 12 .…”