Ligand binding and allostery can emerge simultaneously

Abstract

A heterotropic allosteric effect involves an effector molecule that is distinct from the substrate or ligand of the protein. How heterotropic allostery originates is an unanswered question. We have previously created several heterotropic allosteric enzymes by recombining the genes for TEM1 b-lactamase (BLA) and maltose binding protein (MBP) to create BLAs that are positively or negatively regulated by maltose. We show here that one of these engineered enzymes has ;10 6 M À1 affinity for Zn 2+, a property that neither of the parental proteins possesses. Furthermore, Zn 2+ is a negative effector that noncompetitively switches off b-lactam hydrolysis activity. Mutagenesis experiments indicate that the Zn 2+ -binding site does not involve a histidine or a cysteine, which is atypical of natural Zn 2+ -binding sites. These studies also implicate helices 1 and 12 of the BLA domain in allosteric signal propagation. These results support a model for the evolution of heterotropic allostery in which effector affinity and allosteric signaling emerge simultaneously.

Serine Protease Activity of Calnuc

“…The fact that these two proteins are targeted to the Golgi and the cytoplasm and that calnuc is also secreted to the outside suggests that the interaction might play a role in keeping the enzymatic activity in the off state until calnuc is sequestered from the G protein for its enzymatic function. The functions of the "Zn 2ϩ key" in calnuc, which locks the enzymatic activity, is similar to that observed in several other proteases that are regulated by Zn 2ϩ -mediated conformational switches (35)(36)(37)(38)(39)(40). The intracellular concentration of Zn 2ϩ varies from the picomolar (in the cytosol) to 100 mM in various organelles (e.g.…”

The Promiscuity of Allosteric Regulation of Nuclear Receptors by Retinoid X Receptor

“…21 As shown in Figure 1, the ligand binding domain (LBD) of RXR forms heterodimeric complexes with the corresponding LBD of those NR proteins and influences the ligand-binding properties (and thus the activities) of these proteins through heterotropic allostery. 22 Despite the fact that all LBDs of the NR proteins share a structural fold of 12 helices and other common secondary structural features, 23 the binding of RXR and its cognate ligand 9c has drastically different effects depending on RXR’s binding partners. For example, the heterodimer RXR:TR can be activated upon the binding of cognate ligand t3 to TR, however the transactivation levels decreases in the presence of 9c -bound RXR.…”

Protein Switch Engineering by Domain Insertion

“…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). …”