Design of a Flexible, Zn-Selective Protein Scaffold that Displays Anti-Irving–Williams Behavior

Abstract

Selective metal binding is a key requirement not only for the functions of natural metalloproteins but also for the potential applications of artificial metalloproteins in heterogeneous environments such as cells and environmental samples. The selection of transition-metal ions through protein design can, in principle, be achieved through the appropriate choice and the precise positioning of amino acids that comprise the primary metal coordination sphere. However, this task is made difficult by the intrinsic flexibility of proteins and the fact that protein design approaches generally lack the sub-Å precision required for the steric selection of metal ions. We recently introduced a flexible/ probabilistic protein design strategy (MASCoT) that allows metal ions to search for optimal coordination geometry within a flexible, yet covalently constrained dimer interface. In an earlier proof-of-principle study, we used MASCoT to generate an artificial metalloprotein dimer, (AB) 2 , which selectively bound Co II and Ni II over Cu II (as well as other first-row transition-metal ions) through the imposition of a rigid octahedral coordination geometry, thus countering the Irving−Williams trend. In this study, we set out to redesign (AB) 2 to examine the applicability of MASCoT to the selective binding of other metal ions. We report here the design and characterization of a new flexible protein dimer, B 2 , which displays Zn II selectivity over all other tested metal ions including Cu II both in vitro and in cellulo. Selective, anti-Irving−Williams Zn II binding by B 2 is achieved through the formation of a unique trinuclear Zn coordination motif in which His and Glu residues are rigidly placed in a tetrahedral geometry. These results highlight the utility of protein flexibility in the design and discovery of selective binding motifs.

Homologous acetone carboxylases select Fe(II) or Mn(II) as the catalytic cofactor

“…These invoke biological constraints leading to metal selectivities such as pre-organized first coordination spheres that favor Mn(II) (octahedral) or Fe(II) (tetrahedral), the participation of one metal to template incorporation of the second, or specific side-chain based post-translational modifications that prohibit entry of one metal or the other. Recent work with designed metalloproteins that incorporate metals with preferences that do not follow the Irving-Williams series order do so through the participation of two metal binding sites, one of which dynamically biases the conformation of the protein to bind lower Irving-Williams metals in the second site, preferentially over Cu(II) ( 41 , 42 ). The mononuclear sites in the ACs, however, lack either a pre-organized, Mn(II)-favoring ligand set or post-translational modifications.…”

Quantifying metal ion specificity of the nickel-binding proteinCcNikZ-II fromClostridium carboxidivoransin the presence of competing metal ions

“…This illustrates how mechanistic models could suggest design principles to guide the improvement of a metalloprotein’s binding specificity and that these principles depend on the composition of solutions in prospective bio-remediation applications. With emerging research on de novo designed proteins [35, 36] that have anti-Irving-William series properties, our work here lays the groundwork for understanding how these synthetic proteins could be further engineered towards specific recovery of target metals from wastewater samples that contain more competing metals higher in the Irving-William series.…”

Primary and Secondary Coordination Sphere Effects on the Structure and Function of S-Nitrosylating Azurin

“…The properties of these metalloenzymes are largely governed not only by the primary metal-coordinating ligand sphere (PCS), but often complex secondary coordination sphere (SCS) environments that provide weak noncovalent effects, such as electrostatic, hydrophobic, steric, and hydrogen bonding interactions. Artificial metalloenzymes, designed using both de novo and re-engineering approaches, have provided an excellent basis for systematically understanding how the SCS modulates the properties of metal centers. − In particular, extensive investigations of the influence of the SCS on metal redox potentials have resulted in the ability to rationally and precisely tune redox potentials over wide ranges. − However, understanding the impact of these effects on tuning the reactivity of metal centers still remains at the frontier of metalloenzyme design.…”