Functional characterization of iron-substituted tristetraprolin-2D (TTP-2D, NUP475-2D): RNA binding affinity and selectivity

The protein tristetraprolin ( TTP, also known as NUP475 and TIS11) is a nonclassical zinc finger protein that is involved in regulating the inflammatory response. Specifically, TTP binds to AUrich sequence elements located at the 3' - untranslated region of cytokine mRNAs forming a complex that is degraded by the exosome. The nucleic acid binding region of TTP is comprised of two CysX8CysX5CysX3His domains that are activated in the presence of zinc. A two-domain construct of TTP (TTP-2D) has been cloned and overexpressed in E. coli. TTP-2D picks up visible red coloration from the expression media, unless it is expressed under iron-restricted conditions. The iron- binding properties of TTP- 2D and the effect of iron substitution on RNA recognition have been investigated. Both Fe( II) and Fe( III) bind to TTP- 2D and a full titration of Fe( III) with TTP- 2D revealed that this metal ion binds with micromolar affinity. Upon reconstitution of TTP- 2D with either Fe( II) or Fe( III), the protein recognizes a canonical RNA- binding sequence, UUUAUUUAUUU, with nanomolar affinity. Substitution of a single adenine or both adenines results in a decreased affinity of TTP- 2D for the RNA molecule, demonstrating that both Fe( II)- TTP- 2D and Fe( III)- TTP- 2D selectively recognize a physiologically relevant RNA sequence. The relative affinities of Fe( II)- TTP- 2D and Fe( III)- TTP- 2D for the series of RNA sequences mirror those observed for Zn( II)- TTP- 2D and suggest that iron is a viable substitute for zinc in this protein.