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 AU-rich 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 CysX(8)CysX(5)CysX(3)His 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.
diTargiani
Robert C
RC
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201-1180, USA.
Lee
Seung Jae
SJ
Wassink
Sarah
S
Michel
Sarah L J
SL
D000818:Animals; D016880:Anisotropy; D001483:Base Sequence; D003001:Cloning, Molecular; D003035:Cobalt / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D051379:Mice; D012333:RNA, Messenger / Q000378:metabolism*; D013053:Spectrophotometry; D051816:Tristetraprolin / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
D005290:Ferric Compounds; D005296:Ferrous Compounds; D012333:RNA, Messenger; D051816:Tristetraprolin; D003035:Cobalt; D007501:Iron; D015032:Zinc