Structural insight and characterization of human Twinkle helicase in mitochondrial disease.

Twinkle is the mammalian helicase vital for replication and integrity of mitochondrial DNA. Over 90 Twinkle helicase disease variants have been linked to progressive external ophthalmoplegia and ataxia neuropathies among other mitochondrial diseases. Despite the biological and clinical importance, Twinkle represents the only remaining component of the human minimal mitochondrial replisome that has yet to be structurally characterized. Here, we present 3-dimensional structures of human Twinkle W315L. Employing cryo-electron microscopy (cryo-EM), we characterize the oligomeric assemblies of human full-length Twinkle W315L, define its multimeric interface, and map clinical variants associated with Twinkle in inherited mitochondrial disease. Cryo-EM, crosslinking-mass spectrometry, and molecular dynamics simulations provide insight into the dynamic movement and molecular consequences of the W315L clinical variant. Collectively, this ensemble of structures outlines a framework for studying Twinkle function in mitochondrial DNA replication and associated disease states.

Design of an Ara h 2 hypoallergen from conformational epitopes.

INTRODUCTION: Adverse reactions are relatively common during peanut oral immunotherapy. To reduce the risk to the patient, some researchers have proposed modifying the allergen to reduce IgE reactivity, creating a putative hypoallergen. Analysis of recently cloned human IgG from patients treated with peanut immunotherapy suggested that there are three common conformational epitopes for the major peanut allergen Ara h 2. We sought to test if structural information on these epitopes could indicate mutagenesis targets for designing a hypoallergen and evaluated the reduction in IgE binding via immunochemistry and a mouse model of passive cutaneous anaphylaxis (PCA). METHODS: X-ray crystallography characterized the conformational epitopes in detail, followed by mutational analysis of key residues to modify monoclonal antibody (mAb) and serum IgE binding, assessed by ELISA and biolayer interferometry. A designed Ara h 2 hypoallergen was tested for reduced vascularization in mouse PCA experiments using pooled peanut allergic patient serum. RESULTS: A ternary crystal structure of Ara h 2 in complex with patient antibodies 13T1 and 13T5 was determined. Site-specific mutants were designed that reduced 13T1, 13T5, and 22S1 mAbs binding by orders of magnitude. By combining designed mutations from the three major conformational bins, a hexamutant (Ara h 2 E46R, E89R, E97R, E114R, Q146A, R147E) was created that reduced IgE binding in serum from allergic patients. Further, in the PCA model where mice were primed with peanut allergic patient serum, reactivity upon allergen challenge was significantly decreased using the hexamutant. CONCLUSION: These studies demonstrate that prior knowledge of common conformational epitopes can be used to engineer reduced IgE reactivity, an important first step in hypoallergen design.

Characterization of spironolactone and metabolites derivatized using Girard's reagent P using mass spectrometry and ion mobility spectrometry.

RATIONALE: Spironolactone is a steroidal drug prescribed for a variety of medical conditions and is extensively metabolized quickly after administration. Measurement of spironolactone and its metabolites remains challenging using mass spectrometry (MS) due to in-source fragmentation and relatively poor ionization using electrospray ionization. Therefore, improved methods of measurements are needed, particularly in the case of small sample volumes. METHODS: Girard's reagent P (GP) derivatization of spironolactone was employed to improve response and provide an MS-based solution to the measurement of spironolactone and its metabolites. We performed ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) and ion mobility spectrometry (IMS)-high-resolution mass spectrometry (HRMS) to fully characterize the GP derivatization products. Analytes were studied in positive ionization mode, and MS/MS was performed using nonresonance and resonance excitation collision-induced dissociation. RESULTS: We observed the successful GP derivatization of spironolactone and its metabolites using authentic chemical standards. A signal enhancement of 1-2 orders of magnitude was observed for GP-derivatized versions of spironolactone and its metabolites. Further, GP derivatization eliminated in-source fragmentation. Finally, we performed GP derivatization and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) in a small volume of murine serum (20 µL) from spironolactone-treated and control animals and observed multiple spironolactone metabolites only in the spironolactone-treated group. CONCLUSIONS: GP derivatization was proven to have advantageous mass spectral performance (e.g., limiting in-source fragmentation, enhancing signals, and eliminating isobaric analytes) for spironolactone and its metabolites. This work and the detailed characterization using ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) and IMS serve as the foundation for future developments in reaction optimization and/or quantitative assay development.

Probing the mechanisms of two exonuclease domain mutators of DNA polymerase ϵ.

We report the properties of two mutations in the exonuclease domain of the Saccharomyces cerevisiae DNA polymerase ϵ. One, pol2-Y473F, increases the mutation rate by about 20-fold, similar to the catalytically dead pol2-D290A/E290A mutant. The other, pol2-N378K, is a stronger mutator. Both retain the ability to excise a nucleotide from double-stranded DNA, but with impaired activity. pol2-Y473F degrades DNA poorly, while pol2-N378K degrades single-stranded DNA at an elevated rate relative to double-stranded DNA. These data suggest that pol2-Y473F reduces the capacity of the enzyme to perform catalysis in the exonuclease active site, while pol2-N378K impairs partitioning to the exonuclease active site. Relative to wild-type Pol ϵ, both variants decrease the dNTP concentration required to elicit a switch between proofreading and polymerization by more than an order of magnitude. While neither mutation appears to alter the sequence specificity of polymerization, the N378K mutation stimulates polymerase activity, increasing the probability of incorporation and extension of a mismatch. Considered together, these data indicate that impairing the primer strand transfer pathway required for proofreading increases the probability of common mutations by Pol ϵ, elucidating the association of homologous mutations in human DNA polymerase ϵ with cancer.

The mosquito protein AEG12 displays both cytolytic and antiviral properties via a common lipid transfer mechanism.

The mosquito protein AEG12 is up-regulated in response to blood meals and flavivirus infection though its function remained elusive. Here, we determine the three-dimensional structure of AEG12 and describe the binding specificity of acyl-chain ligands within its large central hydrophobic cavity. We show that AEG12 displays hemolytic and cytolytic activity by selectively delivering unsaturated fatty acid cargoes into phosphatidylcholine-rich lipid bilayers. This property of AEG12 also enables it to inhibit replication of enveloped viruses such as Dengue and Zika viruses at low micromolar concentrations. Weaker inhibition was observed against more distantly related coronaviruses and lentivirus, while no inhibition was observed against the nonenveloped virus adeno-associated virus. Together, our results uncover the mechanistic understanding of AEG12 function and provide the necessary implications for its use as a broad-spectrum therapeutic against cellular and viral targets.

A post-transcriptional regulon controlled by TtpA, the single tristetraprolin family member expressed in Dictyostelium discoideum.

Post-transcriptional processes mediated by mRNA binding proteins represent important control points in gene expression. In eukaryotes, mRNAs containing specific AU-rich motifs are regulated by binding of tristetraprolin (TTP) family tandem zinc finger proteins, which promote mRNA deadenylation and decay, partly through interaction of a conserved C-terminal CNOT1 binding (CNB) domain with CCR4-NOT protein complexes. The social ameba Dictyostelium discoideum shared a common ancestor with humans more than a billion years ago, and expresses only one TTP family protein, TtpA, in contrast to three members expressed in humans. Evaluation of ttpA null-mutants identified six transcripts that were consistently upregulated compared to WT during growth and early development. The 3'-untranslated regions (3'-UTRs) of all six 'TtpA-target' mRNAs contained multiple TTP binding motifs (UUAUUUAUU), and one 3'-UTR conferred TtpA post-transcriptional stability regulation to a heterologous mRNA that was abrogated by mutations in the core TTP-binding motifs. All six target transcripts were upregulated to similar extents in a C-terminal truncation mutant, in contrast to less severe effects of analogous mutants in mice. All six target transcripts encoded probable membrane proteins. In Dictyostelium, TtpA may control an 'RNA regulon', where a single RNA binding protein, TtpA, post-transcriptionally co-regulates expression of several functionally related proteins.

Characterization of SARS2 Nsp15 nuclease activity reveals it's mad about U.

Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3' of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here, we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3' of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA, and will aid in the development of new anti-viral therapeutics.

Pharmacophore optimization of imidazole chalcones to modulate microtubule dynamics.

We recently reported a new class of imidazole-based chalcones as potential antimitotic agents. In view of their promising cytotoxic activity, a comprehensive structure-activity relationship (SAR) of these compounds was undertaken focusing on four major structural variations: the length of the molecule, the Michael acceptor character, the nature and substitution pattern of ring B, and the nature of the amide functionality tethering ring B. These second-generation analogs (IBCs) demonstrated a superior bioactivity profile than the previously reported imidazole chalcones (referred to as IPEs). The analog IBC-2 with one less methylene group (nor series) and para-fluoro substituted ring B demonstrated the best cytotoxicity profile among the library of compounds. A computational analysis of the NCI-60 data associated both IBCs and the previously reported IPEs with the privileged pharmacological pharmacophore of chalcones. Interestingly, biological studies suggest that the imidazole ring is essential for cytotoxic activity of the elongated chalcone analogues. Immunofluorescence studies revealed that IBC-2, unlike IPEs, has the ability to induce microtubule catastrophe independently of Aurora-B inhibition. The effects of IBC-2 on microtubule dynamics are similar to those of Nocodazole, but the cell cycle effects appear to be different. In-silico studies demonstrate that the members of the new series have the ability to bind to the colchicine binding site of ß-tubulin with binding scores similar to those of IPEs, corresponding chalcones and Nocodazole. Although tubulin binding can partially explain the biological effects of IBC-2, on-going target identification studies are aimed at further investigation of its biological targets.

An Ancient Family of RNA-Binding Proteins: Still Important!

RNA-binding proteins are important modulators of mRNA stability, a crucial process that determines the ultimate cellular levels of mRNAs and their encoded proteins. The tristetraprolin (TTP) family of RNA-binding proteins appeared early in the evolution of eukaryotes, and has persisted in modern eukaryotes. The domain structures and biochemical functions of family members from widely divergent lineages are remarkably similar, but their mRNA 'targets' can be very different, even in closely related species. Recent gene knockout studies in species as distantly related as plants, flies, yeasts, and mice have demonstrated crucial roles for these proteins in a wide variety of physiological processes. Inflammatory and hematopoietic phenotypes in mice have suggested potential therapeutic approaches for analogous human disorders.

A new class of cytotoxic agents targets tubulin and disrupts microtubule dynamics.

Despite the advances in treatment strategies, cancer is still the second leading cause of death in the USA. A majority of the currently used cancer drugs have limitations in their clinical use due to poor selectivity, toxic side effects and multiple drug resistance, warranting the development of new anticancer drugs of different mechanisms of action. Here we describe the design, synthesis and initial biological evaluation of a new class of antimitotic agents that modulate tubulin polymerization. Structurally, these compounds are chalcone mimics containing a 1-(1H-imidazol-2-yl)ethan-1-one moiety, which was initially introduced to act as a metal-binding group and inhibit histone deacetylase enzymes. Although several analogues selectively inhibited purified HDAC8 with IC50 values in low micromolar range, tissue culture studies suggest that HDAC inhibition is not a major mechanism responsible for cytotoxicity. The compounds demonstrated cell growth inhibition with GI50 values of upper nanomolar to low micromolar potency with significant selectively for cancer over normal cells. Interestingly, several compounds arrested HeLaM cells in mitosis and seem to target tubulin to cause mitotic arrest. For example, when combined with inhibitors of Aurora B kinase, they led to dramatic disassembly of the mitotic spindle. In-vitro tubulin polymerization studies showed that the compounds reduced the rate of polymerization of microtubules during the elongation phase and lowered the amount of polymerized tubulin during the plateau phase. Finally, in silico docking studies identified binding of IPE-7 to the colchicine site with similar affinity as the test compound D64131. These compounds represent a new antimitotic pharmacophore with limited HDAC inhibitory activity.

Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide.

Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol ß, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

Revealing the role of the product metal in DNA polymerase ß catalysis.

DNA polymerases catalyze a metal-dependent nucleotidyl transferase reaction during extension of a DNA strand using the complementary strand as a template. The reaction has long been considered to require two magnesium ions. Recently, a third active site magnesium ion was identified in some DNA polymerase product crystallographic structures, but its role is not known. Using quantum mechanical/ molecular mechanical calculations of polymerase ß, we find that a third magnesium ion positioned near the newly identified product metal site does not alter the activation barrier for the chemical reaction indicating that it does not have a role in the forward reaction. This is consistent with time-lapse crystallographic structures following insertion of Sp-dCTPαS. Although sulfur substitution deters product metal binding, this has only a minimal effect on the rate of the forward reaction. Surprisingly, monovalent sodium or ammonium ions, positioned in the product metal site, lowered the activation barrier. These calculations highlight the impact that an active site water network can have on the energetics of the forward reaction and how metals or enzyme side chains may interact with the network to modulate the reaction barrier. These results also are discussed in the context of earlier findings indicating that magnesium at the product metal position blocks the reverse pyrophosphorolysis reaction.

A Bioactive Resveratrol Trimer from the Stem Bark of the Sri Lankan Endemic Plant Vateria copallifera.

A new resveratrol trimer, vateriferol (1), having four cis-oriented methine protons and constituting four contiguous stereocenters, was isolated from the bark extract of Vateria copallifera by bioassay-guided fractionation using a combination of normal, reversed phase, and size exclusion column chromatography. The structure was established based on its spectroscopic data. Vateriferol (1) was evaluated in vitro for its antioxidant capacity, enzyme inhibitory activity, growth inhibitory activity on a number of cancer cell lines, neuroprotective activity, and anti-inflammatory activity. Vateriferol (1) exhibited AChE inhibitory activity (IC50 8.4 ± 0.2 µM), ORAC activity (2079 ± 0.20 TE/g), and neuroprotective activity at 1.5 µM using PC12 cells deprived of oxygen and glucose and lowered NO levels in lipopolysaccharide-stimulated SIM-A9 microglial cells at 14.7 and 73.6 µM. Vateriferol (1) exhibited weak cytotoxic potency (<50% growth inhibition) against the tested cell lines at 147.2 µM.

Identification of drivers for the metamorphic transition of HIV-1 reverse transcriptase.

Recent structural characterizations of the p51 and p66 monomers have established an important starting point for understanding the maturation pathway of the human immunodeficiency virus (HIV)-1 reverse transcriptase p66/p51 heterodimer. This process requires a metamorphic transition of the polymerase domain leading to formation of a p66/p66' homodimer that exists as a structural heterodimer. To better understand the drivers for this metamorphic transition, we have performed NMR studies of 15N-labeled RT216 - a construct that includes the fingers and most of the palm domains. These studies are consistent with the conclusion that the p66 monomer exists as a spring-loaded complex. Initial dissociation of the fingers/palm : connection complex allows the fingers/palm to adopt an alternate, more stable structure, reducing the rate of reassociation and facilitating subsequent maturation steps. One of the drivers for an initial extension of the fingers/palm domains is identified as a straightening of helix E relative to its conformation in the monomer by eliminating a bend of ∼50° near residue Phe160. NMR and circular dichroism data also are consistent with the conclusion that a hydrophobic surface of palm domain that becomes exposed after the initial dissociation, as well as the intrinsic conformational preferences of the palm domain C-terminal segment, facilitates the formation of the ß-sheet structure that is unique to the active polymerase subunit. Spectral comparisons based on 15N-labeled constructs are all consistent with previous structural conclusions based on studies of 13C-methyl-labeled constructs.

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA-protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg(2+)-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg(2+) binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.

Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse.

DNA polymerases facilitate faithful insertion of nucleotides, a central reaction occurring during DNA replication and repair. DNA synthesis (forward reaction) is "balanced," as dictated by the chemical equilibrium by the reverse reaction of pyrophosphorolysis. Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic metal lowers the pKa of O3' of the growing primer terminus, and the nucleotide-binding metal facilitates substrate binding. Recent time-lapse crystallographic studies of DNA polymerases have identified an additional metal ion (product metal) associated with pyrophosphate formation, leading to the suggestion of its possible involvement in the reverse reaction. Here, we establish a rationale for a role of the product metal using quantum mechanical/molecular mechanical calculations of the reverse reaction in the confines of the DNA polymerase ß active site. Additionally, site-directed mutagenesis identifies essential residues and metal-binding sites necessary for pyrophosphorolysis. The results indicate that the catalytic metal site must be occupied by a magnesium ion for pyrophosphorolysis to occur. Critically, the product metal site is occupied by a magnesium ion early in the pyrophosphorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal ions is consistent with crystallographic structures. The transition barrier for pyrophosphorolysis was estimated to be significantly higher than that for the forward reaction, consistent with kinetic activity measurements of the respective reactions. These observations provide a framework to understand how ions and active site changes could modulate the internal chemical equilibrium of a reaction that is central to genome stability.

Hiding in Plain Sight: The Bimetallic Magnesium Covalent Bond in Enzyme Active Sites.

The transfer of phosphate groups is an essential function of many intracellular biological enzymes. The transfer is in many cases facilitated by a protein scaffold involving two closely spaced magnesium "ions". It has long been a mystery how these "ions" can retain their closely spaced positions throughout enzymatic phosphate transfer: Coulomb's law would dictate large repulsive forces between these ions at the observed distances. Here we show, however, that the electron density can be borrowed from nearby electron-rich oxygens to populate a bonding molecular orbital that is largely localized between the magnesium "ions". The result is that the Mg-Mg core of these phosphate transfer enzymes is surprisingly similar to a metastable [Mg2]2+ ion in the gas phase, an ion that has been identified experimentally and studied with high-level quantum-mechanical calculations. This similarity is confirmed by comparative computations of the electron densities of [Mg2]2+ in the gas phase and the Mg-Mg core in the structures derived from QM/MM studies of high-resolution X-ray crystal structures. That there is a level of covalent bonding between the two Mg "ions" at the core of these enzymes is a novel concept that enables an improved vision of how these enzymes function at the molecular level. The concept is broader than magnesium-other biologically relevant metals (e.g., Mn and Zn) can also form similar stabilizing covalent Me-Me bonds in both organometallic and inorganic crystals.

Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation.

Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop-binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA.

Functional equivalence of an evolutionarily conserved RNA binding module.

Members of the tristetraprolin (TTP) family of proteins participate in the regulation of mRNA turnover after initially binding to AU-rich elements in target mRNAs. Related proteins from most groups of eukaryotes contain a conserved tandem zinc finger (TZF) domain consisting of two closely spaced, similar CCCH zinc fingers that form the primary RNA binding domain. There is considerable sequence variation within the TZF domains from different family members within a single organism and from different organisms, raising questions about sequence-specific effects on RNA binding and decay promotion. We hypothesized that TZF domains from evolutionarily distant species are functionally interchangeable. The single family member expressed in the fission yeast Schizosaccharomyces pombe, Zfs1, promotes the turnover of several dozen transcripts, some of which are involved in cell-cell interactions. Using knockin techniques, we replaced the TZF domain of S. pombe Zfs1 with the equivalent domains from human TTP and the single family member proteins expressed in the silkworm Bombyx mori, the pathogenic yeast Candida guilliermondii, and the plant Chromolaena odorata. We found that the TZF domains from these widely disparate species could completely substitute for the native S. pombe TZF domain, as determined by measurement of target transcript levels and the flocculation phenotype characteristic of Zfs1 deletion. Recombinant TZF domain peptides from several of these species bound to an AU-rich RNA oligonucleotide with comparably high affinity. We conclude that the TZF domains from TTP family members in these evolutionarily widely divergent species are functionally interchangeable in mRNA binding and decay.

Mutational and structural analysis of the tandem zinc finger domain of tristetraprolin.

Tristetraprolin (TTP), the best known member of a class of tandem (R/K)YKTELCX8CX5CX3H zinc finger proteins, can destabilize target mRNAs by first binding to AU-rich elements (AREs) in their 3'-untranslated regions (UTRs) and subsequently promoting deadenylation and ultimate destruction of those mRNAs. This study sought to determine the roles of selected amino acids in the RNA binding domain, known as the tandem zinc finger (TZF) domain, in the ability of the full-length protein to bind to AREs within the tumor necrosis factor α (TNF) mRNA 3'-UTR. Within the CX8C region of the TZF domain, mutation of some of the residues specific to TTP, not found in other members of the TTP protein family, resulted in decreased binding to RNA as well as inhibited mRNA deadenylation and decay. Evaluation of simulation solution models revealed a distinct structure in the second zinc finger of TTP that was induced by the presence of these TTP-specific residues. In addition, mutations within the lead-in sequences preceding the first C of highly conserved residues within the CX5C or CX3H regions or within the linker region between the two fingers also perturbed both RNA binding and the simulation model of the TZF domain in complex with RNA. We conclude that, although the majority of conserved residues within the TZF domain of TTP are required for productive binding, not all residues at sequence-equivalent positions in the two zinc fingers of the TZF domain of TTP are functionally equivalent.