1. Zinc Finger Proteins

New Families of Non-Classical Zinc Finger Proteins: Zinc Finger proteins (ZFs) are proteins that utilize zinc as a co-factor.1-3 ZFs contain domains with repeats of cysteine and histidine residues that coordinate zinc resulting in a folded protein that can function – e.g. recognize DNA, RNA or another protein. More than 10% of the human genome encodes for ZF type proteins, yet many of these proteins’ mechanisms of metal mediated DNA, RNA or protein recognition are poorly understood. Much of what we know about ZF proteins is based upon ‘classical’ ZFs, which contain zinc-binding domains with a conserved Cys2His2 motif, fold into an alpha helical-beta strand structure when zinc is coordinated and recognize specific DNA targets. However, there are at least 13 other classes of non-classical ZFs, which have distinct domains, structures and functions. My laboratory focuses on two classes of ‘non classical’ ZF proteins: the Cys3His (or CCCH) class and the Cys2His2Cys (or CCHHC) class. We aim to discover how proteins from these families use zinc coordination to fold and to identify these proteins’ biological targets. Our long- term goal is to identify the paradigms of metal mediated DNA/RNA or protein recognition by these new types of zinc fingers. The non-classical ZFs that we are studying play key roles in inflammation, infectious disease and neuronal development. Therefore, we aim to identify strategies to modulate their activity as a means to target inflammation and infectious disease and to understand neuronal development.

r1

Fig. 1. (a) Cartoon diagram of TTP/RNA recognition. (b) Characterization of ZF sites of TTP via Co-binding (c) Fluorescence anisotropy to measure RNA binding of TTP.

Tristetraprolin (CCCH Zinc Finger): Tristetraprolin (TTP) is a CCCH ZF protein that regulates inflammation by targeting cytokine mRNAs.2 TTP binds to adenine and uracil rich repeats located at the 3’ untranslated region of cytokine mRNA, forming a complex that is degraded thereby shutting off inflammation. We were the first laboratory to prepare a construct of the protein that consists of the just the two ZF domains and demonstrate that it specifically recognizes AU-rich mRNA (Fig.1).4 We have also shown that TTP can be activated with either zinc or iron bound suggesting a functional mechanism in which metal ion choice is flexible.4 TTP is present during periods of increased oxidative stress, and we developed a rapid spectroscopic assay to measure ZF oxidation as a function of metal ion, oxidant and time and found that zinc protects TTP from oxidation.5 We demonstrated that this is a generalizable assay by applying the assay to measure oxidation of classical ZF proteins.6 We also reported that Cd affects the function of TTP, suggesting a mechanism for Cd induced toxicity.7 Our work has defined the key sequence elements within TTP that are required for RNA binding, identified the metal ions that activate the protein and determined the roles of ROS in function.

Our current efforts are focused on developing TTP as an anti-inflammatory, in collaboration with G. Wilson (UMB School of Medicine), and investigating how metalllodrugs interact with TTP to mediate inflammation. We are also investigating the interactions of gold complexes, as models of anti-inflammatory gold drugs, with TTP. In addition, we are exploring the role of H2S signaling in TTP function in collaboration with M. Filopovic, U. Bordeaux and the consequences of copper on TTP function in colllaboration with K. Splan, Macalaster College. A collaboration with O. Seneque, U. Grenoble aimed at designing TTP luminescent probes is also ongoing.

research2

Fig. 2. (a) Cartoon diagram of pre-mRNA processing with CPSF30 colored blue. (b) CPSF30 protein is red and exhibits a UV-visible spectrum indicative of iron coordination. (c) XANES data for CPSF30 reveals 2Fe-2S cluster.

Cleavage and Polyadenylation Specificity Factor 30 (CPSF30): CPSF30 is a CCCH type ZF protein that is part of a complex of proteins named CPSF that are involved in pre-mRNA processing. Pre-mRNA processing involves cleavage of a poly uracil tail followed by addition of a poly adenosine tails (Fig. 2a). We were the first laboratory to prepare a construct of CPSF30 that contains its five CCCH domains and identify its functional role – it binds to an AU-rich hexamer present in all pre-mRNA. We also discovered that CPSF30 contains a 2Fe-2S cluster, in addition to four zinc sites (Fig. 2b & c). Both the iron and zinc sites must be occupied, to achieve high affinity RNA binding. The 2Fe-2S site is only the second example of a CCCH ligated 2Fe-2S cluster in biology! Thus, our work was the first to show that CPSF30 binds AU-rich RNA and that the protein contains an unusual 2Fe-2S cluster. We recently published these findings in PNAS.8

The role of the 2Fe-2S cluster in CPSF30 as well as the mechanism of RNA recognition remain unanswered. Our current efforts involve a biophysical/structural/spectroscopic approach to understand the 2Fe-2S cluster’s role and to determine its mechanism of RNA recognition. Our experimental approaches include HDX/MS exchange (in collaboration with Patrick Wintrode, UMB), and resonance Raman, EPR UV-visible and CD spectroscopies (in collaboration with Michael Johnson, U. Ga). We have also begun working on the K. lactis (yeast) version of CPSF30 to correlate our in vitro findings in an in vivo model system.

Neural Zinc Finger Factor (CCHHC Zinc Finger): Neural Zinc Finger Factor 1 (NZF-1) and its homolog Myelin Transcription Factor 1 (MyT1) contain CCHHC repeats, are involved in the regulation of the central nervous system and are critically important for neuronal development and myelin formation. NZF-1 and MyT1 have high sequence homology and contain similar clusters of CCHHC domains; yet recognize completely different DNA targets and regulate different functions. Our work has focused on identifying the ligands involved in Zn coordination, the DNA targets for these proteins and determining the key sequence elements that allow NZF-1 and MyT1 to recognize different DNA targets (Fig. 3).1

Fig. 3. (a) NZF-1 regulates -RAR to promote neuronal development, inset fluorescence anisotropy was utilized to understand DNA binding requirements (b) Paradigm of DNA recognition: sequence logo indicates the amino acid residues that tune DNA recognition for the Cys2His2Cys class of ZFs.

Fig. 3. (a) NZF-1 regulates beta-RAR to promote neuronal development, inset fluorescence anisotropy was utilized to understand DNA binding requirements (b) Paradigm of DNA recognition: sequence logo indicates the amino acid residues that tune DNA recognition for the Cys2His2Cys class of ZFs.

We have performed extensive mutagenesis studies to identify which of the five potentially coordinating residues (Cys2His2Cys) present in each domain bind Zn and have determined that it is always the second histidine, with the first histidine playing a key role in hydrogen bonding during DNA recognition.9,10 We have compared the DNA binding properties of NZF-1 and MyT1 and identified a single amino acid residue that ‘switches’ the DNA binding properties of MyT1 to those of NZF-1.9 These results led us to propose a new paradigm for DNA binding and recognition by the Cys2His2Cys class of ZFs that involves a small subset of non-conserved amino acids dictating the DNA recognition properties of the protein. Our current efforts on NZF-1 and MyT1 involve using high-throughput approaches to identify additional gene targets and examining the function of these proteins in cell models using a cobalt-based probe.

2. Metal speciation in biological systems

Analytical Approaches to measure iron speciation in biological systems: The Michel laboratory is part of a team of researchers on an FDA funded clinical trial that aims to characterize iron speciation in blood plasma from intravenous (IV) iron treatment with iron nanoparticles. Patients with chronic kidney disease develop severe anemia and must be treated with IV iron. IV iron drugs are nanoparticle-based drugs, and in the US there is one generic IV iron drug on the market: ferric iron gluconate (the brand is named Ferrlecit). There are concerns that the generic ferric iron gluconate has adverse (toxic) effects in some patients. The hypothesis is that iron releases differently to the plasma in the generic drug. This causes iron overload, resulting in increased labile iron (or non-transferrin bound iron) in the plasma. The Michel laboratory is developing high throughput LC/ICP-MS based assays to measure the iron speciation in plasma of patients treated with iron nanoparticle drugs (Fig. 4).

Fig. 4. Main iron species in plasma during iron overload: total iron (TI), transferrin bound iron (TBI) and non-transferrin bound iron (NTBI).

Fig. 4. Main iron species in plasma during iron overload: total iron (TI), transferrin bound iron (TBI) and non-transferrin bound iron (NTBI).

We will apply this assay to clinical trial of 52 healthy volunteers. We anticipate that this novel LC/ICP-MS approach will have broad applicability – both for the study of metal-nanoparticle biodistribution as well as for understanding metal ion speciation in biological systems.

3. METALLOREGULATORY PROTEINS

NikR Proteins: Metalloregulatory proteins are a type of transcription factor that coordinate metal ions to control metal ion homeostasis. Metal ion coordination to specific sites on the protein causes a change in the protein’s structure, which then induces binding of the protein to specific DNA sequences.11 One class of metalloregulatory proteins is known as the NikR (‘nickel regulatory) protein family. NikR proteins are a large family of proteins found in a wide variety of bacteria and archaea. The biological role of NikR proteins is to regulate genes that encode for proteins involved in the import, trafficking, use and storage of nickel ions. The long-term goal of our research program in this area is to understand how Nature has adapted the structure/function properties of proteins in response to evolutionary pressures from host bacteria. Our first efforts have focused on NikR proteins, beginning with Helicobacter pylori NikR (HpNikR).

Nickel reg2

Fig. 5. Cartoon diagram of genes regulated by NikR from Helicbacter pylori.

HpNikR: HpNikR is a key transcription factor found in the virulent bacterium, Helicobacter pylori (H.pylori) which causes stomach ulcers in the short term and stomach cancers in the long term. HpNikR regulates multiple genes found in (H.pylori) in a nickel dependent manner (Fig. 5). To understand how HpNikR regulates multiple genes and the role(s) of nickel coordination in this function, we have combined biophysical and structural studies that include fluorescence anisotropy, UV-visible spectroscopy, X-ray crystallography and Small Angle X-ray Scattering (SAXS). Our first discovery was that HpNikR requires two metal ions to recognize target DNA – a ‘high affinity’ Ni(II) ion and a ‘low affinity’ Mg(II) [or Ca(II) or Mn(II)] ion.12 We also discovered that DNA recognition by HpNikR follows a ‘two-tiered’ mechanism in which genes that encode for nickel co-factored proteins are preferentially recognized.13

NIKR structure

Fig. 6. Crystal structure of HpNikR

We then published the first high- resolution crystal structure of holo-HpNikR in collaboration with Professor Edvin Pozharskiy (UMB School of Medicine) (Fig. 6).14 This structure showed that the four Ni(II) ions found in HpNikR coordinate to two distinct sites. In one site, two of the four Ni(II) ions are each bound by three histidines (H74, H88, H101) and 2-3 water molecules in a 5-coordinate square pyramidal geometry (2 water molecules) or a 6-coordinate octahedral geometry (with 3 water molecules). In the other site, the remaining two Ni(II) ions are bound by three histidines (H88, H99, H101) and one cysteine (C107) in a four-coordinate square planar site. We proposed that heterogeneous Ni(II) coordination in HpNikR was related to its multi-sequence DNA binding properties. Using a combination of mutagenesis, crystallography and solution based small angle X-ray scattering (SAXS, in collaboration with Profs. Hiro Tsuruta and Britt Hedman, SSRL, Stanford, CA), we found that when Ni(II) is restricted to only 4-coordinate sites (as seen in its homolog from E. coli), that HpNikR’s conformational flexibility is altered and DNA binding properties are affected. From these studies, we proposed a model of nickel mediated DNA recognition by HpNikR (Fig. 6).15

nikr mech

Fig. 7. Proposed mechanism of Ni(II) mediated DNA recognition by HpNikR.

The mechanism of DNA recognition by HpNikR is drastically different than the mechanism that has been determined for NikR from E. coli, which is the only homolog for which substantial mechanistic work has been obtained. We hypothesize that these difference can be attributed to bacterial evolution. Bacterial evolution involves a complex series of events that that leads to the exquisite adaptation of bacteria to specific environmental niches. A key component of this adaptation involves changes in the patterns of regulation in the bacteria. This regulatory control occurs at the level of transcription factors, and as more transcription factors are characterized, we are learning that evolutionary changes can be observed at the molecular level. Thus, our current research efforts are focused on understanding how NikR proteins have evolved in different bacteria. Our approach include biochemical, biophysical and structural studies in conjunction with in vivo studies in collaboration with Professor D. Scott Merrell (Dept. of Microbiology, USUHS).16 These studies will contribute to our emerging understanding of how bacterial evolution in general is manifest at the protein level.

References:

(1) Besold, A. N.; Michel, S. L. Neural Zinc Finger Factor/Myelin Transcription Factor Proteins: Metal Binding, Fold, and Function. Biochemistry 2015, 54, 4443-4452.

(2) Lee, S. J.; Michel, S. L. Structural metal sites in nonclassical zinc finger proteins involved in transcriptional and translational regulation. Acc Chem Res 2014, 47, 2643-2650.

(3) Michalek, J. L.; Besold, A. N.; Michel, S. L. Cysteine and histidine shuffling: mixing and matching cysteine and histidine residues in zinc finger proteins to afford different folds and function. Dalton Trans 2011, 40, 12619-12632.

(4) diTargiani, R. C.; Lee, S. J.; Wassink, S.; Michel, S. L. Functional characterization of iron-substituted tristetraprolin-2D (TTP-2D, NUP475-2D): RNA binding affinity and selectivity. Biochemistry 2006, 45, 13641-13649.

(5) Lee, S. J.; Michel, S. L. Cysteine oxidation enhanced by iron in tristetraprolin, a zinc finger peptide. Inorg Chem 2010, 49, 1211-1219.

(6) Lee, S. J.; Michalek, J. L.; Besold, A. N.; Rokita, S. E.; Michel, S. L. Classical Cys2His2 zinc finger peptides are rapidly oxidized by either H2O2 or O2 irrespective of metal coordination. Inorg Chem 2011, 50, 5442-5450.

(7) Michalek, J. L.; Lee, S. J.; Michel, S. L. Cadmium coordination to the zinc binding domains of the non-classical zinc finger protein Tristetraprolin affects RNA binding selectivity. J Inorg Biochem 2012, 112, 32-38.

(8) Shimberg, G. D.; Michalek, J. L.; Oluyadi, A. A.; Rodrigues, A. V.; Zucconi, B. E.; Neu, H. M.; Ghosh, S.; Sureschandra, K.; Wilson, G. M.; Stemmler, T. L.; Michel, S. L. Cleavage and polyadenylation specificity factor 30: An RNA-binding zinc-finger protein with an unexpected 2Fe-2S cluster. Proc Natl Acad Sci U S A 2016, 113, 4700-4705.

(9) Besold, A. N.; Oluyadi, A. A.; Michel, S. L. Switching metal ion coordination and DNA Recognition in a Tandem CCHHC-type zinc finger peptide. Inorg Chem 2013, 52, 4721-4728.

(10) Besold, A. N.; Amick, D. L.; Michel, S. L. A role for hydrogen bonding in DNA recognition by the non-classical CCHHC type zinc finger, NZF-1. Mol Biosyst 2014, 10, 1753-1756.

(11) Dosanjh N.S., Michel S.L.J. Microbial nickel metalloregulation: NikRs for nickel ions. Curr. Opin. Chem. Biol. 2006 10, 123-30

(12) Dosanjh N.S., Hammerbacher N.A., Michel S.L.J. Characterization of the Helicobacter pylori NikR-P(ureA) DNA interaction: metal ion requirements and sequence specificity. Biochemistry 2007 46,2520-9

(13) Dosanjh N.S., West A.L., Michel S.L.J. Helicobacter pylori NikR’s interaction with DNA: a two-tiered mode of recognition. Biochemistry 2009 48, 527-36.

(14) West A.L., St John F., Lopes P.E., MacKerell A.D. Jr, Pozharski E., Michel S.L.J.
Holo-Ni(II)HpNikR is an asymmetric tetramer containing two different nickel-binding sites. J Am Chem Soc 2010 20, 14447-56

(15) West A.L., Evans S.E., González J.M., Carter L.G., Tsuruta H., Pozharski E., Michel S.L.J., Ni(II) coordination to mixed sites modulates DNA binding of HpNikR via a long-range effect Proc Natl Acad Sci U S A, 2012 109, 5633-8

(16) Williams C.L., Neu H.M., Gilbreath J.J., Michel S.L.J., Zurawski D.V., Merrell D.S. Characterization of Copper Resistance in Acinetobacter baumannii Appl Environ Microbiol. 2016 Aug 12. pii: AEM.01813-16. [Epub ahead of print]