Non-Conserved Amino Acid Residues Modulate the Thermodynamics of Zn(II) Binding to Classical ßßα Zinc Finger Domains.

Classical zinc fingers domains (ZFs) bind Zn(II) ion by a pair of cysteine and histidine residues to adopt a characteristic and stable ßßα fold containing a small hydrophobic core. As a component of transcription factors, they recognize specific DNA sequences to transcript particular genes. The loss of Zn(II) disrupts the unique structure and function of the whole protein. It has been shown that the saturation of ZFs under cellular conditions is strictly related to their affinity for Zn(II). High affinity warrants their constant saturation, while medium affinity results in their transient structurization depending on cellular zinc availability. Therefore, there must be factors hidden in the sequence and structure of ZFs that impact Zn(II)-to-protein affinities to control their function. Using molecular dynamics simulations and experimental spectroscopic and calorimetric approaches, we showed that particular non-conserved residues derived from ZF sequences impact hydrogen bond formation. Our in silico and in vitro studies show that non-conserved residues can alter metal-coupled folding mechanisms and overall ZF stability. Furthermore, we show that Zn(II) binding to ZFs can also be entropically driven. This preference does not correlate either with Zn(II) binding site or with the extent of the secondary structure but is strictly related to a reservoir of interactions within the second coordination shell, which may loosen or tighten up the structure. Our findings shed new light on how the functionality of ZFs is modulated by non-coordinating residues diversity under cellular conditions. Moreover, they can be helpful for systematic backbone alteration of native ZF ßßα scaffold to create artificial foldamers and proteins with improved stability.

Structures of Silver Fingers and a Pathway to Their Genotoxicity.

Recently, we demonstrated that AgI can directly replace ZnII in zinc fingers (ZFs). The cooperative binding of AgI to ZFs leads to a thermodynamically irreversible formation of silver clusters destroying the native ZF structure. Thus, a reported loss of biological function of ZF proteins is a likely consequence of such replacement. Here, we report an X-ray absorption spectroscopy (XAS) study of Agn Sn clusters formed in ZFs to probe their structural features. Selective probing of the local environment around AgI by XAS showed the predominance of digonal AgI coordination to two sulfur donors, coordinated with an average Ag-S distance at 2.41 Å. No Ag-N bonds were present. A mixed AgS2 /AgS3 geometry was found solely in the CCCH AgI -ZF. We also show that cooperative replacement of ZnII ions with the studied Ag2 S2 clusters occurred in a three-ZF transcription factor protein 1MEY#, leading to a dissociation of 1MEY# from the complex with its cognate DNA.

High Ambrosia pollen concentrations in Poland respecting the long distance transport (LDT).

High Ambrosia pollen concentrations in Poland rather rarely come from the local sources. The aim of this study was to define the temporal and spatial differences of the high Ambrosia pollen concentrations by creating models for the pollen transport from the distant sources. This study was thought to determine the direction of the air masses inflow into Poland, carrying Ambrosia pollen, from areas of the bordering countries with the pollen concentrations higher than iSTOTEN_n Poland. Pollen and meteorological datasets at 8 monitoring sites in Poland, and daily pollen concentrations at 11 sites in the Czech Republic, 5 sites in Slovakia and 3 sites in Ukraine were analysed recently. Days with concentrations ≥10 Pollen/m3 and concurrent meteorological situations were analysed in great deal. The HYSPLIT model was applied to compute backward trajectories up to 4 days backward (96 h) and at three altitudes: 20, 500 and 1000 m above ground level (a.g.l.). High pollen concentrations occur most frequently when the air masses inflow into Poland from southerly (S, SE, SW, 44%) and easterly (E, 6%) directions and in no advection situations (25%). In years with the highest frequency of days over 10 Pollen/m3, the prevailing directions of the pollen influx into Poland were from the South (2004-2006, 2008, 2011) but in one year (2014) from the East. Trajectories for the studied period show that air masses come most frequently from Slovakia and the Czech Republic. Sometimes, the Ambrosia pollen transport happens from Ukraine.

Formation of highly stable multinuclear AgnSn clusters in zinc fingers disrupts their structure and function.

Silver (Ag(i)) binding to consensus zinc fingers (ZFs) causes Zn(ii) release inducing a gradual disruption of the hydrophobic core, followed by an overall conformational change and formation of highly stable AgnSn clusters. A compact eight-membered Ag4S4 structure formed by a CCCC ZF is the first cluster example reported for a single biological molecule. Ag(i)-induced conformational changes of ZFs can, as a consequence, affect transcriptional regulation and other cellular processes.

Structural zinc binding sites shaped for greater works: Structure-function relations in classical zinc finger, hook and clasp domains.

Metal ions are essential elements present in biological systems able to facilitate many cellular processes including proliferation, signaling, DNA synthesis and repair. Zinc ion (Zn(II)) is an important cofactor for numerous biochemical reactions. Commonly, structural zinc sites demonstrate high Zn(II) affinity and compact architecture required for sequence-specific macromolecule binding. However, how Zn(II)-dependent proteins fold, how their dissociation occurs, and which factors modulate zinc protein affinity as well as stability remains not fully understood. The molecular rules governing precise regulation of zinc proteins function are hidden in the relationship between sequence and structure, and hence require deep understanding of their folding mechanism under metal load, reactivity and metal-to-protein affinity. Even though, this sequence-structure relationship has an impact on zinc proteins function, it has been shown that other biological factors including cellular localization and Zn(II) availability influence overall protein behavior. Taking into account all of the mentioned factors, in this review, we aim to describe the relationship between structure-function-stability of zinc structural sites, found in a zinc finger, zinc hook and zinc clasps, and reach far beyond a structural point of view in order to appreciate the balance between chemistry and biology that govern the protein world.

Metal binding properties of zinc fingers with a naturally altered metal binding site.

Zinc fingers (ZFs) are among the most abundant motifs found in proteins, and are commonly known for their structural role. Classical ZFs (CCHH) are part of the transcription factors that participate in DNA binding. Although biochemical studies of classical ZFs have a long history, there is limited knowledge about the sequential and structural diversity of ZFs. We have found that classical ZFs, with metal binding sites consisting of amino acids other than conserved Cys or His residues, are frequently encoded in the human genome, and we refer to these peptides as ZFs with a naturally altered metal binding site. The biological role of the altered ZFs remains undiscovered. In this study, we characterized nine natural XCHH, CXHH, CCXH and CCHX ZFs in terms of their Zn(ii) and Co(ii) binding properties, such as complex stoichiometry, spectroscopic properties and metal-to-peptide affinity. We revealed that XCHH and CXHH ZFs form ML complexes that are 4-5 orders of magnitude weaker in comparison to CCHH ZFs. Nevertheless, spectroscopic studies demonstrate that, depending on the altered position, they may adopt an open coordination geometry with one or two water molecules bound to a central metal ion, which has not been demonstrated in natural ZFs before. Stability data show that both CCXH and CCHX peptides have high Zn(ii) affinity (with a Kd of 10-9 to 10-11 M), suggesting their potential biological function. This study is a comprehensive overview of the relationship between the sequence, structure, and stability of ZFs.