Thermodynamics of Zn²⁺Binding to Cys₂His₂and Cys₂HisCys Zinc Fingers and a Cys₄Transcription Factor Site

Abstract

The thermodynamics of Zn(2+) binding to three peptides corresponding to naturally occurring Zn-binding sequences in transcription factors have been quantified with isothermal titration calorimetry (ITC). These peptides, the third zinc finger of Sp1 (Sp1-3), the second zinc finger of myelin transcription factor 1 (MyT1-2), and the second Zn-binding sequence of the DNA-binding domain of glucocorticoid receptor (GR-2), bind Zn(2+) with Cys(2)His(2), Cys(2)HisCys, and Cys(4) coordination, respectively. Circular dichroism confirms that Sp1-3 and MyT1-2 have considerable and negligible Zn-stabilized secondary structure, respectively, and indicate only a small amount for GR-2. The pK(a)'s of the Sp1-3 cysteines and histidines were determined by NMR and used to estimate the number of protons displaced by Zn(2+) at pH 7.4. ITC was also used to determine this number, and the two methods agree. Subtraction of buffer contributions to the calorimetric data reveals that all three peptides have a similar affinity for Zn(2+), which has equal enthalpy and entropy components for Sp1-3 but is more enthalpically disfavored and entropically favored with increasing Cys ligands. The resulting enthalpy-entropy compensation originates from the Zn-Cys coordination, as subtraction of the cysteine deprotonation enthalpy results in a similar Zn(2+)-binding enthalpy for all three peptides, and the binding entropy tracks with the number of displaced protons. Metal and protein components of the binding enthalpy and entropy have been estimated. While dominated by Zn(2+) coordination to the cysteines and histidines, other residues in the sequence affect the protein contributions that modulate the stability of these motifs.

Dissecting ITC data of metal ions binding to ligands and proteins

“…Since ligand pKa values are not known, the total number of protons that are displaced upon metal binding (a + 2b in Scheme 1) cannot be calculated using the approach described above. An alternate method [73,74], which takes advantage of the change in ΔH HB if the experimental buffer is altered, has been widely used in ITC experiments [4,10,11,24,26,41,75]. Practically, this strategy requires multiple titrations to be carried out at the same pH in at least three different buffers under otherwise identical experimental conditions.…”

“…As such, only indirect experiments that rely on competition with another metal ion or spectroscopic probe can be used to investigate Zn 2+ -protein interactions. Alternatively, Zn 2+ binding events are characterized by a quantifiable heat signature which can be exploited by ITC to gain insight into Zn 2 + -dependent systems such as zinc finger domains [4][5][6][7], carbonic anhydrase [8][9][10], and insulin [11]. Additionally, ITC has played a role in understanding countless important bioinorganic systems including ferritin and transferrin [12][13][14][15][16][17], amyloid beta [18,19], nucleases [20][21][22][23] and cardiac troponin C [24].…”

“…Ideally, all of these reactions should be accounted for in order to obtain condition independent data that is comparable among various metal-ligand systems. However, significant effort is required to determine the necessary information for all linked equilibria in protein-based systems [4]; as such, condition independence is nearly impossible to achieve for these systems. There are many cases when a thorough deconvolution of metal and proton speciation is not required to reach a meaningful conclusion [12][13][14][15][16][17]26,27].…”

The thermodynamics of protein interactions with essential first row transition metals

“…A more recent study using ITC to quantify the thermodynamics of Zn 2+ binding to peptides corresponding to three naturally occurring Zn-binding sequences in transcription factor proteins supported these early findings and concluded that zinc binding to the classical Cys 2 His 2 zinc finger is both enthalpically and entropically favored but less so (i.e. more enthalpically disfavored and entropically favored with increasing Cys ligands) with two derivatives containing three Cys and one His (Cys 2 HisCys) or four Cys (Cys 4 ) metal coordinating residues (21). Additionally, the study found that upon binding, Zn 2+ displaces a proton from each coordinated Cys residue negating the notion that Cys thiols in zinc fingers and corresponding proteins remain protonated upon Zn 2+ binding.…”

“…This trend is attributed to an increased enthalpic penalty to deprotonate the thiols of peptide with increasing number of cysteines. This is accompanied by a concomitant entropic gain due to protons release and this enthalpic-entropic compensation process is perhaps a common feature of tetrahedral Zn 2+ coordination in these types of peptides (21). …”