Zinc finger protein binding to DNA: an energy perspective using molecular dynamics simulation and free energy calculations on mutants of both zinc finger domains and their specific DNA bases

Energy calculations based on MM-GBSA were employed to study various zinc finger protein (ZF) motifs binding to DNA. Mutants of both the DNA bound to their specific amino acids were studied. Calculated energies gave evidence for a relationship between binding energy and affinity of ZF motifs to their sites on DNA. ΔG values were ?15.82(12), ?3.66(12), and ?12.14(11.6) kcal/mol for finger one, finger two, and finger three, respectively. The mutations in the DNA bases reduced the value of the negative energies of binding (maximum value for ΔΔG = 42Kcal/mol for F1 when GCG mutated to GGG, and ΔΔG = 22 kcal/mol for F2, the loss in total energy of binding originated in the loss in electrostatic energies upon mutation (r = .98). The mutations in key amino acids in the ZF motif in positions-1, 2, 3, and 6 showed reduced binding energies to DNA with correlation coefficients between total free energy and electrostatic was .99 and with Van der Waal was .93. Results agree with experimentally found selectivity which showed that Arginine in position-1 is specific to G, while Aspartic acid (D) in position 2 plays a complicated role in binding. There is a correlation between the MD calculated free energies of binding and those obtained experimentally for prepared ZF motifs bound to triplet bases in other reports (), our results may help in the design of ZF motifs based on the established recognition codes based on energies and contributing energies to the total energy.     

Functional analyses of the Dof domain, a zinc finger DNA-binding domain, in a pumpkin DNA-binding protein AOBP

AOBP, a DNA-binding protein in pumpkin, contains a Dof domain that is composed of 52 amino acid residues and is highly conserved in several DNA-binding proteins of higher plants. The Dof domain has a significant resemblance to Cys2/Cys2 zinc finger DNA-binding domains of steroid hormone receptors and GATA1, but has a longer putative loop where an extra Cys residue is conserved. We show that the Dof domain in AOBP functions as a zinc finger DNA-binding domain and suggest that the Cys residue uniquely conserved in the putative loop might negatively regulate the binding to DNA.     

Mechanism of DNA binding by the ADR1 zinc finger transcription factor as determined by SPR

The ADR1 protein recognizes a six base-pair consensus DNA sequence using two zinc fingers and an adjacent accessory motif. Kinetic measurements were performed on the DNA-binding domain of ADR1 using surface plasmon resonance. Binding by ADR1 was characterized to two known native binding sequences from the ADH2 and CTA1 promoter regions, which differ in two of the six consensus positions. In addition, non-specific binding by ADR1 to a random DNA sequence was measured. ADR1 binds the native sites with nanomolar affinities. Remarkably, ADR1 binds non-specific DNA with affinities only approximately tenfold lower than the native sequences. The specific and non-specific binding affinities are conferred mainly by differences in the association phase of DNA binding. The association rate for the complex is strongly influenced by the proximal accessory region, while the dissociation reaction and specificity of binding are controlled by the two zinc fingers. Binding kinetics of two ADR1 mutants was also examined. ADR1 containing an R91K mutation in the accessory region bound with similar affinity to wild-type, but with slightly less sequence specificity. The R91K mutation was observed to increase binding affinity to a suboptimal sequence by decreasing the complex dissociation rate. L146H, a change-of-specificity mutation at the +3 position of the second zinc finger, bound its preferred sequence with a slightly higher affinity than wild-type. The L146H mutant indicates that beneficial protein-DNA contacts provide similar levels of stabilization to the complex, whether they are hydrogen-bonding or van der Waals interactions.     

Molecular strategies to replace the structural metal site in the prokaryotic zinc finger domain

The specific arrangement of secondary elements in a local motif often totally relies on the formation of coordination bonds between metal ions and protein ligands. This is typified by the ~ 30 amino acid eukaryotic zinc finger motif in which a β-sheet and an α-helix are clustered around a zinc ion by various combinations of four ligands.     

Importance of long-time simulations for rare event sampling in zinc finger proteins

Molecular dynamics (MD) simulation methods have seen significant improvement since their inception in the late 1950s. Constraints of simulation size and duration that once impeded the field have lessened with the advent of better algorithms, faster processors, and parallel computing. With newer techniques and hardware available, MD simulations of more biologically relevant timescales can now sample a broader range of conformational and dynamical changes including rare events. One concern in the literature has been under which circumstances it is sufficient to perform many shorter timescale simulations and under which circumstances fewer longer simulations are necessary. Herein, our simulations of the zinc finger NEMO (2JVX) using multiple simulations of length 15, 30, 1000, and 3000 ns are analyzed to provide clarity on this point.     

Zinc binding of Tim10: evidence for existence of an unstructured binding intermediate for a zinc finger protein

Zinc-finger proteins are among the most abundant proteins in eukaryotic genomes. Tim10 and all the small Tim proteins of the mitochondrial intermembrane space contain a consensus twin CX(3)C zinc-finger motif. Zn(2+) can bind to the reduced Tim10, but not disulphide bonded (oxidized) protein. However, the zinc-binding reaction of Tim10 and of zinc-finger proteins, in general, is ill-defined. In this study, the thermodynamic and kinetic properties of zinc-binding to reduced Tim10 were investigated using circular dichroism (CD), fluorescence spectrometry, and stopped-flow fluorescence techniques. At equilibrium, coupled with the use of protein fluorescence and metal chelators, the zinc-binding affinity was determined for Tim10 to be about 8 x 10(-10)M. Then, far UV CD was used to investigate the secondary structure change upon zinc-binding of the same set of protein samples at various free Zn(2+) concentrations. Comparison between the results of CD and fluorescence studies showed that the zinc-binding reaction is not a simple one-step process. It involves formation of a binding intermediate that is structurally as unfolded as the apoTim10; subsequently, a degree of folding is induced at increased zinc concentrations in the final complex. Next, the stopped-flow fluorescence technique was used to investigate the kinetic process of the binding reaction. Data analysis shows that the reaction has a single kinetic phase at a low free Zn(2+) concentration ( approximately 1 nM), and a double kinetic phase at a high free Zn(2+) concentration. The kinetic result is consistent with that of the studies at equilibrium. Therefore, a two-step reaction model mechanism is proposed, in which zinc-binding is regulated by the initial selective-binding of Zn(2+) to Cys followed by folding. Implication of the two-step zinc-binding mechanism for Zn(2+) trafficking in the cell is discussed.     

Structure and DNA binding characteristics of the three‐Cys2His2 domain of mouse testis zinc finger protein

The C‐terminal three‐Cys₂His₂ zinc‐finger domain (TZD) of mouse testis zinc‐finger protein binds to the 5′‐TGTACAGTGT‐3′ at the Aie1 (aurora‐C) promoter with high specificity. Interestingly, the primary sequence of TZD is unique, possessing two distinct linkers, TGEKP and GAAP, and distinct residues at presumed DNA binding sites at each finger, especially finger 3. A K_d value of ～10^?8 M was obtained from surface plasmon resonance analysis for the TZD‐DNA complex. NMR structure of the free TZD showed that each zinc finger forms a typical ββα fold. On binding to DNA, chemical shift perturbations and the R₂ transverse relaxation rate in finger 3 are significantly smaller than those in fingers 1 and 2, which indicates that the DNA binding affinity in finger 3 is weaker. Furthermore, the shift perturbations between TZD in complex with the cognate DNA and its serial mutants revealed that both ADE7 and CYT8, underlined in 5′‐ATATGTACAGTGTTAT‐3′, are critical in specific binding, and the DNA binding in finger 3 is sequence independent. Remarkably, the shift perturbations in finger 3 on the linker mutation of TZD (GAAP mutated to TGEKP) were barely detected, which further indicates that finger 3 does not play a critical role in DNA sequence‐specific recognition. The complex model showed that residues important for DNA binding are mainly located on positions ?1, 2, 3, and 6 of α‐helices in fingers 1 and 2. The DNA sequence and nonsequence‐specific bindings occurring simultaneously in TZD provide valuable information for better understanding of protein–DNA recognition. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

人工锌指核酸酶介导的基因组定点修饰技术

锌指核酸酶(ZFN)由锌指蛋白(ZFP)结构域和Fok I核酸内切酶的切割结构域人工融合而成,是近年来发展起来的一种可用于基因组定点改造的分子工具。ZFN可识别并结合特定的DNA序列,并通过切割这一序列的特定位点造成DNA的双链断裂(DSB)。在此基础上,人们可以对基因组的特定位点进行各种遗传操作,包括基因打靶、基因定点插入、基因修复等,从而能够方便快捷地对基因组实现靶向遗传修饰。这种新的基因组定点修饰方法的突出优势是适用性好,对物种没有选择性,并且可以在细胞和个体水平进行遗传操作。文章综述了ZFN技术的研究进展及应用前景,重点介绍ZFN的结构与作用机制、现有的靶点评估及锌指蛋白库的构建与筛选方法、基因组定点修饰的策略,以及目前利用这一技术已成功实现突变的物种及内源基因,为开展这一领域的研究工作提供参考。     

DNA inhibits catalysis by the carboxyltransferase subunit of acetyl-CoA carboxylase: implications for active site communication

Acetyl-CoA carboxylase (ACC) catalyzes the first committed step in the synthesis of long-chain fatty acids. The crystal structure of the Escherichia coli carboxyltransferase component of ACC revealed an alpha(2)beta(2) subunit composition with two active sites and, most importantly, a unique zinc domain in each alphabeta pair that is absent in the eukaryotic enzyme. We show here that carboxyltransferase binds DNA. Half-maximal saturation of different single-stranded or double-stranded DNA constructs is seen at 0.5-1.0 muM, and binding is cooperative and nonspecific. The substrates (malonyl-CoA and biocytin) inhibit DNA:carboxyltransferase complex formation. More significantly, single-stranded DNA, double-stranded DNA, and heparin inhibit the reaction catalyzed by carboxyltransferase, with single-stranded DNA and heparin acting as competitive inhibitors. However, double-inhibition experiments revealed that both DNA and heparin can bind the enzyme in the presence of a bisubstrate analog (BiSA), and the binding of BiSA has a very weak synergistic effect on the binding of the second inhibitor (DNA or heparin) and vice versa. In contrast, DNA and heparin can also bind to the enzyme simultaneously, but the binding of either molecule has a strong synergistic effect on binding of the other. An important mechanistic implication of these observations is that the dual active sites of ACC are functionally connected.     

锌指蛋白核酸酶的作用原理及其应用

锌指蛋白核酸酶(Zinc finger nucleases,ZFN)因其能特异性识别并切割DNA序列以及可设计性,被用于基因定点突变和外源基因定点整合。目前,ZFN技术以其准确的靶位点设计能力和诱发高效率基因打靶的优势,越来越受到基因改造研究者的重视,已经成功应用于动植物细胞、胚胎的基因改造。随着鉴定靶DNA高亲和力的锌指蛋白(Zinc finger protein,ZFP)实验技术日渐成熟,可以预见到不久的将来这项技术会在基因工程和育种中得到广泛应用。文章介绍了锌指蛋白识别DNA靶位点和ZFN介导的基因打靶(Double strand break gene targeting,DSB-GT)的原理,同时还综述了目前ZFN技术用于基因改造的研究进展。     

Solution structure of the C4 zinc finger domain of HDM2

HDM2 is a ubiquitin E3 ligase that is a key negative regulator of the tumor suppressor p53. Here, we report the determination of the solution structure of the C4 zinc finger domain of HDM2 using multidimensional NMR. The HDM2 C4 zinc finger domain has a fold consisting of a 3(10) helix followed by four beta-strands, which shares significant structural similarity to the zinc ribbon protein family. Family based sequence analysis identified two putative binding sites, one of which resembles an RNA binding motif.