Keep Your Fingers Off My DNA: Protein–Protein Interactions Mediated by C2H2 Zinc Finger Domains

Cys2-His2 (C2H2) zinc finger domains (ZFs) were originally identified as DNA-binding domains, and uncharacterized domains are typically assumed to function in DNA binding. However, a growing body of evidence suggests an important and widespread role for these domains in protein binding. There are even examples of zinc fingers that support both DNA and protein interactions, which can be found in well-known DNA-binding proteins such as Sp1, Zif268, and Ying Yang 1 (YY1). C2H2 protein–protein interactions (PPIs) are proving to be more abundant than previously appreciated, more plastic than their DNA-binding counterparts, and more variable and complex in their interactions surfaces. Here we review the current knowledge of over 100 C2H2 zinc finger-mediated PPIs, focusing on what is known about the binding surface, contributions of individual fingers to the interaction, and function. An accurate understanding of zinc finger biology will likely require greater insights into the potential protein interaction capabilities of C2H2 ZFs.     

Deep sequencing of large library selections allows computational discovery of diverse sets of zinc fingers that bind common targets

The Cys₂His₂ zinc finger (ZF) is the most frequently found sequence-specific DNA-binding domain in eukaryotic proteins. The ZF’s modular protein–DNA interface has also served as a platform for genome engineering applications. Despite decades of intense study, a predictive understanding of the DNA-binding specificities of either natural or engineered ZF domains remains elusive. To help fill this gap, we developed an integrated experimental-computational approach to enrich and recover distinct groups of ZFs that bind common targets. To showcase the power of our approach, we built several large ZF libraries and demonstrated their excellent diversity. As proof of principle, we used one of these ZF libraries to select and recover thousands of ZFs that bind several 3-nt targets of interest. We were then able to computationally cluster these recovered ZFs to reveal several distinct classes of proteins, all recovered from a single selection, to bind the same target. Finally, for each target studied, we confirmed that one or more representative ZFs yield the desired specificity. In sum, the described approach enables comprehensive large-scale selection and characterization of ZF specificities and should be a great aid in furthering our understanding of the ZF domain.     

Solution NMR structure of zinc finger 4 and 5 from human INSM1, an essential regulator of neuroendocrine differentiation

Human INSM1 containing five C‐terminal C2H2‐type zinc fingers (ZFs), is a key regulator of neuroendocrine development. Previous research reported that full‐length INSM1 containing all five ZFs recognized a consensus DNA sequence. Structure elucidation of human INSM1 ZFs is currently insufficient to understand the DNA binding mechanism. Herein, we present the solution NMR structure of ZF4‐5, in which the two ZFs adopt a head‐to‐tail arrangement and each ZF features a canonical ββα fold. NMR titrations and isothermal titration calorimetry experiments showed that ZF4‐5 binds weakly to the consensus DNA sequence. Proteins 2017; 85:957–962. © 2016 Wiley Periodicals, Inc.     

Recognition mechanism of Wilms’ tumour suppressor protein and DNA triplets: insights from molecular dynamics simulation and free energy analysis

The Wilms’ tumour suppressor protein (WT1) plays a multifaceted role in human cancer processes. Mutations on its DNA recognition domain could lead to Denys–Drash syndrome, and alternate splicing results in insertion of the tripeptide Lys–Thr–Ser (KTS) between the third and fourth zinc fingers (ZFs), leading to changes in the DNA-binding function. However, detailed recognition mechanisms of the WT1–DNA complex have not been explored. To clarify the mutational effects upon WT1 towards DNA binding at the atomic level, molecular dynamics simulations and the molecular mechanics/Poisson Boltzmann surface area (MM/PBSA) method were employed. The simulation results indicate that mutations in ZF domains (E427Q and Q369H) may weaken the binding affinity, and the statistical analyses of the hydrogen bonds and hydrophobic interactions show that eight residues (Lys351, Arg366, Arg375, Arg376, Lys399, Arg403, Arg424 and Arg430) have a significant influence on recognition and binding to DNA. Insertion of the tripeptide KTS could form an immobilized hydrogen-bonding network with Arg403, affecting the flexibility and angle of the linker between ZF3 and ZF4, thus influencing the recognition between the protein and the DNA triplet at its 5′ terminus. These results represent the first step towards a thorough characterization of the WT1 recognition mechanisms, providing a better understanding of the structure–function relationship of WT1 and its mutants.     

Solution structure of the RNA binding domain in the human muscleblind‐like protein 2

The muscleblind‐like (MBNL) proteins 1, 2, and 3, which contain four CCCH zinc finger motifs (ZF1–4), are involved in the differentiation of muscle inclusion by controlling the splicing patterns of several pre‐mRNAs. Especially, MBNL1 plays a crucial role in myotonic dystrophy. The CCCH zinc finger is a sequence motif found in many RNA binding proteins and is suggested to play an important role in the recognition of RNA molecules. Here, we solved the solution structures of both tandem zinc finger (TZF) motifs, TZF12 (comprising ZF1 and ZF2) and TZF34 (ZF3 and ZF4), in MBNL2 from Homo sapiens. In TZF12 of MBNL2, ZF1 and ZF2 adopt a similar fold, as reported previously for the CCCH‐type zinc fingers in the TIS11d protein. The linker between ZF1 and ZF2 in MBNL2 forms an antiparallel β‐sheet with the N‐terminal extension of ZF1. Furthermore, ZF1 and ZF2 in MBNL2 interact with each other through hydrophobic interactions. Consequently, TZF12 forms a single, compact global fold, where ZF1 and ZF2 are approximately symmetrical about the C2 axis. The structure of the second tandem zinc finger (TZF34) in MBNL2 is similar to that of TZF12. This novel three‐dimensional structure of the TZF domains in MBNL2 provides a basis for functional studies of the CCCH‐type zinc finger motifs in the MBNL protein family.     

Weird fingers: Functional analysis of WIP domain proteins

WIP proteins form a plant specific subfamily of C2H2 zinc finger (ZF) proteins. In this study, we functionally characterized the WIP domain, which consists of four ZF motifs, and discuss molecular functions for WIP proteins. Mutations in each of the ZFs lead to loss of function of the TT1/WIP1 protein in Arabiopsis thaliana. SV40 type nuclear localisation signals were detected in two of the ZFs and functionally characterized using GFP fusions as well as new mutant alleles identified by TILLING. Promoter swap experiments showed that selected WIP proteins are partially able to take over TT1 function. Activity of the AtBAN promoter, a potential TT1 target, could be increased by the addition of TT1 to the TT2-TT8-TTG1 regulatory complex.     

Characterization of the tandem CWCH2 sequence motif: a hallmark of inter-zinc finger interactions

Background  

The C2H2 zinc finger (ZF) domain is widely conserved among eukaryotic proteins. In Zic/Gli/Zap1 C2H2 ZF proteins, the two N-terminal ZFs form a single structural unit by sharing a hydrophobic core. This structural unit defines a new motif comprised of two tryptophan side chains at the center of the hydrophobic core. Because each tryptophan residue is located between the two cysteine residues of the C2H2 motif, we have named this structure the tandem CWCH2 (tCWCH2) motif.     

Stability of folding structure of Zic zinc finger proteins

Zic family proteins have five C₂H₂-type zinc finger (ZF) motifs. We physicochemically characterized the folding properties of Zic ZFs. Alteration of chelation with zinc ions and of hydrophobic interactions changed circular dichroism spectra, suggesting that they caused structural changes. The motifs were heat stable, but electrostatic interactions had little effect on structural stability. These results highlight the importance of chelating interactions and hydrophobic interactions for the stability of the folding structure of Zic ZF proteins.