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.     

A role of basic residues and the putative intercalating phenylalanine of the HMG-1 box B in DNA supercoiling and binding to four-way DNA junctions

HMG (high mobility group) 1 is a chromosomal protein with two homologous DNA-binding domains, the HMG boxes A and B. HMG-1, like its individual HMG boxes, can recognize structural distortion of DNA, such as four-way DNA junctions (4WJs), that are very likely to have features common to their natural, yet unknown, cellular binding targets. HMG-1 can also bend/loop DNA and introduce negative supercoils in the presence of topoisomerase I in topologically closed DNAs. Results of our gel shift assays demonstrate that mutation of Arg(97) within the extended N-terminal strand of the B domain significantly (>50-fold) decreases affinity of the HMG box for 4WJs and alters the mode of binding without changing the structural specificity for 4WJs. Several basic amino acids of the extended N-terminal strand (Lys(96)/Arg(97)) and helix I (Arg(110)/Lys(114)) of the B domain participate in DNA binding and supercoiling. The putative intercalating hydrophobic Phe(103) of helix I is important for DNA supercoiling but dispensable for binding to supercoiled DNA and 4WJs. We conclude that the B domain of HMG-1 can tolerate substitutions of a number of amino acid residues without abolishing the structure-specific recognition of 4WJs, whereas mutations of most of these residues severely impair the topoisomerase I-mediated DNA supercoiling and change the sign of supercoiling from negative to positive.     

The M.EcoRV DNA-(adenine N6)-methyltransferase uses DNA bending for recognition of an expanded EcoDam recognition site

The M.EcoRV DNA methyltransferase recognizes GATATC sites. It is related to EcoDam, which methylates GATC sites. The DNA binding domain of M.EcoRV is similar to that of EcoDam suggesting a similar mechanism of DNA recognition. We show that amino acid residue Lys11 of M.EcoRV is involved in recognition of Gua1 and Arg128 contacts the Gua in base pair 6. These residues correspond to Lys9 and Arg124 in EcoDam, which recognize the Gua residues in both strands of the Dam recognition sequence, indicating that M.EcoRV and EcoDam make similar contacts to outermost base pairs of their recognition sequences and M.EcoRV recognizes its target site as an expanded GATC site. In contrast to EcoDam, M.EcoRV considerably bends the DNA (59+/-4 degrees) suggesting indirect readout of the AT-rich inner sequence. Recognition of an expanded target site by DNA bending is a new principle for changing DNA recognition specificity of proteins during molecular evolution. R128A is inefficient in DNA bending and binding, whereas K11A bends DNA with relaxed sequence specificity. These results suggest a temporal order of the formation of protein-DNA contacts in which the Gua6-Arg128 contact forms early followed by DNA bending and, finally, the formation of the Lys11-Gua1 contact.     

Mechanism of DNA-binding loss upon single-point mutation in p53

Over 50% of all human cancers involve p53 mutations, which occur mostly in the sequence-specific DNA-binding central domain (p53c), yielding little/non-detectable affinity to the DNA consensus site. Despite our current understanding of protein-DNA recognition, the mechanism(s) underlying the loss in protein-DNA binding affinity/specificity upon single-point mutation are not well understood. Our goal is to identify the common factors governing the DNA-binding loss of p53c upon substitution of Arg 273 to His or Cys, which are abundant in human tumours. By computing the free energies of wild-type and mutant p53c binding to DNA and decomposing them into contributions from individual residues, the DNA-binding loss upon charge/noncharge-conserving mutation of Arg 273 was attributed not only to the loss of DNA phosphate contacts, but also to longer-range structural changes caused by the loss of the Asp 281 salt-bridge. The results herein and in previous works suggest that Asp 281 plays a critical role in the sequence-specific DNA-binding function of p53c by (i) orienting Arg 273 and Arg 280 in an optimal position to interact with the phosphate and base groups of the consensus DNA, respectively, and (ii) helping to maintain the proper DNA-binding protein conformation.     

Mutation of the Oct-1 POU-specific recognition helix leads to altered DNA binding and influences enhancement of adenovirus DNA replication.

To assess which residues of Oct-1 POU-specific (POUs) are important for DNA recognition and stimulation of adenovirus DNA replication we have mutated 10 residues of the POUs helix-turn-helix motif implicated in DNA contact. Seven of these turned out to have reduced DNA binding affinity. Of these, three alanine substituted proteins were found to have a changed specificity using a binding site selection procedure. Mutation of the first residue in the recognition helix, Gln44, to alanine led to a loss of specificity for the first two bases, TA, of the wild-type recognition site TATGC(A/T)AAT. Instead of the A, a T was selected, suggesting a new contact and a novel specificity. A change in specificity was also observed for the T45A mutant, which could bind to TATAC(A/T)AAT, a site hardly recognized by the wild-type protein. Mutation of residue Arg49 led to a relaxed specificity for three consecutive bases, TGC. This residue, which is critical for high affinity binding, is absent from the structurally homologous lambdoid helix-turn-helix motifs. Employing a reconstituted system all but two mutants could stimulate adenovirus DNA replication upon saturation. Mutation of residues Gln27 and Arg49 impairs the ability of the Oct-1 POU domain protein to enhance replication, with a concomitant loss of DNA contacts. Since the POU domain binds the precursor terminal protein-DNA polymerase complex and guides it to the origin, lack of stimulation may be caused by incorrect targetting of the DNA polymerase due to loss of specificity.     

Highly organized but pliant active site of DNA polymerase beta: compensatory mechanisms in mutant enzymes revealed by dynamics simulations and energy analyses

To link conformational transitions noted for DNA polymerases with kinetic results describing catalytic efficiency and fidelity, we investigate the role of key DNA polymerase beta residues on subdomain motion through simulations of five single-residue mutants: Arg-283-Ala, Tyr-271-Ala, Asp-276-Val, Arg-258-Lys, and Arg-258-Ala. Since a movement toward a closed state was only observed for R258A, we suggest that Arg(258) is crucial in modulating motion preceding chemistry. Analyses of protein/DNA interactions in the mutant active site indicate distinctive hydrogen bonding and van der Waals patterns arising from compensatory structural adjustments. By comparing closed mutant complexes with the wild-type enzyme, we interpret experimentally derived nucleotide binding affinities in molecular terms: R283A (decreased), Y271A (increased), D276V (increased), and R258A (decreased). Thus, compensatory interactions (e.g., in Y271A with adjacent residues Phe(272), Asn(279), and Arg(283)) increase the overall binding affinity for the incoming nucleotide although direct interactions may decrease. Together with energetic analyses, we predict that R258G might increase the rate of nucleotide insertion and maintain enzyme fidelity as R258A; D276L might increase the nucleotide binding affinity more than D276V; and R283A/K280A might decrease the nucleotide binding affinity and increase misinsertion more than R283A. The combined observations regarding key roles of specific residues (e.g., Arg(258)) and compensatory interactions echo the dual nature of polymerase active site, namely versatility (to accommodate various basepairs) and specificity (for preserving fidelity) and underscore an organized but pliant active site essential to enzyme function.     

Binding studies with mutants of Zif268. Contribution of individual side chains to binding affinity and specificity in the Zif268 zinc finger-DNA complex.

The Zif268 zinc finger-DNA complex has served as a model system for understanding how Cys2His2 type zinc fingers recognize DNA. Structural studies of the Zif268-DNA complex revealed that residues at four positions in the alpha helix of each zinc finger play key roles in recognition, but there has been no information about the precise contributions of individual residues. Here we report the results of binding studies involving five mutants of Zif268 that have changes in the base-contacting residues of finger one. These studies let us evaluate the contributions that Arg18 (position -1 of the alpha helix), Asp20 (position 2), Glu21 (position 3), and Arg24 (position 6) make to the overall energy of DNA binding. Our results confirm the important role played by these arginines. By comparing the affinities of the wild type and mutant peptides for various sites, we also prove that Asp20 and Glu21 play important roles in determining binding site specificity.     

Relationship between folding and function in a sequence-specific miniature DNA-binding protein

Previously, we have described a miniature protein-based approach to the design of molecules that bind DNA or protein surfaces with high affinity and specificity. In this approach, the small, well-folded protein avian pancreatic polypeptide acts as a scaffold to present and stabilize an alpha-helical or PPII-helical recognition epitope. The first miniature protein designed in this way, a molecule called p007, presents the alpha-helical recognition epitope found on the bZIP protein GCN4 and binds DNA with nanomolar affinity and exceptional specificity. In this work we use alanine-scanning mutagenesis to explore the contributions of 29 p007 residues to DNA affinity, specificity, and secondary structure. Virtually every residue within the p007 alpha-helix, and most residues within the p007 PPII helix, contribute to both DNA affinity and specificity. These residues include those introduced to make specific and nonspecific DNA contacts, as well as those that complete the miniature protein core. Moreover, there exists a direct correlation between the affinity of a p007 variant for specific DNA and the ability of that variant to select for specific DNA over nonspecific DNA. Although we observe no correlation between alpha-helicity and affinity, we observe a limited correlation between alpha-helicity and sequence specificity that emphasizes the role of coupled binding/folding in the function of p007. Our results imply that formation of a highly evolved set of protein.DNA contacts in the context of a well-packed hydrophobic core, and not the extent of intrinsic alpha-helical structure, is the primary determinant of p007 function.