ABSTRACT
Cys2-His2 zinc fingers are one of the most common types of DNA-binding domains. Modifications to zinc-finger binding specificity have recently enabled custom DNA-binding proteins to be designed to a wide array of target sequences. We present here a 1.96 A structure of Aart, a designed six-zinc finger protein, bound to a consensus DNA target site. This is the first structure of a designed protein with six fingers, and was intended to provide insights into the unusual affinity and specificity characteristics of this protein. Most protein-DNA contacts were found to be consistent with expectations, while others were unanticipated or insufficient to explain specificity. Several were unexpectedly mediated by glycerol, water molecules or amino acid-base stacking interactions. These results challenge some conventional concepts of recognition, particularly the finding that triplets containing 5'A, C, or T are typically not specified by direct interaction with the amino acid in position 6 of the recognition helix.
Subject(s)
DNA/metabolism , Peptides/chemistry , Peptides/metabolism , Protein Conformation , Zinc Fingers , Amino Acid Sequence , Base Sequence , Crystallography, X-Ray , DNA/chemistry , Glycerol/chemistry , Models, Molecular , Molecular Sequence Data , Molecular Structure , Nucleic Acid Conformation , Peptides/chemical synthesis , Peptides/genetics , Protein BindingABSTRACT
Using MacroModel, peptide, peptidomimetic and non-peptidomimetic inhibitors of the zinc metalloenzyme, farnesyltransferase (FTase), were docked into the enzyme binding site. Inhibitor flexibility, farnesyl pyrophosphate substrate flexibility, and partial protein flexibility were taken into account in these docking studies. In addition to CVFM and CVIM, as well as our own inhibitors FTI-276 and FTI-2148, we have docked other farnesyltransferase inhibitors (FTIs) including Zarnestra, which presently is in advanced clinical trials. The AMBER* force field was employed, augmented with parameters that were derived for zinc. A single binding site model that was derived from the crystal structure of CVFM complexed with farnesyltransferase and farnesylpyrophosphate was used for these studies. The docking results using the lowest energy structure from the simulation, or one of the lowest energy structures, were generally in excellent agreement with the X-ray structures. One of the most important findings of this study is that numerous alternative conformations for the methionine side chain can be accommodated by the enzyme suggesting that the methionine pocket can tolerate groups larger than methionine at the C-terminus of the tetrapeptide and suggesting alternative locations for the placement of side chains that may improve potency.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Alkyl and Aryl Transferases/chemistry , Computational Biology , Computer Simulation , Alkyl and Aryl Transferases/metabolism , Animals , Models, Molecular , Protein Binding , Protein Prenylation , RatsABSTRACT
Crystals of a designed six-finger zinc-finger protein, Aart, bound to a 22-base-pair duplex DNA containing a consensus binding site have been obtained. Crystals grew by hanging-drop vapor diffusion from solutions containing polyethylene glycol 4000 as the precipitating agent. The irregularly shaped crystals belong to space group P1, with unit-cell parameters a = 41.95, b = 71.76, c = 74.73 A, alpha = 100.87, beta = 96.22, gamma = 106.33 degrees . There are most likely to be two protein-DNA complexes in the asymmetric unit. A complete native data set has been collected from a high-energy synchrotron source to a resolution of 2.95 A at 100 K, with an Rmerge of 9.3%.
