Malcolm D. Walkinshaw

MeCP2 binding to DNA depends upon hydration at methyl-CpG

MeCP2 is an essential transcriptional repressor that mediates gene silencing through binding to methylated DNA. Binding specificity has been thought to depend on hydrophobic interactions between cytosine methyl groups and a hydrophobic patch within the methyl-CpG-binding domain (MBD). X-ray analysis of a methylated DNA-MBD cocrystal reveals, however, that the methyl groups make contact with a predominantly hydrophilic surface that includes tightly bound water molecules. This suggests that MeCP2 recognizes hydration of the major groove of methylated DNA rather than cytosine methylation per se. The MeCP2-DNA complex also identifies a unique structural role for T158, the residue most commonly mutated in Rett syndrome.

Structure of Ocr from Bacteriophage T7, a Protein that Mimics B-Form DNA

We have solved, by X-ray crystallography to a resolution of 1.8 A, the structure of a protein capable of mimicking approximately 20 base pairs of B-form DNA. This ocr protein, encoded by gene 0.3 of bacteriophage T7, mimics the size and shape of a bent DNA molecule and the arrangement of negative charges along the phosphate backbone of B-form DNA. We also demonstrate that ocr is an efficient inhibitor in vivo of all known families of the complex type I DNA restriction enzymes. Using atomic force microscopy, we have also observed that type I enzymes induce a bend in DNA of similar magnitude to the bend in the ocr molecule. This first structure of an antirestriction protein demonstrates the construction of structural mimetics of long segments of B-form DNA.

The X‐ray structure of a tetrapeptide bound to the active site of human cyclophilin A

Human cyclophilin A (165 residues) has peptidyl‐prolyl cis‐trans isomerase activity. Here we report a high‐resolution three‐dimensional X‐ray structure of a substrate, ac‐Ala‐Ala‐Pro‐Ala‐amc (ac. acetyl: amc. amidomethylcoumarin) bound to the active‐site of cyclophilin. The structure consisting of a dimer of complexes and 135 water molecules was refined to a crystallographic R‐factor of 17.7% for all data in the range 8 Å‐2.3 Å.

Crystal Structure of a Thermostable Lipase from Bacillus stearothermophilus P1

We describe the first lipase structure from a thermophilic organism. It shares less than 20% amino acid sequence identity with other lipases for which there are crystal structures, and shows significant insertions compared with the typical alpha/beta hydrolase canonical fold. The structure contains a zinc-binding site which is unique among all lipases with known structures, and which may play a role in enhancing thermal stability. Zinc binding is mediated by two histidine and two aspartic acid residues. These residues are present in comparable positions in the sequences of certain lipases for which there is as yet no crystal structural information, such as those from Staphylococcal species and Arabidopsis thaliana. The structure of Bacillus stearothermophilus P1 lipase provides a template for other thermostable lipases, and offers insight into mechanisms used to enhance thermal stability which may be of commercial value in engineering lipases for industrial uses.

Discovery of a novel family of CDK inhibitors with the program LIDAEUS: structural basis for ligand-induced disordering of the activation loop

A family of 4-heteroaryl-2-amino-pyrimidine CDK2 inhibitor lead compounds was discovered with the new database-mining program LIDAEUS through in silico screening. Four compounds with IC(50) values ranging from 17 to 0.9 microM were selected for X-ray crystal analysis. Two distinct binding modes are observed, one of which resembles the hydrogen bonding pattern of bound ATP. In the second binding mode, the ligands trigger a conformational change in the activation T loop by inducing movement of Lys(33) and Asp(145) side chains. The family of molecules discovered provides an excellent starting point for the design and synthesis of tight binding inhibitors, which may lead to a new class of antiproliferative drugs.

Molecular architecture of the Mos1 paired-end complex: the structural basis of DNA transposition in a eukaryote

A key step in cut-and-paste DNA transposition is the pairing of transposon ends before the element is excised and inserted at a new site in its host genome. Crystallographic analyses of the paired-end complex (PEC) formed from precleaved transposon ends and the transposase of the eukaryotic element Mos1 reveals two parallel ends bound to a dimeric enzyme. The complex has a trans arrangement, with each transposon end recognized by the DNA binding region of one transposase monomer and by the active site of the other monomer. Two additional DNA duplexes in the crystal indicate likely binding sites for flanking DNA. Biochemical data provide support for a model of the target capture complex and identify Arg186 to be critical for target binding. Mixing experiments indicate that a transposase dimer initiates first-strand cleavage and suggest a pathway for PEC formation.

Structures of Immunophilins and their Ligand Complexes

This review includes an analysis of available X-ray and NMR structures of both members of the immunophilin family; cyclophilins and the FK-506 binding proteins (FKBPs). Available structures are compared and contrasted to highlight different structural features seen both within and between species. Each immunophilin family has been structurally characterised with a variety of small molecule ligands, principally immunosuppressive drugs and their analogues and an overview of these complexes is also presented. Currently the Protein Data Base contains over 60 entries for cyclophilins and over 40 entries for FKBPs. A number of FKBP related structures are also available including structures of MIP (Macrophage Infectivity Potentiator protein) from Legionella pneumophila and Trypanosoma cruzi and Trigger Factor from Mycoplasma genitalium. For all structures discussed in the review a summary of the available biological data is also presented.

Metals in protein structures: a review of their principal features

Metals are present in more than one-third of all proteins as they occur in nature, and are usually important in biological function or maintenance of the structure. Some are present as cations, directly associated with amino acid functional groups of the protein, others within small molecule cofactors associated with the protein. For the 10 metals commonly found as cations, Na, Mg, K, Ca, Mn, Fe, Co, Ni, Cu and Zn, a survey is given of occurrence, relative frequencies of both metal and donor atom or group type, and geometry of coordination. The survey is based on crystal structure information deposited in the Protein Data Bank (PDB) [Berman, H.; Henrick, K.; Nakamura, H.; Markley, J.L. The Worldwide Protein Data Bank (wwPDB): Ensuring a Single, Uniform Archive of PDB Data. Nucleic Acids Res. 2007, 35, D301–D303]. The precision and reliability of this information is assessed in detail. Illustrative examples are given for each metal, including, usually, details of the structure of the metal site in relation to the whole protein and to its function; there are comparisons between metals and descriptions of features such as binding to carboxylate and multiple metal sites close to each other. Metals found within cofactors which associate with the protein, most notably Mo, are included within these examples. Also included briefly are the prediction of metal sites in proteins resulting from genomic synthesis, information which can be derived from methods other than X-ray diffraction of crystals, and metal–protein systems which do not occur naturally.

Cyclosporin A—cyclophilin complex formation A model based on X-ray and NMR data

The previously determined 3D NMR solution structure of cyclophilin‐bound cyclosporin A (CsA) was docked onto the X‐ray crystal structure of cyclophilin. Intermolecular nuclear Overhauser effects (NOE) between CsA and cyclophilin were used as constraints in a restrained energy minimization to generate a model of the complex which satisfied all the NOE distance constraints. The model shows that the residues 9 to 11 and 1 to 5 of the cyclic CsA molecule are in contact with cyclophilin. Comparing the model of the CsA—cyclophilin complex to the X‐ray crystal structure of a complex of cyclophilin with a substrate for peptidyl‐proline cis‐trans isomerase activity, i.e. the linear tetrapeptide substrate ae‐Ala‐Ala‐Pro‐Ala‐amc (ac. acetyl; amc. amidomethylcoumarin), one notices that the contacting peptide segments in the two ligands are oriented in opposite directions, and that the side chain or MeVal‐11 of CsA superposes rather precisely with the position of the prolyl residue in ae‐Ala‐Ala‐Pro‐Ala‐amc.

Consensus Docking: Improving the Reliability of Docking in a Virtual Screening Context

Structure-based virtual screening relies on scoring the predicted binding modes of compounds docked into the target. Because the accuracy of this scoring relies on the accuracy of the docking, methods that increase docking accuracy are valuable. Here, we present a relatively straightforward method for improving the probability of identifying accurately docked poses. The method is similar in concept to consensus scoring schemes, which have been shown to increase ranking power and thus hit rates, but combines information about predicted binding modes rather than predicted binding affinities. The pose prediction success rate of each docking program alone was found in this trial to be 55% for Autodock, 58% for DOCK, and 64% for Vina. By using more than one docking program to predict the binding pose, correct poses were identified in 82% or more of cases, a significant improvement. In a virtual screen, these more reliably posed compounds can be preferentially advanced to subsequent scoring stages to improve hit rates. Consensus docking can be easily introduced into established structure-based virtual screening methodologies.