Geoff Kneale

Insoluble aggregates and protease-resistant conformers of prion protein in uninfected human brains.

Aggregated prion protein (PrPSc), which is detergent-insoluble and partially proteinase K (PK)-resistant, constitutes the major component of infectious prions that cause a group of transmissible spongiform encephalopathies in animals and humans. PrPSc derives from a detergent-soluble and PK-sensitive cellular prion protein (PrPC) through an α-helix to β-sheet transition. This transition confers on the PrPSc molecule unique physicochemical and biological properties, including insolubility in nondenaturing detergents, an enhanced tendency to form aggregates, resistance to PK digestion, and infectivity, which together are regarded as the basis for distinguishing PrPSc from PrPC. Here we demonstrate, using sedimentation and size exclusion chromatography, that small amounts of detergent-insoluble PrP aggregates are present in uninfected human brains. Moreover, PK-resistant PrP core fragments are detectable following PK treatment. This is the first study that provides experimental evidence supporting the hypothesis that there might be silent prions lying dormant in normal human brains.

Influence of pH on the conformation and stability of mismatch base-pairs in DNA

A series of self-complementary dodecanucleotide duplexes containing two symmetrically disposed mismatches have been studied by pH-dependent, ultraviolet light melting techniques. The results indicate that A.C, and C.C mismatches are strongly stabilized by protonation and that the degree of stabilization of the A.C mismatch depends greatly on the flanking bases. In one case, a duplex containing two A.C mismatches is more stable than the native sequence below pH 5.5. The G.A mismatch displays conformational flexibility, with a protonated G(syn).A(anti) base-pair occurring in certain base stacking environments but not in others. The A.A and T.C mismatches are not stabilized at low pH. These solution studies correlate well with predictions based on X-ray crystallographic data.

PrP conformational transitions alter species preference of a PrP-specific antibody

The epitope of the 3F4 antibody most commonly used in human prion disease diagnosis is believed to consist of residues Met-Lys-His-Met (MKHM) corresponding to human PrP-(109–112). This assumption is based mainly on the observation that 3F4 reacts with human and hamster PrP but not with PrP from mouse, sheep, and cervids, in which Met at residue 112 is replaced by Val. Here we report that, by brain histoblotting, 3F4 did not react with PrP of uninfected transgenic mice expressing elk PrP; however, it did show distinct immunoreactivity in transgenic mice infected with chronic wasting disease. Compared with human PrP, the 3F4 reactivity with the recombinant elk PrP was 2 orders of magnitude weaker, as indicated by both Western blotting and surface plasmon resonance. To investigate the molecular basis of these species- and conformer-dependent preferences of 3F4, the epitope was probed by peptide membrane array and antigen competition experiments. Remarkably, the 3F4 antibody did not react with MKHM but reacted strongly with KTNMK (corresponding to human PrP-(106–110)), a sequence that is also present in cervids, sheep, and cattle. 3F4 also reacted with elk PrP peptides containing KTNMKHV. We concluded that the minimal sequence for the 3F4 epitope consists of residues KTNMK, and the species- and conformer-dependent preferences of 3F4 arise largely from the interactions between Met112 (human PrP) or Val115 (cervid PrP) and adjacent residues.

Structure and operation of the DNA-translocating type I DNA restriction enzymes.

Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes.

Methods for the analysis of DNA-protein interactions

The interaction of proteins with DNA is a central theme of molecular biology. In this article, we review some of the principal techniques currently used for the identification and characterization of DNA binding proteins, and for investigation of the molecular interactions that are responsible for the recognition of specific DNA sequences.

High-level expression of the cloned genes encoding the subunits of and intact DNA methyltransferase, M·EcoR124

We have cloned the genes coding for the two subunits (HsdM and HsdS) of the type-I DNA methyltransferase (MTase), M.EcoR124, into the specially constructed expression vector, pJ119. These subunits have been synthesized together as an intact MTase. We have also cloned the individual subunit-encoding genes under the control of the T7 gene 10 promoter or the lacUV5 promoter. High levels of expression have been obtained in all cases. While HsdM was found to be soluble, HsdS was insoluble. However, in the presence of the co-produced HsdM subunit, HsdS was found in the soluble fraction as part of an active MTase. We have partially purified the cloned multi-subunit enzyme and shown that it is capable of DNA methylation both in vivo and in vitro.

Accessibility of a critical prion protein region involved in strain recognition and its implications for the early detection of prions

Abstract.Human prion diseases are characterized by the accumulation in the brain of proteinase K (PK)-resistant prion protein designated PrP27-30 detectable by the 3F4 antibody against human PrP109-112. We recently identified a new PK-resistant PrP species, designated PrP*20, in uninfected human and animal brains. It was preferentially detected with the 1E4 antibody against human PrP 97-108 but not with the anti-PrP 3F4 antibody, although the 3F4 epitope is adjacent to the 1E4 epitope in the PrP*20 molecule. The present study reveals that removal of the N-terminal amino acids up to residue 91 significantly increases accessibility of the 1E4 antibody to PrP of brains and cultured cells. In contrast to cells expressing wild-type PrP, cells expressing pathogenic mutant PrP accumulate not only PrP*20 but also a small amount of 3F4-detected PK-resistant PrP27-30. Remarkably, during the course of human prion disease, a transition from an increase in 1E4-detected PrP*20 to the occurrence of the 3F4-detected PrP27-30 was observed. Our study suggests that an increase in the level of PrP*20 characterizes the early stages of prion diseases.

Structural analysis of the genetic switch that regulates the expression of restriction-modification genes

Controller (C) proteins regulate the timing of the expression of restriction and modification (R–M) genes through a combination of positive and negative feedback circuits. A single dimer bound to the operator switches on transcription of the C-gene and the endonuclease gene; at higher concentrations, a second dimer bound adjacently switches off these genes. Here we report the first structure of a C protein–DNA operator complex, consisting of two C protein dimers bound to the native 35 bp operator sequence of the R–M system Esp1396I. The structure reveals a role for both direct and indirect DNA sequence recognition. The structure of the DNA in the complex is highly distorted, with severe compression of the minor groove resulting in a 50° bend within each operator site, together with a large expansion of the major groove in the centre of the DNA sequence. Cooperative binding between dimers governs the concentration-dependent activation–repression switch and arises, in part, from the interaction of Glu25 and Arg35 side chains at the dimer–dimer interface. Competition between Arg35 and an equivalent residue of the σ70 subunit of RNA polymerase for the Glu25 site underpins the switch from activation to repression of the endonuclease gene.

HsdR Subunit of the Type I Restriction-Modification Enzyme EcoR124I: Biophysical Characterisation and Structural Modelling

Type I restriction-modification (RM) systems are large, multifunctional enzymes composed of three different subunits. HsdS and HsdM form a complex in which HsdS recognizes the target DNA sequence, and HsdM carries out methylation of adenosine residues. The HsdR subunit, when associated with the HsdS-HsdM complex, translocates DNA in an ATP-dependent process and cleaves unmethylated DNA at a distance of several thousand base-pairs from the recognition site. The molecular mechanism by which these enzymes translocate the DNA is not fully understood, in part because of the absence of crystal structures. To date, crystal structures have been determined for the individual HsdS and HsdM subunits and models have been built for the HsdM–HsdS complex with the DNA. However, no structure is available for the HsdR subunit. In this work, the gene coding for the HsdR subunit of EcoR124I was re-sequenced, which showed that there was an error in the published sequence. This changed the position of the stop codon and altered the last 17 amino acid residues of the protein sequence. An improved purification procedure was developed to enable HsdR to be purified efficiently for biophysical and structural analysis. Analytical ultracentrifugation shows that HsdR is monomeric in solution, and the frictional ratio of 1.21 indicates that the subunit is globular and fairly compact. Small angle neutron-scattering of the HsdR subunit indicates a radius of gyration of 3.4 nm and a maximum dimension of 10 nm. We constructed a model of the HsdR using protein fold-recognition and homology modelling to model individual domains, and small-angle neutron scattering data as restraints to combine them into a single molecule. The model reveals an ellipsoidal shape of the enzymatic core comprising the N-terminal and central domains, and suggests conformational heterogeneity of the C-terminal region implicated in binding of HsdR to the HsdS–HsdM complex.

Predicting the Effects of Basepair Mutations in DNA-Protein Complexes by Thermodynamic Integration

Thermodynamically rigorous free energy methods in principle allow the exact computation of binding free energies in biological systems. Here, we use thermodynamic integration together with molecular dynamics simulations of a DNA-protein complex to compute relative binding free energies of a series of mutants of a protein-binding DNA operator sequence. A guanine-cytosine basepair that interacts strongly with the DNA-binding protein is mutated into adenine-thymine, cytosine-guanine, and thymine-adenine. It is shown that basepair mutations can be performed using a conservative protocol that gives error estimates of ∼10% of the change in free energy of binding. Despite the high CPU-time requirements, this work opens the exciting opportunity of being able to perform basepair scans to investigate protein-DNA binding specificity in great detail computationally.