Structural analysis of DNA binding by C.Csp231I, a member of a novel class of R-M controller proteins regulating gene expression.

In a wide variety of bacterial restriction-modification systems, a regulatory `controller' protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.8 Šresolution, and the molecular basis of sequence recognition in this class of proteins was elucidated. An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal.

The structural basis of differential DNA sequence recognition by restriction-modification controller proteins.

Controller (C) proteins regulate the expression of restriction-modification (RM) genes in a wide variety of RM systems. However, the RM system Esp1396I is of particular interest as the C protein regulates both the restriction endonuclease (R) gene and the methyltransferase (M) gene. The mechanism of this finely tuned genetic switch depends on differential binding affinities for the promoters controlling the R and M genes, which in turn depends on differential DNA sequence recognition and the ability to recognize dual symmetries. We report here the crystal structure of the C protein bound to the M promoter, and compare the binding affinities for each operator sequence by surface plasmon resonance. Comparison of the structure of the transcriptional repression complex at the M promoter with that of the transcriptional activation complex at the R promoter shows how subtle changes in protein-DNA interactions, underpinned by small conformational changes in the protein, can explain the molecular basis of differential regulation of gene expression.

Recognition of dual symmetry by the controller protein C.Esp1396I based on the structure of the transcriptional activation complex.

The controller protein C.Esp1396I regulates the timing of gene expression of the restriction-modification (RM) genes of the RM system Esp1396I. The molecular recognition of promoter sequences by such transcriptional regulators is poorly understood, in part because the DNA sequence motifs do not conform to a well-defined symmetry. We report here the crystal structure of the controller protein bound to a DNA operator site. The structure reveals how two different symmetries within the operator are simultaneously recognized by the homo-dimeric protein, underpinned by a conformational change in one of the protein subunits. The recognition of two different DNA symmetries through movement of a flexible loop in one of the protein subunits may represent a general mechanism for the recognition of pseudo-symmetric DNA sequences.

Structural analysis of a novel class of R-M controller proteins: C.Csp231I from Citrobacter sp. RFL231.

Controller proteins play a key role in the temporal regulation of gene expression in bacterial restriction-modification (R-M) systems and are important mediators of horizontal gene transfer. They form the basis of a highly cooperative, concentration-dependent genetic switch involved in both activation and repression of R-M genes. Here we present biophysical, biochemical, and high-resolution structural analysis of a novel class of controller proteins, exemplified by C.Csp231I. In contrast to all previously solved C-protein structures, each protein subunit has two extra helices at the C-terminus, which play a large part in maintaining the dimer interface. The DNA binding site of the protein is also novel, having largely AAAA tracts between the palindromic recognition half-sites, suggesting tight bending of the DNA. The protein structure shows an unusual positively charged surface that could form the basis for wrapping the DNA completely around the C-protein dimer.

Overexpression, purification and preliminary X-ray diffraction analysis of the controller protein C.Csp231I from Citrobacter sp. RFL231.

Restriction-modification controller proteins play an essential role in regulating the temporal expression of restriction-modification genes. The controller protein C.Csp231I represents a new class of controller proteins. The gene was sublconed to allow overexpression in Escherichia coli. The protein was purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.0 A resolution and belonged to space group P2(1). An electrophoretic mobility-shift assay provided evidence of strong binding of C.Csp231I to a sequence located upstream of the csp231IC start codon.

Structure of the restriction-modification controller protein C.Esp1396I.

The controller protein of the Esp1396I restriction-modification (R-M) system binds differentially to three distinct operator sequences upstream of the methyltransferase (M) and endonuclease (R) genes to regulate the timing of gene expression. The crystal structure of a complex of the protein with two adjacent operator DNA sequences has been reported; however, the structure of the free protein has not yet been determined. Here, the crystal structure of the free protein is reported, with seven dimers in the asymmetric unit. Two of the 14 monomers show an alternative conformation to the major conformer in which the side chains of residues 43-46 in the loop region flanking the DNA-recognition helix are displaced by up to 10 A. It is proposed that the adoption of these two conformational states may play a role in DNA-sequence promiscuity. The two alternative conformations are also found in the R35A mutant structure, which is otherwise identical to the native protein. Comparison of the free and bound protein structures shows a 1.4 A displacement of the recognition helices when the dimer is bound to its DNA target.

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

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.

Cooperative binding of the C.AhdI controller protein to the C/R promoter and its role in endonuclease gene expression.

The controller (C) proteins of a wide variety of restriction-modification (R-M) systems are thought to regulate expression of the endonuclease (R) gene by a genetic switch that ensures that methylation precedes endonuclease expression. Previous DNA footprinting experiments with C.AhdI have located the binding site upstream of the C and R genes in the AhdI R-M system, and the structure of C.AhdI has recently been determined. Here, we provide evidence that the binding site can accommodate either one or two dimers of C.AhdI in a concentration-dependent manner. The dimer binding site is adjacent to the -35 hexamer site required for the interaction with RNA polymerase (RNAP); however, co-operative binding of a second dimer blocks this site. Optimum DNA binding site sizes for dimer and tetramer formation were determined to be ca 21 bp and 34 bp, respectively. The stoichiometry and affinities of relevant DNA-protein complexes have been characterised by sedimentation velocity and EMSA using native and mutant promoter sequences. Molecular models of the dimer and tetramer complexes have been constructed that are consistent with the hydrodynamic data. Our results suggest a mechanism for both positive and negative regulation of endonuclease expression, whereby at moderate levels of C.AhdI, the protein binds to the promoter as a dimer and stimulates transcription by the interaction with RNAP. As the levels of C.AhdI increase further, binding of the second dimer competes with RNAP, thus down-regulating transcription of its own gene, and hence that of the endonuclease.

High-resolution crystal structure of the restriction-modification controller protein C.AhdI from Aeromonas hydrophila.

Restriction-modification (R-M) systems serve to protect the host bacterium from invading bacteriophage. The multi-component system includes a methyltransferase, which recognizes and methylates a specific DNA sequence, and an endonuclease which recognises the same sequence and cleaves within or close to this site. The endonuclease will only cleave DNA that is unmethylated at the specific site, thus host DNA is protected while non-host DNA is cleaved. However, following DNA replication, expression of the endonuclease must be delayed until the host DNA is appropriately methylated. In many R-M systems, this regulation is achieved at the transcriptional level via the controller protein, or C-protein. We have solved the first X-ray structure of an R-M controller protein, C.AhdI, to 1.69 A resolution using selenomethionine MAD. C.AhdI is part of a Type IIH R-M system from the pathogen Aeromonas hydrophila. The structure reveals an all-alpha protein that contains a classical helix-turn-helix (HTH) domain and can be assigned to the Xre family of transcriptional regulators. Unlike its monomeric structural homologues, an extended helix generates an interface that results in dimerisation of the free protein. The dimer is electrostatically polarised and a positively charged surface corresponds to the position of the DNA recognition helices of the HTH domain. Comparison with the structure of the lambda cI ternary complex suggests that C.AhdI activates transcription through direct contact with the sigma70 subunit of RNA polymerase.

Reach extension in 10-Gb/s directly modulated transmission systems using asymmetric and narrowband optical filtering.

Optical filtering has been used to extend the reach of directly modulated laser in 10Gb/s WDM systems via two separate mechanisms: narrowing the broadened spectrum, and converting frequency modulation into useful amplitude modulation. We investigate in detail, the impact of asymmetric and narrowband optical filtering at the transmitter or receiver. Experimental demonstrations for both shorter distance and long-haul like transmission using optical filtering are performed. The transmission reach is nearly doubled from <25-km to >45-km without dispersion compensation. 1400-km error-free transmission (Q > 15.6-dB) is further achieved over dispersion-managed link for a directly modulated DFB laser within an 8x10-Gb/s WDM system.

DNA footprinting and biophysical characterization of the controller protein C.AhdI suggests the basis of a genetic switch.

We have cloned and expressed the ahdIC gene of the AhdI restriction-modification system and have purified the resulting controller (C) protein to homogeneity. The protein sequence shows a HTH motif typical of that found in many transcriptional regulators. C.AhdI is found to form a homodimer of 16.7 kDa; sedimentation equilibrium experiments show that the dimer dissociates into monomers at low concentration, with a dissociation constant of 2.5 microM. DNase I and Exo III footprinting were used to determine the C.AhdI DNA-binding site, which is found approximately 30 bp upstream of the ahdIC operon. The intact homodimer binds cooperatively to a 35 bp fragment of DNA containing the C-protein binding site with a dissociation constant of 5-6 nM, as judged both by gel retardation analysis and by surface plasmon resonance, although in practice the affinity for DNA is dominated by protein dimerization as DNA binding by the monomer is negligible. The location of the C-operator upstream of both ahdIC and ahdIR suggests that C.AhdI may act as a positive regulator of the expression of both genes, and could act as a molecular switch that is critically dependent on the K(d) for the monomer-dimer equilibrium. Moreover, the structure and location of the C.AhdI binding site with respect to the putative -35 box preceding the C-gene suggests a possible mechanism for autoregulation of C.AhdI expression.

Crystallization and preliminary X-ray analysis of the controller protein C.AhdI from Aeromonas hydrophilia.

Single crystals of purified homodimeric controller protein from Aeromonas hydrophilia (C.AhdI) have been grown under several different conditions using vapour diffusion. X-ray diffraction data have been collected using synchrotron radiation from crystals of both the native and a selenomethionine (SeMet) derivative of the protein. The native crystal form belongs to space group P2(1) and data were collected to a resolution of 2.2 A. Two crystal forms of the SeMet protein have been obtained and were found to belong to space groups P1 and P2(1); data have been recorded to 2.0 and 1.7 A resolution, respectively, for the two crystal forms. Three-wavelength MAD data were collected to 1.7 A for the SeMet derivative crystal, which is isomorphous with the native P2(1) crystal.

Crystallization and preliminary crystallographic analysis of deoxyuridine 5'-triphosphate nucleotidohydrolase from Bacillus subtilis.

Single crystals of purified homotrimeric deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) from Bacillus subtilis have been grown under several different conditions using vapour diffusion. X-ray diffraction data have been collected using synchrotron radiation from three crystal forms of the unliganded enzyme and from enzyme cocrystallized with a substrate analogue and inhibitor, dUDP, and a metal ion, Sr(2+). The three crystal forms of unliganded enzyme belong to hexagonal (P6(3)), orthorhombic (P2(1)2(1)2) and cubic (P2(1)3) space groups and data have been recorded to 1.75, 1.90 and 2.50 A spacing, respectively. Crystals grown in the presence of dUDP and Sr(2+) belong to the orthorhombic space group P2(1)2(1)2(1) and data were measured to 1.90 A spacing. Solution of the hexagonal crystal form by molecular replacement using the dUTPase from feline immunodeficiency virus as a search model is in progress.

Evolution of the dUTPase gene of mammalian and avian herpesviruses.

Sequences of dUTPases encoded by Alpha- and Gammaherpesviruses resemble other dUTPases in their possession of five conserved motifs, but differ in having greater chain lengths (about twice as long) and in the location of Motif 3 at an N terminal location relative to the other motifs. It was proposed that the herpesvirus gene arose by intragenic duplication of a standard dUTPase coding sequence and subsequent loss of one copy of each motif from the double length chain, and that the resulting enzyme was active as a monomer. With knowledge of the trimeric 3D structure of standard dUTPases, it is possible to suggest transformations that occurred in evolutionary development of the herpesvirus dUTPase. The distinct location of Motif 3 can indeed be seen to be consistent with it contributing to a single intramolecular active site with the other motifs. Separately, the occurrence in herpesvirus dUTPases of around 20 to 40 additional residues between Motifs 4 and 5 allows the C-terminal Motif 5 to reach the active site intramolecularly. The driving force behind these evolutionary changes remains obscure. We speculate that they may have allowed acquisition of a novel, presently unknown function by the protein. Consistent with this idea is the observation that in Alpha- and Gammaherpesvirus dUTPases the original locus of Motif 3 is occupied by a distinct conserved sequence (Motif 6); perhaps this element constitutes part of a separate functional capability. Notably, the apparently orthologous protein in Betaherpesviruses lacks the standard motifs while Motif 6 is still present.

Whole-blood test for total cholesterol by a self-metering, self-timing disposable device with built-in quality control.

A whole-blood test for total cholesterol has been developed that is performed in a low-cost disposable flow device without user intervention (after sample addition). The device meters the sample, separates plasma from erythrocytes, and precisely times plasma flow into various reagent compartments, including a quality-assurance chamber. Test results are displayed as a well-defined and easily readable color bar. A quality-control window attests to the integrity of the test components. Here, we describe the assembly and individual functions of the device and report its performance characteristics. Precision and accuracy studies in four clinical studies at independent locations yielded total imprecision of <5% and an average bias of 1.35% vs the Abell-Kendall method.

Rapid optimization of enzyme substrates using defined substrate mixtures.

A strategy is described for the rapid optimization of kcat/Km for protease substrates. Selected positions of a given peptide substrate sequence are varied through synthesis with mixtures of amino acids. Incubation of the resulting peptide mixture with the enzyme of interest and analysis by high pressure liquid chromatography provides a direct measure of analogs with enhanced kcat/Km. High performance liquid chromatography/continuous flow fast atom bombardment mass spectrometry is used to assign structure to each peak in the chromatogram. As an example of the utility and efficiency of "substrate mapping" we describe optimization of the collagenase substrate Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 (where Dnp is dinitrophenyl) at the P'1 and P'2 positions. Six different mixtures were prepared for evaluation, representing the synthesis of 128 different synthetic substrates. "Substrate mapping" has led to Dnp-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-NH2, a substrate that possesses a 10-fold better kcat/Km than Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2.