Assembly of a 20-nm protein cage by Escherichia coli 2-hydroxypentadienoic acid hydratase.

The pentameric Escherichia coli enzyme 2-hydroxypentadienoic acid hydratase assembles to form a 20-nm-diameter particle comprising 60 protein subunits, arranged with 532 symmetry when crystallised at low pH in the presence of phosphate or sulphate ions. The particles form rapidly and are stable in solution during gel filtration at low pH. They are probably formed through trimers of pentamers, which are stabilised by the interaction of two phosphate ions with residues of the N-terminal domains of subunits at the 3-fold axis. Once the particles are formed at high concentrations of phosphate (or sulphate), they remain stable in solution at 20-fold lower concentrations of the anion. Guest molecules can be trapped within the hollow protein shell during assembly. The C-termini of the subunits are freely accessible on the surface of the protein cage and thus are ideal sites for addition of affinity tags or other modifications. These particles offer a convenient model system for studying the assembly of large symmetrical structures and a novel protein nanoparticle for encapsulation and cargo delivery.

The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms.

Forkhead-associated (FHA) domains are a class of ubiquitous signaling modules that appear to function through interactions with phosphorylated target molecules. We have used oriented peptide library screening to determine the optimal phosphopeptide binding motifs recognized by several FHA domains, including those within a number of DNA damage checkpoint kinases, and determined the X-ray structure of Rad53p-FHA1, in complex with a phospho-threonine peptide, at 1.6 A resolution. The structure reveals a striking similarity to the MH2 domains of Smad tumor suppressor proteins and reveals a mode of peptide binding that differs from SH2, 14-3-3, or PTB domain complexes. These results have important implications for DNA damage signaling and CHK2-dependent tumor suppression, and they indicate that FHA domains play important and unsuspected roles in S/T kinase signaling mechanisms in prokaryotes and eukaryotes.

Preferential binding of fd gene 5 protein to tetraplex nucleic acid structures.

The gene 5 protein of filamentous bacteriophage fd is a single-stranded DNA-binding protein that binds non-specifically to all single-stranded nucleic acid sequences, but in addition is capable of specific binding to the sequence d(GT(5)G(4)CT(4)C) and the RNA equivalent r(GU(5)G(4)CU(4)C), the latter interaction being important for translational repression. We show that this sequence preference arises from the formation of a tetraplex structure held together by a central block of G-quartets, the structure of which persists in the complex with gene 5 protein. Binding of gene 5 protein to the tetraplex leads to formation of a approximately 170 kDa nucleoprotein complex consisting of four oligonucleotide strands and eight gene 5 protein dimers, with a radius of gyration of 45 A and an overall maximum dimension of 120-130 A. A model of the complex is presented that is consistent with the data obtained. It is proposed that the G-quartet may act as a nucleation site for binding gene 5 protein to adjacent single-stranded regions, suggesting a novel mechanism for translational repression.

The influence of DNA binding on the backbone dynamics of the yeast cell-cycle protein Mbp1.

Mbp1 is a transcription factor involved in the regulation of the cell cycle in yeast. The N-terminus of this protein contains a DNA binding domain that includes a winged helix-turn-helix motif. The C-terminal 24 residues of this domain (the 'tail') are disordered in the crystal state, but are important for DNA binding. We have measured 15N NMR relaxation rates at 11.75 and 14.1 T to determine the dynamics of the free protein and in its complex with a specific DNA duplex. The dynamics data were quantitatively analysed using both spectral density mapping and the Lipari-Szabo formalism including the effects of chemical exchange and rotational anisotropy. A detailed analysis has been made of the effect of anisotropy, exchange and experimental precision on the recovered motional parameters. The backbone NH relaxation is affected by motions on a variety of time scales from millisecond to tens of picoseconds. The relaxation data show a structured core of 100 residues corresponding to that observed in the crystal state. Within the core of the protein, two regions on either side of the putative recognition helix (helix B) show slow (ca. 0.2 ms) conformational exchange dynamics that are quenched upon DNA binding. The C-terminal 24 residues are generally more dynamic than in the core. However, in the free protein, a stretch of approximately 8 residues in the middle of the tail show relaxation behaviour similar to that in the core, indicating a structured region. NOEs between Ala 114 in this structured part of the tail and residues in the N-terminal beta strand of the core of the protein demonstrate that the tail folds back onto the core of the protein. In the complex with DNA, the structured part of the tail extends by ca. 3 residues. These data provide a framework for understanding the biochemical data on the mechanism and specificity of DNA binding.

Characterization of the DNA-binding domains from the yeast cell-cycle transcription factors Mbp1 and Swi4.

The minimal DNA-binding domains of the Saccharomyces cerevisiae transcription factors Mbp1 and Swi4 have been identified and their DNA binding properties have been investigated by a combination of methods. An approximately 100 residue region of sequence homology at the N-termini of Mbp1 and Swi4 is necessary but not sufficient for full DNA binding activity. Unexpectedly, nonconserved residues C-terminal to the core domain are essential for DNA binding. Proteolysis of Mbp1 and Swi4 DNA-protein complexes has revealed the extent of these sequences, and C-terminally extended molecules with substantially enhanced DNA binding activity compared to the core domains alone have been produced. The extended Mbp1 and Swi4 proteins bind to their cognate sites with similar affinity [K(A) approximately (1-4) x 10(6) M(-)(1)] and with a 1:1 stoichiometry. However, alanine substitution of two lysine residues (116 and 122) within the C-terminal extension (tail) of Mbp1 considerably reduces the apparent affinity for an MCB (MluI cell-cycle box) containing oligonucleotide. Both Mbp1 and Swi4 are specific for their cognate sites with respect to nonspecific DNA but exhibit similar affinities for the SCB (Swi4/Swi6 cell-cycle box) and MCB consensus elements. Circular dichroism and (1)H NMR spectroscopy reveal that complex formation results in substantial perturbations of base stacking interactions upon DNA binding. These are localized to a central 5'-d(C-A/G-CG)-3' region common to both MCB and SCB sequences consistent with the observed pattern of specificity. Changes in the backbone amide proton and nitrogen chemical shifts upon DNA binding have enabled us to experimentally define a DNA-binding surface on the core N-terminal domain of Mbp1 that is associated with a putative winged helix-turn-helix motif. Furthermore, significant chemical shift differences occur within the C-terminal tail of Mbp1, supporting the notion of two structurally distinct DNA-binding regions within these proteins.

X-ray structural analysis of the yeast cell cycle regulator Swi6 reveals variations of the ankyrin fold and has implications for Swi6 function.

Swi6 is a 92,000 Mr protein common to two distinct transcriptional activation complexes (SBF and MBF) that coordinate gene expression at the G1-S boundary of the yeast cell cycle. The X-ray structure of a central 36,000 Mr fragment has been determined and refined at 2.1 A resolution. The structure reveals a basic framework of five ankyrin repeat modules that is elaborated through a series of helical insertions distinguishing it from structures of other ankyrin repeat proteins. A second domain contains an approximately 30-residue region of extended structure that interacts with the ankyrin repeat core over a substantial proportion of its surface. Conservation of residues buried by these interactions indicates that all members of the Swi6/Cdc10 family share a similar architecture. Several temperature-sensitive mutations within Swi6 and Cdc10 appear to disrupt these interdomain contacts rather than destabilize the ankyrin repeat core. The unusual domain arrangement may be crucial for the modulation of interactions with other co-regulatory molecules such as cyclin-CDK complexes, and has implications for the quaternary interactions within the multisubunit SBF and MBF transcription complexes.

Interaction of the type I methyltransferase M.EcoR124I with modified DNA substrates: sequence discrimination and base flipping.

We have analysed the DNA-protein contacts made between the type I DNA methyltransferase M.EcoR124I and its recognition sequence. The effects of base modifications have been probed by measuring the affinity of M.EcoR124I for the modified sequences relative to that for the wild-type sequence by using gel-retardation competition assays. These results, along with those from methylation interference footprinting and photo-affinity cross-linking have identified the location of potential DNA contacts within the DNA recognition site. Substitution of 6-thioguanosine for each of the three specific guanines in the recognition sequence leads to a large (10-20-fold) decrease in the strength of DNA binding, indicating the importance of hydrogen-bonding interactions in the major groove of DNA. In contrast, replacement of either (or both) of the adenines at the target site for methylation by the enzyme, to produce either a base pair mismatch or loss of the base, leads to a marked increase in DNA-binding affinity. The results strongly support the proposal that type I methyltransferases employ a base-flipping mechanism to methylate their target base.

The crystal structure of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 A resolution.

The structure of the L1 metallo-beta-lactamase from the opportunistic pathogen Stenotrophomonas maltophilia has been determined at 1.7 A resolution by the multiwavelength anomalous dispersion (MAD) approach exploiting both the intrinsic binuclear zinc centre and incorporated selenomethionine residues. L1 is unique amongst all known beta-lactamases in that it exists as a tetramer. The protein exhibits the alphabeta/betaalpha fold found only in the metallo-beta-lactamases and displays several unique features not previously observed in these enzymes. These include a disulphide bridge and two substantially elongated loops connected to the active site of the enzyme. Two closely spaced zinc ions are bound at the active site with tetrahedral (Zn1) and trigonal bipyramidal (Zn2) co-ordination, respectively; these are bridged by a water molecule which we propose acts as the nucleophile in the hydrolytic reaction. Ligation of the second zinc ion involves both residues and geometry which have not been previously observed in the metallo-beta-lactamases. Simulated binding of the substrates ampicillin, ceftazidime and imipenem suggests that the substrate is able to bind to the enzyme in a variety of different conformations whose common features are direct interactions of the beta-lactam carbonyl oxygen and nitrogen with the zinc ions and of the beta-lactam carboxylate with Ser187. We describe a catalytic mechanism whose principal features are a nucleophilic attack of the bridging water on the beta-lactam carbonyl carbon, electrostatic stabilisation of a negatively charged tetrahedral transition state and protonation of the beta-lactam nitrogen by a second water molecule co-ordinated by Zn2. Further, we propose that direct metal:substrate interactions provide a substantial contribution to substrate binding and that this may explain the lack of specificity which is a feature of this class of enzyme.

Structural and functional architecture of the yeast cell-cycle transcription factor swi6.

The structural and functional organisation of Swi6, a transcriptional regulator of the budding yeast cell cycle has been analysed by a combination of biochemical, biophysical and genetic methods. Limited proteolysis indicates the presence of a approximately 15 kDa N-terminal domain which is dispensable for Swi6 activity in vivo and which is separated from the rest of the molecule by an extended linker of at least 43 residues. Within the central region, a 141 residue segment that is capable of transcriptional activation encompasses a structural domain of approximately 85 residues. In turn, this is tightly associated with an adjacent 28 kDa domain containing at least four ankyrin-repeat (ANK) motifs. A second protease sensitive region connects the ANK domain to the remaining 30 kDa C-terminal portion of Swi6 which contains a second transcriptional activator and sequences required for heteromerisation with Swi4 or Mbp1. Transactivation by the activating regions of Swi6 is antagonised when either are combined with the central ankyrin repeat motifs. Hydrodynamic measurements indicate that an N-terminal 62 kDa fragment comprising the first three domains is monomeric in solution and exhibits an unusually high frictional coefficient consistent with the extended, multi-domain structure suggested by proteolytic analysis.

The X-ray structure of the DNA-binding domain from the Saccharomyces cerevisiae cell-cycle transcription factor Mbp1 at 2.1 A resolution.

The structure of the DNA-binding domain of the Saccharomyces cerevisiae cell-cycle transcription factor Mbp1 has been solved using the multiwavelength anomalous diffraction (MAD) technique on crystals of selenomethionyl protein and refined at 2.1 A resolution. The molecule is globular, consisting of a twisted, six-stranded beta-barrel that is packed against a loose bundle of four alpha-helices. Two of the beta-strands in combination with two of the helices form a structure characteristic of the DNA-binding motif found in the CAP family of helix-turn-helix transcription factors. In Mbp1, this beta2/alpha2 motif is associated with regions of both positive electrostatic potential and sequence conservation within the Mbp1/Swi4 family, suggesting a role in DNA-binding in these proteins. A combination of structural and biochemical data further indicate a similarity to HNF3gamma/fork head, a member of the family of "winged" helix-turn-helix proteins. We propose a model for DNA-binding involving a recognition helix in the major groove, phosphodiester backbone interactions through the beta-hairpin and further base and/or phosphate interactions mediated by a C-terminal, positively charged loop.

Expression, purification, and crystallization of the DNA-binding domain from the Saccharomyces cerevisae cell-cycle transcription factor MBP-1.

A 124-residue N-terminal fragment corresponding to the DNA-binding domain of the Saccharomyces cerevisae cell-cycle transcription factor MBP-1 has been expressed with a hexahistidine affinity tag in E. coli and purified to apparent homogeneity. Crystals have been grown using PEG 3350 as precipitant which diffract x-rays to greater than 2.6 A resolution. The space group is tetragonal, P4(3)2(1)2 or P4(1)2(1)2 with unit cell dimensions a = b = 42.2 A, c = 123.2 A and a monomer in the asymmetric unit.

Surface labelling of the type I methyltransferase M.EcoR124I reveals lysine residues critical for DNA binding.

The type IC methyltransferase M.EcoR124I consists of a specificity subunit (HsdS) and two methylation subunits (HsdM). Using chemical modifications, we have investigated the accessibility of lysine residues in the free enzyme and in the complex with its DNA recognition sequence. A total of 41 of the 109 lysine residues in the enzyme are susceptible to modification, of which 19 are located in the HsdS subunit and 11 in each of the two HsdM subunits. DNA binding results in extensive protection of lysine residues in the HsdS subunit, while those in the HsdM subunit are only protected weakly. The DNA binding activity of the methylase is abolished when a small fraction of the accessible lysine residues are modified. Peptide mapping and N-terminal sequencing has been used to locate the rapidly modified lysine residues in HsdS that are critical for DNA binding. Highly modified residues (K297, K261 and K327) are found in the C-terminal variable domain that is responsible for DNA recognition, but others (K196, K203 and K210) are found in the conserved regions that had not previously been implicated in DNA binding.

Probing the domain structure of the type IC DNA methyltransferase M.EcoR124I by limited proteolysis.

Limited proteolysis has been used to probe the domain structure of the type I DNA methyltransferase M.EcoR124I. Trypsin digestion of the methyltransferase generates two fragments derived from the HsdS subunit, a 28 kDa N-terminal domain and a 19 kDa C-terminal domain, leaving the HsdM subunit intact. Extensive digestion by chymotrypsin, however, removes 59 amino acid residues from the N terminus of the HsdM subunit to leave a 52 kDa C-terminal domain. Binding of the cofactor S-adenosyl methionine has no appreciable effect on the rate of cleavage, but binding of a 30 bp DNA duplex containing the cognate recognition sequence confers almost total protection. Following trypsin cleavage of the methyltransferase, a stable proteolytic product is produced which has been purified for biochemical characterisation. The trypsinised enzyme is shown to be a multimeric complex containing two intact HsdM subunits and both fragments of the HsdS subunit, consistent with the circular model proposed for the organisation of domains in the specificity subunit in type IC methyltransferases. Gel retardation studies show that the proteolysed enzyme still retains DNA binding activity, but its specificity for the DNA recognition sequence is dramatically reduced.

DNA-binding induces a major structural transition in a type I methyltransferase.

The type IC DNA methyltransferase M.EcoR124I is a complex multisubunit enzyme that recognizes the non-palindromic DNA sequence GAAN6RTCG. Small angle X-ray scattering has been used to investigate the solution structure of the methyltransferase and of complexes of the enzyme with unmethylated and hemimethylated 30 bp DNA duplexes containing the specific recognition sequence. A major change in the quaternary structure of the enzyme is observed following DNA binding, based on a decrease in the radius of gyration from 56 to 40 A and a reduction in the maximum dimension of the enzyme from 180 to 112 A. The structural transition observed is independent of the methylation state of the DNA. CD shows that there is no change in the secondary structure of the protein subunits when DNA is bound. In contrast, there is a large increase in the CD signal arising from the DNA, suggesting considerable structural distortion which may allow access to the bases targeted for methylation. We propose that DNA binding induces a large rotation of the two HsdM subunits towards the DNA, mediated by hinge bending domains in the specificity subunit HsdS.

Effects of creative and noncreative problem-solving on anxiety.

This study has investigated whether the type of problem in creative performance increases anxiety more than the type of problem in noncreative performance. Subjects were 9 male and 48 female undergraduate students in psychology, selected from a voluntary pool and assigned (3 males, 16 females) nonsystematically to either a divergent creative problem-solving condition, a convergent noncreative problem-solving condition, or a control condition involving a neutral problem-solving condition. The Spielberger State-Trait Anxiety Inventory was administered to each group before and after the experimental conditions. It was hypothesized: (a) that divergent creative problem-solving would increase state anxiety significantly more than both the convergent noncreative problem-solving task and the neutral problem-solving task and (b) that trait anxiety would not be significantly affected by any of the conditions. Only the latter hypothesis was confirmed. Divergent creative problem-solving did not significantly increase state anxiety, perhaps because the employed subjects were students and may have felt more comfortable with divergent problems than the average population.

Effective functioning of chronic psychiatric patients in aftercare settings.

The purpose of this study was to determine personality and environmental characteristics as they relate to effective functioning of chronic psychiatric patients in aftercare settings in North Western Ontario. The guiding assumption was that indices of health are better predictors of effective functioning than the absence of pathology.