Structural and functional characterization of TesB from Yersinia pestis reveals a unique octameric arrangement of hotdog domains.

Acyl-CoA thioesterases catalyse the hydrolysis of the thioester bonds present within a wide range of acyl-CoA substrates, releasing free CoASH and the corresponding fatty-acyl conjugate. The TesB-type thioesterases are members of the TE4 thioesterase family, one of 25 thioesterase enzyme families characterized to date, and contain two fused hotdog domains in both prokaryote and eukaryote homologues. Only two structures have been elucidated within this enzyme family, and much of the current understanding of the TesB thioesterases has been based on the Escherichia coli structure. Yersinia pestis, a highly virulent bacterium, encodes only one TesB-type thioesterase in its genome; here, the structural and functional characterization of this enzyme are reported, revealing unique elements both within the protomer and quaternary arrangements of the hotdog domains which have not been reported previously in any thioesterase family. The quaternary structure, confirmed using a range of structural and biophysical techniques including crystallography, small-angle X-ray scattering, analytical ultracentrifugation and size-exclusion chromatography, exhibits a unique octameric arrangement of hotdog domains. Interestingly, the same biological unit appears to be present in both TesB structures solved to date, and is likely to be a conserved and distinguishing feature of TesB-type thioesterases. Analysis of the Y. pestis TesB thioesterase activity revealed a strong preference for octanoyl-CoA and this is supported by structural analysis of the active site. Overall, the results provide novel insights into the structure of TesB thioesterases which are likely to be conserved and distinguishing features of the TE4 thioesterase family.

Structural and dynamic requirements for optimal activity of the essential bacterial enzyme dihydrodipicolinate synthase.

Dihydrodipicolinate synthase (DHDPS) is an essential enzyme involved in the lysine biosynthesis pathway. DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface. Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer evolved to optimise the dynamics within the tight-dimer. In order to gain insights into DHDPS flexibility and its relationship to quaternary structure and function, we performed comparative Molecular Dynamics simulation studies of native tetrameric and dimeric forms of DHDPS from E. coli and also the native dimeric form from methicillin-resistant Staphylococcus aureus (MRSA). These reveal a striking contrast between the dynamics of tetrameric and dimeric forms. Whereas the E. coli DHDPS tetramer is relatively rigid, both the E. coli and MRSA DHDPS dimers display high flexibility, resulting in monomer reorientation within the dimer and increased flexibility at the tight-dimer interface. The mutant E. coli DHDPS dimer exhibits disorder within its active site with deformation of critical catalytic residues and removal of key hydrogen bonds that render it inactive, whereas the similarly flexible MRSA DHDPS dimer maintains its catalytic geometry and is thus fully functional. Our data support the hypothesis that in both bacterial species optimal activity is achieved by fine tuning protein dynamics in different ways: E. coli DHDPS buttresses together two dimers, whereas MRSA dampens the motion using an extended tight-dimer interface.

Characterisation of dihydrodipicolinate synthase (DHDPS) from Bacillus anthracis.

Bacillus anthracis is a Gram-positive spore-forming bacterium that is the causative agent of anthrax disease. The use of anthrax as a bioweapon has increased pressure for the development of an effective treatment. Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the biosynthetic pathway yielding two essential bacterial metabolites, meso-diaminopimelate (DAP) and (S)-lysine. DHDPS is therefore a potential antibiotic target, as microbes require either lysine or DAP as a component of the cell wall. This paper is the first biochemical description of DHDPS from B. anthracis. Enzyme kinetic analyses, isothermal titration calorimetry (ITC), mass spectrometry and differential scanning fluorimetry (DSF) were used to characterise B. anthracis DHDPS and compare it with the well characterised Escherichia coli enzyme. B. anthracis DHDPS exhibited different kinetic behaviour compared with E. coli DHDPS, in particular, substrate inhibition by (S)-aspartate semi-aldehyde was observed for the B. anthracis enzyme (K(si(ASA))=5.4+/-0.5 mM), but not for the E. coli enzyme. As predicted from a comparison of the X-ray crystal structures, the B. anthracis enzyme was not inhibited by lysine. The B. anthracis enzyme was thermally stabilised by the first substrate, pyruvate, to a greater extent than its E. coli counterpart, but has a weaker affinity for pyruvate based on enzyme kinetics and ITC studies. This characterisation will provide useful information for the design of inhibitors as new antibiotics targeting B. anthracis.

Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands.

Apoptosis is an important part of the host's defense mechanism for eliminating invading pathogens. Some viruses express proteins homologous in sequence and function to mammalian pro-survival Bcl-2 proteins. Anti-apoptotic F1L expressed by vaccinia virus is essential for survival of infected cells, but it bears no discernable sequence homology to proteins other than its immediate orthologues in related pox viruses. Here we report that the crystal structure of F1L reveals a Bcl-2-like fold with an unusual N-terminal extension. The protein forms a novel domain-swapped dimer in which the alpha1 helix is the exchanged domain. Binding studies reveal an atypical BH3-binding profile, with sub-micromolar affinity only for the BH3 peptide of pro-apoptotic Bim and low micromolar affinity for the BH3 peptides of Bak and Bax. This binding interaction is sensitive to F1L mutations within the predicted canonical BH3-binding groove, suggesting parallels between how vaccinia virus F1L and myxoma virus M11L bind BH3 domains. Structural comparison of F1L with other Bcl-2 family members reveals a novel sequence signature that redefines the BH4 domain as a structural motif present in both pro- and anti-apoptotic Bcl-2 members, including viral Bcl-2-like proteins.

CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13.

The pathway to cell death in Caenorhabditis elegans is well established. In cells undergoing apoptosis, the Bcl-2 homology domain 3 (BH3)-only protein EGL-1 binds to CED-9 at the mitochondrial membrane to cause the release of CED-4, which oligomerises and facilitates the activation of the caspase CED-3. However, despite many studies, the biophysical features of the CED-4/CED-9 complex have not been fully characterised. Here, we report the purification of a soluble and stable 2 : 2 heterotetrameric complex formed by recombinant CED-4 and CED-9 coexpressed in bacteria. Consistent with previous studies, synthetic peptides corresponding to the BH3 domains of worm BH3-only proteins (EGL-1, CED-13) dissociate CED-4 from CED-9, but not from the gain-of-function CED-9 (G169E) mutant. Surprisingly, the ability of worm BH3 domains to dissociate CED-4 was specific since mammalian BH3-only proteins could not do so.

Inhibiting protein-protein interactions as an emerging paradigm for drug discovery.

Association of proteins into homo- and hetero-oligomers plays an important role in a plethora of biological phenomena. Inhibition of these interactions is increasingly recognized as a valuable new direction in drug design. In this mini-review we consider inhibition of protein misfolding and aggregation, molecules that disrupt enzyme quaternary structure, and signaling inhibitors, as emerging drugs.

Trifluoroethanol induces the self-association of specific amphipathic peptides.

We have examined the effect of trifluoroethanol (TFE) on the solution behaviour of three amphipathic peptides. One of the peptides, containing three heptad repeat units (Ac-YS-(AKEAAKE)3GAR-NH2), remained monomeric under conditions where TFE induced a two-state transition from a random coil to an alpha-helix. In contrast, the TFE-induced alpha-helical formation of two peptides derived from human apolipoproteins C-II and E was accompanied by the formation of discrete dimers and trimers, respectively. The apolipoprotein C-II peptide further aggregated to form beta-sheet at higher concentrations of TFE (50% v/v). The results suggest a class of peptides capable of discrete self-association in the presence of cosolvents which favour intramolecular hydrogen bonding.

Self-association of human apolipoprotein E3 and E4 in the presence and absence of phospholipid.

Human apolipoprotein E (apoE) exists as three main isoforms, differing by single amino acid substitutions, with the apoE4 isoform strongly linked to the incidence of late onset Alzheimer's disease. We have expressed and purified apoE3 and apoE4 from Escherichia coli and compared their hydrodynamic properties by gel permeation liquid chromatography, capillary electrophoresis, circular dichroism, and sedimentation methods. Sedimentation velocity experiments, employing a new method for determining the size distribution of polydisperse macromolecules in solution (Schuck, P. (2000) Biophys. J. 78, 1606-1619), provide direct evidence for the heterogeneous solution structures of apoE3 and apoE4. In a lipid-free environment, apoE3 and apoE4 exist as a slow equilibrium mixture of monomer, tetramer, octamer, and a small proportion of higher oligomers. Both sedimentation velocity and equilibrium experiments indicate that apoE4 has a greater propensity to self-associate. We also demonstrate that apoE3 and apoE4 oligomers dissociate significantly in the presence of dihexanoylphosphatidylcholine micelles (20 mm) and to a lesser extent at submicellar concentrations (4 mm). The alpha-helical content for both isoforms was almost identical (50%) in the presence and absence of dihexanoylphosphatidylcholine. These results reveal that apoE oligomers undergo phospholipid-induced dissociation to folded monomers, suggesting the monomeric form prevails on the lipoprotein surface in vivo.