The structure and mechanism of serine acetyltransferase from Escherichia coli

Serine acetyltransferase (SAT) catalyzes the first step of cysteine synthesis in microorganisms and higher plants. Here we present the 2.2 Angstrom crystal structure of SAT from Escherichia coli, which is a dimer of trimers, in complex with cysteine. The SAT monomer consists of an amino-terminal alpha-helical domain and a carboxyl- terminal left-handed beta-helix. We identify His(158) and Asp(143) as essential residues that form a catalytic triad with the substrate for acetyl transfer. This structure shows the mechanism by which cysteine inhibits SAT activity and thus controls its own synthesis. Cysteine is found to bind at the serine substrate site and not the acetyl-CoA site that had been reported previously. On the basis of the geometry around the cysteine binding site, we are able to suggest a mechanism for the O-acetylation of serine by SAT. We also compare the structure of SAT with other left-handed beta-helical structures.

SARS coronavirus unique domain: three-domain molecular architecture in solution and RNA binding

Nonstructural protein 3 of the severe acute respiratory syndrome (SARS) coronavirus includes a "SARS-unique domain" (SUD) consisting of three globular domains separated by short linker peptide segments. This work reports NMR structure determinations of the C-terminal domain (SUD-C) and a two-domain construct (SUD-MC) containing the middle domain (SUD-M) and the C-terminal domain, and NMR data on the conformational states of the N-terminal domain (SUD-N) and the SUD-NM two-domain construct. Both SUD-N and SUD-NM are monomeric and globular in solution; in SUD-NM, there is high mobility in the two-residue interdomain linking sequence, with no preferred relative orientation of the two domains. SUD-C adopts a frataxin like fold and has structural similarity to DNA-binding domains of DNA-modifying enzymes. The structures of both SUD-M (previously determined) and SUD-C (from the present study) are maintained in SUD-MC, where the two domains are flexibly linked. Gel-shift experiments showed that both SUD-C and SUD-MC bind to single-stranded RNA and recognize purine bases more strongly than pyrimidine bases, whereby SUD-MC binds to a more restricted set of purine-containing RNA sequences than SUD-M. NMR chemical shift perturbation experiments with observations of (15)N-labeled proteins further resulted in delineation of RNA binding sites (i.e., in SUD-M, a positively charged surface area with a pronounced cavity, and in SUD-C, several residues of an anti-parallel beta-sheet). Overall, the present data provide evidence for molecular mechanisms involving the concerted actions of SUD-M and SUD-C, which result in specific RNA binding that might be unique to the SUD and, thus, to the SARS coronavirus.

Conserved hydrophobic clusters on the surface of the Caf1A usher C-terminal domain are important for F1 antigen assembly

The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 (capsular antigen fraction 1) protein subunits. The Caf1A usher promotes polymerization of subunits and secretion of growing polymers to the cell surface. The usher monomer (811 aa, 90.5 kDa) consists of a large transmembrane β-barrel that forms a secretion channel and three soluble domains. The periplasmic N-terminal domain binds chaperone-subunit complexes supplying new subunits for the growing fiber. The middle domain, which is structurally similar to Caf1 and other fimbrial subunits, serves as a plug that regulates the permeability of the usher. Here we describe the identification, characterization, and crystal structure of the Caf1A usher C-terminal domain (Caf1A(C)). Caf1A(C) is shown to be a periplasmic domain with a seven-stranded β-barrel fold. Analysis of C-terminal truncation mutants of Caf1A demonstrated that the presence of Caf1A(C) is crucial for the function of the usher in vivo, but that it is not required for the initial binding of chaperone-subunit complexes to the usher. Two clusters of conserved hydrophobic residues on the surface of Caf1A(C) were found to be essential for the efficient assembly of surface polymers. These clusters are conserved between the FGL family and the FGS family of chaperone-usher systems.

Wavelet filtered modelling applied to measurements of a waveguide's THz time domain response

A quasi-optical de-embedding technique for characterizing waveguides is demonstrated using wideband time-resolved terahertz spectroscopy. A transfer function representation is adopted for the description of the signal in the input and output port of the waveguides. The time domain responses were discretised and the waveguide transfer function was obtained through a parametric approach in the z-domain after describing the system with an ARX as well as with a state space model. Prior to the identification procedure, filtering was performed in the wavelet domain to minimize signal distortion and the noise propagating in the ARX and subspace models. The model identification procedure requires isolation of the phase delay in the structure and therefore the time-domain signatures must be firstly aligned with respect to each other before they are compared. An initial estimate of the number of propagating modes was provided by comparing the measured phase delay in the structure with theoretical calculations that take into account the physical dimensions of the waveguide. Models derived from measurements of THz transients in a precision WR-8 waveguide adjustable short will be presented.

Protein structure prediction servers at University College London

A number of state-of-the-art protein structure prediction servers have been developed by researchers working in the Bioinformatics Unit at University College London. The popular PSIPRED server allows users to perform secondary structure prediction, transmembrane topology prediction and protein fold recognition. More recent servers include DISOPRED for the prediction of protein dynamic disorder and DomPred for domain boundary prediction.

A diabatically-generated potential vorticity structure near the extratropical tropopause in three simulated extratropical cyclones

The structure of near-tropopause potential vorticity (PV) acts as a primary control on the evolution of extratropical cyclones. Diabatic processes such as the latent heating found in ascending moist warm conveyor belts modify PV. A dipole in diabatically-generated PV (hereafter diabatic PV) straddling the extratropical tropopause, with the positive pole above the negative pole, was diagnosed in a recently published analysis of a simulated extratropical cyclone. This PV dipole has the potential to significantly modify the propagation of Rossby waves and the growth of baroclinically-unstable waves. This previous analysis was based on a single case study simulated with 12-km horizontal grid spacing and parameterized convection. Here, the dipole is investigated in three additional cold-season extratropical cyclones simulated in both convection-parameterizing and convection-permitting model configurations. A diabatic PV dipole across the extratropical tropopause is diagnosed in all three cases. The amplitude of the dipole saturates approximately 36 hours from the time diabatic PV is accumulated. The node elevation of the dipole varies between 2-4 PVU in the three cases, and the amplitude of the system-averaged dipole varies between 0.2-0.4 PVU. The amplitude of the negative pole is similar in the convection-parameterizing and convection-permitting simulations. The positive pole, which is generated by long-wave radiative cooling, is weak in the convection-permitting simulations due to the small domain size which limits the accumulation of diabatic tendencies within the interior of the domain. The possible correspondence between the diabatic PV dipole and the extratropical tropopause inversion layer is discussed.

The effects of explicit versus parameterized convection on the MJO in a large-domain high-resolution tropical case study. Part II: Processes leading to differences in MJO development

High-resolution simulations over a large tropical domain (∼20◦S–20◦N and 42◦E–180◦E) using both explicit and parameterized convection are analyzed and compared during a 10-day case study of an active Madden-Julian Oscillation (MJO) event. In Part II, the moisture budgets and moist entropy budgets are analyzed. Vertical subgrid diabatic heating profiles and vertical velocity profiles are also compared; these are related to the horizontal and vertical advective components of the moist entropy budget which contribute to gross moist stability, GMS, and normalized GMS (NGMS). The 4-km model with explicit convection and good MJO performance has a vertical heating structure that increases with height in the lower troposphere in regions of strong convection (like observations), whereas the 12-km model with parameterized convection and a poor MJO does not show this relationship. The 4-km explicit convection model also has a more top-heavy heating profile for the troposphere as a whole near and to the west of the active MJO-related convection, unlike the 12-km parameterized convection model. The dependence of entropy advection components on moisture convergence is fairly weak in all models, and differences between models are not always related to MJO performance, making comparisons to previous work somewhat inconclusive. However, models with relatively good MJO strength and propagation have a slightly larger increase of the vertical advective component with increasing moisture convergence, and their NGMS vertical terms have more variability in time and longitude, with total NGMS that is comparatively larger to the west and smaller to the east.

The atomic structure of low-index surfaces of the intermetallic compound InPd

The intermetallic compound InPd (CsCl type of crystal structure with a broad compositional range) is considered as a candidate catalyst for the steam reforming of methanol. Single crystals of this phase have been grown to study the structure of its three low-index surfaces under ultra-high vacuum conditions, using low energy electron diffraction (LEED), X-ray photoemission spectroscopy (XPS), and scanning tunneling microscopy (STM). During surface preparation, preferential sputtering leads to a depletion of In within the top few layers for all three surfaces. The near-surface regions remain slightly Pd-rich until annealing to ∼580 K. A transition occurs between 580 and 660 K where In segregates towards the surface and the near-surface regions become slightly In-rich above ∼660 K. This transition is accompanied by a sharpening of LEED patterns and formation of flat step-terrace morphology, as observed by STM. Several superstructures have been identified for the different surfaces associated with this process. Annealing to higher temperatures (≥750 K) leads to faceting via thermal etching as shown for the (110) surface, with a bulk In composition close to the In-rich limit of the existence domain of the cubic phase. The Pd-rich InPd(111) is found to be consistent with a Pd-terminated bulk truncation model as shown by dynamical LEED analysis while, after annealing at higher temperature, the In-rich InPd(111) is consistent with an In-terminated bulk truncation, in agreement with density functional theory (DFT) calculations of the relative surface energies. More complex surface structures are observed for the (100) surface. Additionally, individual grains of a polycrystalline sample are characterized by micro-spot XPS and LEED as well as low-energy electron microscopy. Results from both individual grains and “global” measurements are interpreted based on comparison to our single crystals findings, DFT calculations and previous literature.

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha 1 (residues Gly-9 to Arg-21), alpha 2 (residues Glu-27 to Asn-40), alpha 3 (residues Arg-44 to Thr-54), alpha 4 (residues Leu-57 to Tyr-64), and alpha 5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

The leptospiral antigen Lp49 is a two-domain protein with putative protein binding function

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 angstrom resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date. (C) 2008 Elsevier Inc. All rights reserved.

The crystal structure of the leptospiral hypothetical protein LIC12922 reveals homology with the periplasmic chaperone SurA

Leptospirosis is a world spread zoonosis caused by members of the genus Leptospira. Although leptospires were identified as the causal agent of leptospirosis almost 100 years ago, little is known about their biology, which hinders the development of new treatment and prevention strategies. One of the several aspects of the leptospiral biology not yet elucidated is the process by which outer membrane proteins (OMPs) traverse the periplasm and are inserted into the outer membrane. The crystal structure determination of the conserved hypothetical protein LIC12922 from Leptospira interrogans revealed a two domain protein homologous to the Escherichia coli periplasmic chaperone SurA. The LIC12922 NC-domain is structurally related to the chaperone modules of E. coli SurA and trigger factor, whereas the parvulin domain is devoid of peptidyl prolyl cis-trans isomerase activity. Phylogenetic analyses suggest a relationship between LIC12922 and the chaperones PrsA, PpiD and SurA. Based on our structural and evolutionary analyses, we postulate that LIC12922 is a periplasmic chaperone involved in OMPs biogenesis in Leptospira spp. Since LIC12922 homologs were identified in all spirochetal genomes sequenced to date, this assumption may have implications for the OMPs biogenesis studies not only in leptospires but in the entire Phylum Spirochaetes. (C) 2010 Elsevier Inc. All rights reserved.

Investigation by Combined Solid-State NMR and SAXS Methods of the Morphology and Domain Size in Polystyrene-b-Polyethylene Oxide-b-Polystyrene Triblock Copolymers

The microphase structure of a series of polystyrene-b-polyethylene oxide-b-polystyrene (SEOS) triblock copolymers with different compositions and molecular weights has been studied by solid-state NMR, DSC, wide and small angle X-ray scattering (WAXS and SAXS). WAXS and DSC measurements were used to detect the presence of crystalline domains of polyethyleneoxide (PEO) blocks at room temperature as a function of the copolymer chemical composition. Furthermore, DSC experiments allowed the determination of the melting temperatures of the crystalline part of the PEO blocks. SAXS measurements, performed above and below the melting temperature of the PEO blocks, revealed the formation of periodic structures, but the absence or the weakness of high order reflections peaks did not allow a clear assessment of the morphological structure of the copolymers. This information was inferred by combining the results obtained by SAXS and (1)H NMR spin diffusion experiments, which also provided an estimation of the size of the dispersed phases of the nanostructured copolymers. (C) 2009 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48:55-64,2010

The CATH Hierarchy Revisited-Structural Divergence in Domain Superfamilies and the Continuity of Fold Space

This paper explores the structural continuum in CATH and the extent to which superfamilies adopt distinct folds. Although most superfamilies are structurally conserved, in some of the most highly populated superfamilies (4% of all superfamilies) there is considerable structural divergence. While relatives share a similar fold in the evolutionary conserved core, diverse elaborations to this core can result in significant differences in the global structures. Applying similar protocols to examine the extent to which structural overlaps occur between different fold groups, it appears this effect is confined to just a few architectures and is largely due to small, recurring super-secondary motifs (e.g., alpha beta-motifs, alpha-hairpins). Although 24% of superfamilies overlap with superfamilies having different folds, only 14% of nonredundant structures in CATH are involved in overlaps. Nevertheless, the existence of these overlaps suggests that, in some regions of structure space, the fold universe should be seen as more continuous.

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: Insights for a new classification of snake venom metalloprotease subgroups

Bothropasin is a 48 kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys(189) is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from I`ll SVMPs. The ECD motif is stabilized by the Cys(117)_Cys(310) disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu(276) of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other Pill SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other Pill members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain. (C) 2008 Elsevier Ltd. All rights reserved.

Crystallographic Structure of Phosphofructokinase-2 from Escherichia coli in Complex with Two ATP Molecules. Implications for Substrate Inhibition

Phosphofructokinase-1 and -2 (Pfk-1 and Pfk-2, respectively) from Escherichia coli belong to different homologous superfamilies. However, in spite of the lack of a common ancestor, they share the ability to catalyze the same reaction and are inhibited by the substrate MgATP. Pfk-2, an ATP-dependent 6-phosphofructokinase member of the ribokinase-like superfamily, is a homodimer of 66 kDa subunits whose oligomerization state is necessary for catalysis and stability. The presence of MgATP favors the tetrameric form of the enzyme. In this work, we describe the structure of Pfk-2 in its inhibited tetrameric form, with each subunit bound to two ATP molecules and two Mg ions. The present structure indicates that substrate inhibition occurs due to the sequential binding of two MgATP molecules per subunit, the first at the usual site occupied by the nucleotide in homologous enzymes and the second at the allosteric site, making a number of direct and Mg-mediated interactions with the first. Two configurations are observed for the second MgATP, one of which involves interactions with Tyr23 from the adjacent subunit in the dimer and the other making an unusual non-Watson-Crick base pairing with the adenine in the substrate ATP. The oligomeric state observed in the crystal is tetrameric, and some of the structural elements involved in the binding of the Substrate and allosteric ATPs are also participating in the dimer-dimer interface. This structure also provides the grounds to compare analogous features of the nonhomologous phosphofructokinases from E. coli. (C) 2008 Elsevier Ltd. All rights reserved.