Structure and conformational dynamics of fatty acid synthases

Multistep reactions rely on substrate channeling between active sites. Carrier protein-based enzyme systems constitute the most versatile class of shuttling systems due to their capability of linking multiple catalytic centers. In eukaryotes and some bacteria, these systems have evolved to multifunctional enzymes, which integrate all functional domains involved into one or more giant polypeptide chains. The metazoan fatty acid synthase (FAS) is a key paradigm for carrier protein-based multienzymes. It catalyzes the de novo biosynthesis of fatty acids from carbohydrate-derived precursors in more than 40 individual reactions steps. Its seven functional domains are encoded on one polypeptide chain, which assembles into an X-shaped dimer for activity. The dimer features two lateral reaction clefts, each equipped with a full set of active sites and a flexibly tethered carrier protein. Substrate loading and condensation in the condensing region are structurally and functionally separated from the β-carbon processing domains in the modifying region. At the beginning of this thesis, only a single crystal structure of an intact metazoan FAS was known. FAS, in particular its modifying region, displays extensive conformational variability, according to electron microscopy (EM) studies. Thus, the aim was to obtain a crystal structure of the FAS modifying region to identify a ground-state structure of the FAS modifying region and to characterize its structural heterogeneity. The second aim was to establish a method for mapping conformational changes in multienzymes at high spatiotemporal resolution. Chapter 1 introduces FAS and gives a methodological overview of studying conformational dynamics of multienzymes. In chapter 2, the 2.7-Å crystal structure of the entire 250-kDa modifying region of insect FAS is presented. It presents a conserved ground-state conformation adopted by the most divergent member of metazoan FAS. Remarkably, even the V-shape of the central dehydratase dimer is conserved, despite a minimal interface. Structural comparison to polyketide synthases (PKSs) highlights distinct properties of FAS such as strong domain interactions and the absence of an N-terminal β-α-β-α extension of the lateral non-catalytic pseudo-ketoreductase. Chapter 3 presents a novel approach for identifying conformational dynamics of multienzymes in solution by filming with high-speed atomic force microscopy (HS-AFM) at a spatial resolution of 2-5 nm. The temporal resolution of 10 fps correlates with the timescale of large-scale conformational changes in FAS. Varied viewing angles are provided by combining different molecular tethering strategies. Reference-free particle classification enables the quantitative characterization of conformational states and their transitions. Chapter 4 discusses the implications of the results on multienzyme biology with respect to biological questions and biotechnological applications. The results of this thesis provide the tools to understand the role of conformational dynamics in multienzymes with respect to their function. These findings will ultimately advance the engineering of enzymatic assembly lines for tailored compound production

High-Speed Atomic Force Microscopy Visualization of the Dynamics of the Multienzyme Fatty Acid Synthase

Multienzymes, such as the protein metazoan fatty acid synthase (FAS), are giant and highly dynamic molecular machines for critical biosynthetic processes. The molecular architecture of FAS was elucidated by static high-resolution crystallographic analysis, while electron microscopy revealed large-scale conformational variability in FAS with some correlation to functional states in catalysis. However, little is known about time scales of conformational dynamics, the trajectory of motions in individual FAS molecules and the extent of coupling between catalysis and structural changes. Here, we present an experimental single-molecule approach to film immobilized or selectively tethered FAS in solution at different viewing angles and high spatio-temporal resolution using high-speed atomic force microscopy (HS-AFM). Mobility of individual regions of the multienzyme is recognized in video sequences and correlation of shape features implies a convergence of temporal resolution and velocity of FAS dynamics. Conformational variety can be identified and grouped by reference-free 2D class averaging, enabling the tracking of conformational transitions in movies. The approach presented here is suited for comprehensive studies of the dynamics of FAS and other multienzymes in aqueous solution at the single-molecule level

Acceptability, effectiveness and costeffectiveness of blended cognitivebehavioural therapy (bCBT) versus face-toface CBT (ftfCBT) for anxiety disorders in specialised mental health care: A 15-week randomised controlled trial with 1-year followup

BACKGROUND: Anxiety disorders are highly prevalent and cause substantial economic burden. Blended cognitive-behavioural therapy (bCBT), which integrates Internet-based CBT and face-to-face CBT (ftfCBT), is an attractive and potentially cost-saving treatment alternative to conventional CBT for patients with anxiety disorders in specialised mental health care. However, little is known about the effectiveness of bCBT in routine care. We examined the acceptability, effectiveness and cost-effectiveness of bCBT versus ftfCBT in outpatient specialised care to patients with panic disorder, social anxiety disorder and generalised anxiety disorder. METHODS AND FINDINGS: Patients with anxiety disorders were randomised to bCBT (n = 52) or ftfCBT (n = 62). Acceptability of bCBT and ftfCBT were evaluated by assessing treatment preference, adherence, satisfaction and therapeutic alliance. Costs and effects were assessed at post-treatment and one-year follow-up. Primary outcome measure was the Beck Anxiety Inventory (BAI). Secondary outcomes were depressive symptoms, general psychopathology, work and social adjustment, quality of life and mastery. Incremental cost-effectiveness ratios (ICERs) were computed from societal and healthcare perspectives by calculating the incremental costs per incremental quality-adjusted life year (QALY). No significant differences between bCBT and ftfCBT were found on acceptability or effectiveness measures at post-treatment (Cohen's d between-group effect size on BAI = 0.15, 95% CI -0.30 to 0.60) or at one-year follow-up (d = -0.38, 95% CI -0.84 to 0.09). The modelled point estimates of societal costs (bCBT €10945, ftfCBT €10937) were higher and modelled point estimates of direct medical costs (bCBT €3748, ftfCBT €3841) were lower in bCBT. The acceptability curves showed that bCBT was expected to be a cost-effective intervention. Results should be carefully interpreted due to the small sample size. CONCLUSIONS: bCBT appears an acceptable, clinically effective and potentially cost-saving alternative option for treating patients with anxiety disorders. Trials with larger samples are needed to further investigate cost-effectiveness. TRIAL REGISTRATION: Netherlands Trial Register: NTR4912

LPS analysis of <i>S</i>. <i>flexneri</i> expressing wild-type WzzB_SF or the WzzBSFA107P mutant.

<p>Whole-cell lysates of <i>S</i>. <i>flexneri</i> RMA4053 strains expressing either wild-type WzzB_SF or the WzzB_SF^A107P mutant proteins were (A) treated by proteinase-K and electrophoresed on a SDS 15% polyacrylamide gel, followed by detection of LPS by silver-staining (the first 20 Oag repeat units are indicated); or (B) electrophoresed on SDS 15% polyacrylamide gels and then subjected to western immunoblotting with WzzB_SF polyclonal antibodies. The size of the full-length WzzB_SF protein (~36 kDa) is indicated. Each lane corresponds to 5 x 10⁷ bacterial cells.</p

Crystallographic data.

<p>Values in parentheses are for the highest resolution shell.</p><p>¹<i>R</i>_meas =.Σ_<i>hkl</i>{1/[1(<i>hkl</i>)-1]}^1/2Σ_<i>i</i> | I_<i>i</i>(<i>hkl</i>)-(I(<i>hkl</i>))|/Σ_<i>hkl</i>Σ_i I_i(<i>hkl</i>).</p><p>²<i>R</i>_p.i.m. = Σ_<i>hkl</i>{1/[N(<i>hkl</i>)-1]}^1/2Σ_<i>i</i> | I_<i>i</i>(<i>hkl</i>)-(I(<i>hkl</i>))|/Σ_<i>hkl</i>Σ_i I_i(<i>hkl</i>).</p><p>³<i>R</i>_work = Σ_<i>hkl</i>(||Fobs_<i>hkl</i>|-|Fcalc_<i>hkl</i>||)/|Fobs_<i>hkl</i>|, where |Fobs_<i>hkl</i>| and |Fcalc_<i>hkl</i>| represent the observed and calculated structure factor amplitudes. <i>R</i>_free is equivalent to <i>R</i>_work but calculated using 5% of the reflections not used in refinement.</p><p>⁴ Calculated using Molprobity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138266#pone.0138266.ref033" target="_blank">33</a>].</p><p>Crystallographic data.</p

Small-angle X-ray scattering (SAXS) data.

<p>(A-B) Averaged and subtracted SAXS data for a dilution series of WzzB_SF (A) and WzzB_SF^A107P (B), placed on absolute scale by comparison to the scattering of pure water. Data points are shown as circles coloured by dilution. The fits of the corresponding <i>P</i>(<i>r</i>) distributions are shown as black lines. (C-D) Distance distributions calculated from the scattering data by indirect Fourier transformation, showing the frequency, <i>P</i>(<i>r</i>), of intermolecular distances of length <i>r</i> within each particle. Distributions are calculated from each dilution in the concentration series. Plots have been scaled to the maximum of the highest concentration for ease of visualisation. Top panel, WzzB_SF; bottom panel, WzzB_SF^A107P. Considerable concentration dependence can be observed in both, particularly in the highest concentration of the wild-type protein. (E-F) Analysis of concentration-dependence of particle size and total scattering. Radius of gyration, <i>R</i>_<i>g</i> (black diamonds), and the ratio of zero-angle scattering to concentration, <i>I</i>(0)/C_rel (coloured diamonds) are plotted for each dilution of each protein. Both metrics are calculated from Guinier analysis of the low-<i>q</i> region of the data, and would be expected to be stable for a monodisperse, non-interacting species. Approximate non-relative concentrations are 10 mg/ml for 1:1 WzzB_SF, and 5 mg/ml for 1:1 WzzB_SF^A107P.</p

Analysis of colicin E2 sensitivity of WzzBSFA107P.

<p>Spot sensitivity assays with purified colicin E2 were performed using <i>S</i>. <i>flexneri</i> RMA4053 strains expressing either WzzB_SF or WzzB_SF^A107P [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138266#pone.0138266.ref013" target="_blank">13</a>]. The minimum inhibitory concentration (MIC) of colicin E2 (in μg/ml) required to generate a clear zone of bacterial growth inhibition is shown on the <i>y-</i>axis (<i>n</i> = 3). There was no difference in MIC values between replicates for each particular strain. The MIC for each strain expressing pQE30, WzzB_SF and WzzB_SF^A107P were 0.25, 32 and 0.5 μg/ml, respectively.</p

Formaldehyde cross-linking analysis.

<p><i>S</i>. <i>flexneri</i> RMA4053 strains expressing either WzzB_SF or WzzB_SF^A107P were harvested, resuspended in potassium phosphate buffer, and treated with 0.5% formaldehyde (+) at 25°C; controls were incubated without formaldehyde (-). Cells were resuspended in the sample buffer and heated at 60°C for 5 min, electrophoresed on a SDS 12% polyacrylamide gel, followed by western immunoblotting with WzzB_SF antibodies. The black arrow indicates the extra band present in WzzB_SF^A107P non-cross-linked sample; the stars indicate the extra bands present in WzzB_SF^A107P cross-linked sample; and the diamonds indicate the ~30 kDa form of WzzB_SF that has been reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138266#pone.0138266.ref022" target="_blank">22</a>]. Each lane corresponds to ~5 x 10⁷ bacterial cells.</p

VS-COPS binding measurements.

<p>(A) Raw data for the binding between the His-tagged periplasmic domain of WzzB_SF and VS-COPS. The experiment comprised 5 steps: (a) initial baseline stabilization (30 s); (b) loading of 2 μM of His-tagged WzzB_SF to the Ni-NTA biosensor (120 s); (c) stabilization of the baseline with the reaction buffer (30 s); (d) loading of 4.5 μM VS-COPS (association, purple curve) or the reaction buffer alone (control, black curve) (60 s); and (e) wash with reaction buffer (disassociation, 60 s). The increase of signal during the loading of His-tagged WzzB_SF to the Ni-NTA sensor (Step b) indicates that the binding was effective. 4.5 μM VS-COPS gave a significant binding signal at the association step (step d), compared to the control. (B) R_eq values, indicating the biosensor signal shift induced by the binding of WzzB_SF (solid line) and WzzB_SF^A107P (dashed line) to VS-COPS at equilibrium (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138266#sec002" target="_blank">Materials and Methods</a> section), were fitted with the Hill equation for cooperative binding (Y = X^h*R_max /(X^h+K_d)), yielding a Hill coefficient of 2.6, which demonstrates positive cooperativity. The binding of His-tagged WzzB_SF^A107P and VS-COPS could not be analysed at high VS-COPS concentrations due to non-specific binding of VS-COPS to the sensor (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138266#sec002" target="_blank">Materials and Methods</a> section).</p