A structural analysis of the AAA+ domains in Saccharomyces cerevisiae cytoplasmic dynein

AbstractDyneins are large protein complexes that act as microtubule based molecular motors. The dynein heavy chain contains a motor domain which is a member of the AAA+ protein family (ATPases Associated with diverse cellular Activities). Proteins of the AAA+ family show a diverse range of functionalities, but share a related core AAA+ domain, which often assembles into hexameric rings. Dynein is unusual because it has all six AAA+ domains linked together, in one long polypeptide. The dynein motor domain generates movement by coupling ATP driven conformational changes in the AAA+ ring to the swing of a motile element called the linker. Dynein binds to its microtubule track via a long antiparallel coiled-coil stalk that emanates from the AAA+ ring. Recently the first high resolution structures of the dynein motor domain were published. Here we provide a detailed structural analysis of the six AAA+ domains using our Saccharomyces cerevisiae crystal structure. We describe how structural similarities in the dynein AAA+ domains suggest they share a common evolutionary origin. We analyse how the different AAA+ domains have diverged from each other. We discuss how this is related to the function of dynein as a motor protein and how the AAA+ domains of dynein compare to those of other AAA+ proteins

Evidence for a Two-Metal-Ion Mechanism in the Cytidyltransferase KdsB, an Enzyme Involved in Lipopolysaccharide Biosynthesis

Lipopolysaccharide (LPS) is located on the surface of Gram-negative bacteria and is responsible for maintaining outer membrane stability, which is a prerequisite for cell survival. Furthermore, it represents an important barrier against hostile environmental factors such as antimicrobial peptides and the complement cascade during Gram-negative infections. The sugar 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) is an integral part of LPS and plays a key role in LPS functionality. Prior to its incorporation into the LPS molecule, Kdo has to be activated by the CMP-Kdo synthetase (CKS). Based on the presence of a single Mg2+ ion in the active site, detailed models of the reaction mechanism of CKS have been developed previously. Recently, a two-metal-ion hypothesis suggested the involvement of two Mg2+ ions in Kdo activation. To further investigate the mechanistic aspects of Kdo activation, we kinetically characterized the CKS from the hyperthermophilic organism Aquifex aeolicus. In addition, we determined the crystal structure of this enzyme at a resolution of 2.10 Å and provide evidence that two Mg2+ ions are part of the active site of the enzyme

Effect of combination treatment on lung volumes and exercise endurance time in COPD

SummaryBackgroundData comparing two bronchodilators vs. one bronchodilator plus inhaled corticosteroid (ICS) on hyperinflation and exercise endurance in chronic obstructive pulmonary disease (COPD) are scarce, though these therapeutic strategies are widely used in clinical practice.MethodsWe performed a randomized, crossover clinical trial of two × 8 weeks comparing tiotropium (18 μg once daily) + salmeterol (50 μg twice daily) (T + S) to salmeterol + fluticasone (50/500 μg twice daily) (S + F) in COPD (forced expiratory volume in 1 s (FEV1) ≤65% predicted, and thoracic gas volume (TGV) ≥120% predicted). Coprimary endpoints were postbronchodilator TGV and exercise endurance time (EET).ResultsIn 309 patients, at baseline, prebronchodilator FEV1 was 1.36 L (46% predicted), TGV was 5.42 L (165% predicted), and EET = 458 s. Relative to S + F, T + S lowered postdose TGV by 182 ± 44 ml after 4 weeks (p < 0.0001) and 87 ± 44 ml after 8 weeks (p < 0.05). EET was nonsignificantly increased following T + S treatment (20 ± 15 s at 4 weeks, 15 ± 13 s at 8 weeks) vs. S + F. BORG dyspnea score at exercise isotime was reduced in favor of T + S.ConclusionThe two bronchodilators decreased hyperinflation significantly more than one bronchodilator and ICS. This difference was not reflected in EET.(ClinicalTrials.gov number, NCT00530842

The cryoEM structure of the S<i>accharomyces cerevisiae</i> ribosome maturation factor Rea1

The biogenesis of 60S ribosomal subunits is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualise the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide-induced conformational changes that create a docking site for the substrate binding MIDAS domain on the AAA +ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors

In vitro characterization of the full-length human dynein-1 cargo adaptor BicD2

International audienceCargo adaptors are crucial in coupling motor proteins with their respective cargos and regulatory proteins. BicD2 is a prominent example within the cargo adaptor family. BicD2 is able to recruit the microtubule motor dynein to RNA, viral particles and nuclei. The BicD2-mediated interaction between the nucleus and dynein is implicated in mitosis, interkinetic nuclear migration (INM) in radial glial progenitor cells, and neuron precursor migration during embryonic neocortex development. In vitro studies involving full-length cargo adaptors are difficult to perform due to the hydrophobic character, low-expression levels, and intrinsic flexibility of cargo adaptors. Here we report the recombinant production of full-length human BicD2 and confirm its biochemical activity by interaction studies with RanBP2. We also describe pH-dependent conformational changes of BicD2 using cryoEM, template-free structure predictions, and biophysical tools. Our results will help define the biochemical parameters for the in vitro reconstitution of higher order BicD2 protein complexes

Modelled ternary AA-LCKS-CTP-Kdo complex.

<p>The CTP and Kdo substrates are shown in stick representation with cyan carbon and purple phosphate atoms (CTP) or blue carbon atoms (Kdo). Water molecules as well as the Mg²⁺ ions are highlighted as spheres in red and green, respectively. Green dashed lines indicate metal ligand interactions. The side chains of D219 and D95 are depicted as sticks with orange carbon atoms, and nitrogen and oxygen atoms are colored in blue and red, respectively. The black arrow points at steric clashes between water ligands of Mg-A and the hydroxyl and carboxyl groups of the Kdo.</p

Two-metal-ion mechanism.

<p>(A) Schematic cartoon illustration of a two-metal-ion mechanism as exemplified by nucleic-acid polymerases (green arrows). The two Mg²⁺ ions (yellow) are highlighted as circles and interactions with surrounding ligands are depicted as dashed red lines. R₁ represents the nucleoside moiety of the nucleotide triphosphate (blue), R₂ symbolizes the growing 3′-end of a DNA or RNA chain (orange), and E corresponds to the protein environment. The interaction between the 3′-hydroxyl group of the terminal nucleotide and Mg-A lowers the pK_a-value of the ligand and allows for its deprotonation by a general base designated B. Both Mg²⁺ ions share a common α-phosphate oxygen ligand, an interaction also maintained during the pentacovalent transition state that results after the nucleophilic attack onto the α-phosphate accomplished by the deprotonated 3′-hydroxyl group. As indicated by the purple dashed line, a basic amino-acid residue might act as general acid and protonate the additional negative charge at the β-phosphate group. (B) Schematic drawing of the proposed two-metal-ion mechanism in CMP-Kdo synthetases. R₃ represents the cytidine moiety of the CTP and R₄ symbolizes the C₈H₁₃O₇ portion of the Kdo sugar. The symbol and color scheme is the same as in (A). Amino-acid residues are labeled according to AA-LCKS nomenclature. Consistent with (A), the C2 hydroxyl group of the Kdo might coordinate to Mg-A, which could facilitate its subsequent deprotonation, and K19 might play the role of a general acid. In contrast to the situation in nucleic acid polymerases and as evident from the AA-LCKS crystal structure, the two metal ions do not share a common oxygen ligand and Mg-B is not in contact with the protein environment.</p

Data collection, phasing, and refinement statistics.

<p>*The values in the parentheses refer to the highest resolution shell. ^aR_sym (I)  =  Σ_hklΣ_i |<i>I_i(hkl)</i> − <<i>I(hkl)</i>>|/ Σ_hklΣ <i>I_i(hkl)</i> for <i>n</i> independent reflections and <i>i</i> observations of a given reflection. <<i>I(hkl)</i>> is the average intensity of the <i>i</i> observations. ^b<i>/<σI>, where <i> is the average intensity and <σI> is the average intensity standard deviation. ^cPhasing power  =  (<i>F</i>_H(calc)/<i>E</i>), where <i>F</i>_H(calc) is the calculated heavy-atom structure factor and <i>E</i> is the estimated lack-of-closure error (isomorphous/anomalous). ^dFOM  =  mean figure of merit. ^eR_work  =  Σ_hkl || F(obs) | − <i>k</i> | F(calc) || / Σ_hkl | F(obs) |. ^fR_free is calculated analogous to R_work using 5% of the X-ray data, randomly selected for cross-validation. ^gr.m.s.  =  Root-mean-square.</i></i></p

CTP-hydrolysis protection in AA-LCKS.

<p>CTP, water molecules, the Mg²⁺ ions as well as D95 and D219 are represented as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023231#pone-0023231-g003" target="_blank">Figure 3</a>. Green dashed lines indicate metal-ligand interactions. The protein is shown in orange in cartoon and transparent surface representation. The transparent sphere around the C5′-methylene group features the 1.7-Å van-der-Waals radius of a carbon atom. Small nucleophiles have to access the α-phosphate from the groove between the N- and C-terminal domains, as indicated by the black arrows, and are therefore forced to pass the hydrophobic C5′-methylene group, which might lead to unfavorable interactions preventing a subsequent nucleophilic attack. </p