Extensive conformational transitions are required to turn on ATP hydrolysis in myosin

Conventional myosin is representative of biomolecular motors in which the hydrolysis of adenosine triphosphate (ATP) is coupled to large-scale structural transitions both in and remote from the active site. The mechanism that underlies such “mechanochemical coupling,” especially the causal relationship between hydrolysis and allosteric structural changes, has remained elusive despite extensive experimental and computational analyses. In this study, using combined quantum mechanical and molecular mechanical simulations and different conformations of the myosin motor domain, we provide evidence to support that regulation of ATP hydrolysis activity is not limited to residues in the immediate environment of the phosphate. Specifically, we illustrate that efficient hydrolysis of ATP depends not only on the proper orientation of the lytic water but also on the structural stability of several nearby residues, especially the Arg238-Glu459 salt bridge (the numbering of residues follows myosin II in Dictyostelium discoideum) and the water molecule that spans this salt bridge and the lytic water. More importantly, by comparing the hydrolysis activities in two motor conformations with very similar active-site (i.e., Switches I and II) configurations, which distinguished this work from our previous study, the results clearly indicate that the ability of these residues to perform crucial electrostatic stabilization relies on the configuration of residues in the nearby N-terminus of the relay helix and the “wedge loop.” Without the structural support from those motifs, residues in a closed active site in the post-rigor motor domain undergo subtle structural variations that lead to consistently higher calculated ATP hydrolysis barriers than in the pre-powerstroke state. In other words, starting from the post-rigor state, turning on the ATPase activity requires not only displacement of Switch II to close the active site but also structural transitions in the N-terminus of the relay helix and the “wedge loop,” which have been proposed previously to be ultimately coupled to the rotation of the converter subdomain 40 Å away.     

An entirely cell-based system to generate single-chain antibodies against cell surface receptors

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.     

Mutational analysis of the stability of the H2A and H2B histone monomers

The eukaryotic histone heterodimer H2A-H2B folds through an obligatory dimeric intermediate that forms in a nearly diffusion-limited association reaction in the stopped-flow dead time. It is unclear whether there is partial folding of the isolated monomers before association. To address the possible contributions of structure in the monomers to the rapid association, we characterized H2A and H2B monomers in the absence of their heterodimeric partner. By far-UV circular dichroism, the H2A and H2B monomers are 15% and 31% helical, respectively—significantly less than observed in X-ray crystal structures. Acrylamide quenching of the intrinsic Tyr fluorescence was indicative of tertiary structure. The H2A and H2B monomers exhibit free energies of unfolding of 2.5 and 2.9 kcal mol^− 1, respectively; at 10 μM, the sum of the stability of the monomers is ∼ 60% of the stability of the native dimer. The helical content, stability, and m values indicate that H2B has a more stable, compact structure than H2A. The monomer m values are larger than expected for the extended histone fold motif, suggesting that the monomers adopt an overly collapsed structure. Stopped-flow refolding—initiated from urea-denatured monomers or the partially folded monomers populated at low denaturant concentrations—yielded essentially identical rates, indicating that monomer folding is productive in the rapid association and folding of the heterodimer. A series of Ala and Gly mutations were introduced into H2A and H2B to probe the importance of helix propensity on the structure and stability of the monomers. The mutational studies show that the central α-helix of the histone fold, which makes extensive intermonomer contacts, is structured in H2B but only partially folded in H2A.     

Structural basis of the honey bee PBP pheromone and pH-induced conformational change

The behavior of insects and their perception of their surroundings are driven, in a large part, by odorants and pheromones. This is especially true for social insects, such as the honey bee, where the queen controls the development and the caste status of the other individuals. Pheromone perception is a complex phenomenon relying on a cascade of recognition events, initiated in antennae by pheromone recognition by a pheromone-binding protein and finishing with signal transduction at the axon membrane level. With to the objective of deciphering this initial step, we have determined the structures of the bee antennal pheromone-binding protein (ASP1) in the apo form and in complex with the main component of the queen mandibular pheromonal mixture, 9-keto-2(E)-decenoic acid (9-ODA) and with nonpheromonal components. In the apo protein, the C terminus obstructs the binding site. In contrast, ASP1 complexes have different open conformations, depending on the ligand shape, leading to different volumes of the binding cavity. The binding site integrity depends on the C terminus (111-119) conformation, which involves the interplay of two factors; i.e. the presence of a ligand and a low pH. Ligand binding to ASP1 is favored by low pH, opposite to what is observed with other pheromone-binding proteins, such as those of Bombyx mori and Anopheles gambiae.     

Solution structure of NEMO zinc finger and impact of an anhidrotic ectodermal dysplasia with immunodeficiency-related point mutation

The regulatory NEMO (NF-κB essential modulator) protein has a crucial role in the canonical NF-κB signaling pathway notably involved in immune and inflammatory responses, apoptosis and oncogenesis. The regulatory domain is located in the C-terminal half of NEMO and contains a classical CCHC-type zinc finger (ZF). We have investigated the structural and functional effects of a cysteine to phenylalanine point mutation (C417F) in the ZF motif, identified in patients with anhidrotic ectodermal dysplasia with immunodeficiency. The solution structures of the wild type and mutant ZF were determined by NMR. Remarkably, the mutant adopts a global ββα fold similar to that of the wild type and retains thermodynamic stability, i.e., the ability to bind zinc with a native-like affinity, although the last zinc-chelating residue is missing. However, the mutation induces enhanced dynamics in the motif and leads to an important loss of stability. A detailed analysis of the wild type solution structure and experimental evidences led to the identification of two possible protein-binding surfaces that are largely destabilized in the mutant. This is sufficient to alter NEMO function, since functional complementation assays using NEMO-deficient pre-B and T lymphocytes show that full-length C417F pathogenic NEMO leads to a partial to strong defect in LPS, IL-1β and TNF-α-induced NF-κB activation, respectively, as compared to wild type NEMO. Altogether, these results shed light onto the role of NEMO ZF as a protein-binding motif and show that a precise structural integrity of the ZF should be preserved to lead to a functional protein-recognition motif triggering full NF-κB activation.     

Early Crop Root Destruction for Management of Tobacco Cyst Nematodes

Prompt tillage after crop harvest was investigated as a cultural control for the tobacco cyst nematode, Globodera tabacum tabacum, on stalk-cut broadleaf cigar wrapper tobacco. Stalk stumps and roots remaining after harvest were destroyed by tilling immediately or from 2 to 6 wk after harvest in field experiments over 4 yr. Cyst nematode Pf/Pi ratios ranged from 0.65 to 1.62 when plants were tilled immediately after harvest and 1.13 to 5.88 when tillage was delayed. Nematode population development was monitored by inoculating plants in pots placed in fields with J2 in eggs and sampling over time (8 to 18 wk). Three generations per year were observed, and G. t. tabacum generation time was as short as 6 wk for each generation. Destroying stalks and root systems remaining after harvesting stalk-cut broadleaf cigar wrapper tobacco removes the host to preclude development of nematodes at the end of the second and entire third generation. Early tillage resulted in consistently lower tobacco cyst nematode populations than allowing viable roots to remain in fields for an additional 8 to 18 wk. This management tactic reduces the need for nematicide application to slow nematode population increases over time and can reduce losses due to infection by G. t. tabacum.     

Shallow-soil endemics: adaptive advantages and constraints of a specialized root-system morphology

Worldwide, many rare plant species occur in shallow-soil, drought-prone environments. For most of these species, the adaptations required to be successful in their own habitats, as well as their possible consequences for establishment and persistence in others, are unknown. Here, two rare Hakea (Proteaceae) species confined to shallow-soil communities in mediterranean-climate south-western Australia were compared with four congeners commonly occurring on deeper soils. Seedlings were grown for 7 months in a glasshouse in individual 1.8 x 0.2-m containers, to allow for unconstrained root development. In addition, a reciprocal transplant experiment was carried out. The rare Hakea species differed consistently from their common congeners in their spatial root placement. They invested more in deep roots and explored the bottom of the containers much more quickly. In the reciprocal transplant experiment they showed increased survival in their own habitat, but not in others. This research suggests that shallow-soil endemics have a specialized root system that allows them to explore a large rock surface area, thereby presumably increasing their chance to locate cracks in the underlying rock. However, this root-system morphology may be maladaptive on deeper soils, providing a possible explanation for the restricted distribution of many shallow-soil endemics.