Proposed mechanism of intracellular H⁺ access.

<p>Cartoon depicts (a) homodimeric CLC-ec1, with subunits colored grey or white. In each subunit, Cl⁻ (green spheres) and crystallographic water molecules (blue dots) are shown. The proposed water-mediated interfacial H⁺ pathway connecting bulk intracellular water to the protein interior is indicated by blue arrows. Also shown are Glu_in (red sticks) and the serine, tyrosine, and extracellular glutamate residues that coordinate the central Cl⁻. (b) E202Y mutant, with its substituted side chain pointing out to the blocked interfacial pathway and recruiting I201_N (N denotes residue of neighboring subunit). Stick thickness represents vertical location of side chains. The polar pathway is also indicated as capped by the E117-R209 salt bridge.</p

Ion transport pathways and solvent accessibility of CLC-ec1.

<p>(a) CLC-ec1 (PDB #1OTS) is shown in surface view, with subunits of the homodimer differently colored and drawn to indicate different aspects of the antiport mechanism. Key mechanistic residues are space-filled. Bifurcated Cl⁻ and H⁺ pathways are indicated as dashed lines on right subunit. Separation of Glu_in from the intracellular solution is shown with a blue arrow, central region between Glu_in and Glu_ex with a red arrow, and the internal and central Cl⁻ ions as green spheres. In subsequent figures, the internal Cl⁻ ion is omitted, since this binds weakly and is unlikely to be directly involved in the transport mechanism. (b) Close-up view of the intracellular surface of CLC-ec1 near Glu_in. Aqueous clefts are shown as dots, and the twin subunit is shown in greyscale to visualize the subunit interface. Polar and interfacial pathways—possible routes for H⁺ access to Glu_in—are indicated with arrows.</p

Effect of E202 substitutions on Cl⁻ pathway.

<p>(a, b) Equilibrium Cl⁻ binding isotherms determined by ITC for wild type or E202Y, respectively. Solid curves represent single-site binding curves with K_D = 0.74 and 1.6 mM, respectively (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001441#pbio.1001441.s009" target="_blank">Table S3</a>). (c) Effect of E202F mutation on Cl⁻ efflux traces in fully H⁺-uncoupled transporter, E148A. For comparison, dashed lines reprise the effect of E202F on the H⁺-coupled wild type. (d) Summary of E202F effect on Cl⁻ efflux rates γ_o on coupled (WT) and uncoupled (E148A) backgrounds. Rates are normalized to the background value for comparison.</p

Structural changes in E202Y.

<p>The mutant backbone is basically unaltered from wild type (Cα rmsd 0.9 Å). (a) Structural comparison between wild-type (ΔNC) and E202Y mutant near the E202 residue. Residues are colored in <i>yellow</i> (for wild type) or <i>green</i> (for E202Y), <i>red</i> (oxygen), and <i>blue</i> (nitrogen). Cytoplasmic side view into the apex region of the interfacial pathway for wild type (b) and E202Y (c). E202_N and I201_N indicate residues coming from the neighboring subunit of the homodimer. Crystallographic water molecules are shown in <i>blue</i> dots. 2F_o-F_c maps are contoured at 1.0 σ.</p

Test of E202 mechanism in a monomeric transporter.

<p>Effects of the E202Y mutation on transport were tested on a monomeric variant of CLC-ec1 in which a double-mutant (I201W/I422W) disrupts the homodimer interface <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001441#pbio.1001441-Robertson1" target="_blank">[8]</a>. Rigorous, complete monomer formation requires phosphatidylcholine/phosphatidylglycerol liposomes, in which transport rates are 2–4-fold slower than with <i>E. coli</i> phospholipids. (a) Representative H⁺ transport traces for WT and E202Y on the monomeric background construct. (b) Comparison of inhibitory effect of E202Y substitution on H⁺ uptake by dimeric versus monomeric transporters. (c) Cl⁻/H⁺ exchange stoichiometry (3.1, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001441#pbio.1001441.s008" target="_blank">Table S2</a>) for monomeric E202Y was determined from initial rates (dashed lines).</p

Molecular Interactions between a Fluoride Ion Channel and Synthetic Protein Blockers

Fluoride ion channels of the Fluc family selectively export F^– ions to rescue unicellular organisms from acute F^– toxicity. Crystal structures of bacterial Fluc channels in complex with synthetic monobodies, fibronectin-derived soluble β-sandwich fold proteins, show 2-fold symmetric homodimers with an antiparallel transmembrane topology. Monobodies also block Fluc F^– current via a pore blocking mechanism. However, little is known about the energetic contributions of individual monobody residues to the affinity of the monobody–channel complex or whether the structural paratope corresponds to functional reality. This study seeks to structurally identify and compare residues interacting with Fluc between two highly similar monobodies and subjects them to mutagenesis and functional measurements of equilibrium affinities via a fluorescence anisotropy binding assay to determine their energetic contributions. The results indicate that the functional and structural paratopes strongly agree and that many Tyr residues at the interface, while playing a key role in affinity, can be substituted with Phe and Trp without large disruptions

Radiocarbon dates related to the phase 2 of the settlement Oppeano 4D.

Asterisks mark the dates coming from structures E and F, studied here (see Figs 2 and 5). (DOCX)</p

Archaeological structure interpreted as byre-houses in Europe (Neolithic-Middle Ages).

Archaeological sites where Neolithic to Middle Age byre-houses were identified (the relative chronology of each site refers to the one published in “References”). “Key features” indicates the proxies employed to identify the structures as byre-houses. Proxies are numbered as follows: 1) Architectural elements (see below); 2) Animal dung; 3) Animal bones; 4) Finds; 5) High P concentration; 6) Staining; 7) Cattle hoof prints; 8) Presence of a sunken floor area with organic infill; 9) Coexistence of hearth and macroscopically observed dung layers; 10) Micromorphology; 11) Macrofossils (i.e., plant remains, seeds, insects); 12) Loss-on-ignition. Following Waterbolk [151], the label “architectural elements” (number 1 in the column “Key Features”) synthetizes several features that are: transversal or longitudinal partitions, separating single or double “boxes” for the animals; structural elements typically observed in byres (i.e., thinner and shallower extra posts in the line of the roof-bearing uprights; regular dense spacing of upright pairs); ditch in the longitudinal axis of the structure to collect manure; stone floors or evidence for matting; extra posts placed at regular distances near the side wall, possibly serving for fixing the heads of the animals with a rope; door at one of the short sides of the structure. (DOCX)</p

PPL and XPL scans and interpreted version (SMT) of the studied thin sections.

For each thin section, the different scans (PPL and XPL), the subdivision in sub-units, and the interpreted version are available as overlapping layers that can be turned on and off using a PDF file reader. For a correct visualization, Adobe Acrobat is recommended. If one prefers a browser visualization, the plug-in Adobe PDF reader (free download) offers the same a correct visualization. Information on how to install and use the plug-in Adobe PDF reader can be find at this link: https://helpx.adobe.com/acrobat/using/display-pdf-in-browser.html (updated on May 4th, 2022). In Adobe PDF software and plug-in, the “layers” panel can be found and works as it follows: Choose View > Show/Hide > Navigation Panes > Layers.To hide a layer, click the eye icon. To show a hidden layer, click the empty box (a layer is visible when the eye icon is present, and hidden when the eye icon is absent).From the options menu, choose one of the following: List Layers For All Pages. Additional information can be found at this link: https://helpx.adobe.com/acrobat/using/pdf-layers.html (updated on May 4th, 2022). Choose View > Show/Hide > Navigation Panes > Layers. To hide a layer, click the eye icon. To show a hidden layer, click the empty box (a layer is visible when the eye icon is present, and hidden when the eye icon is absent). From the options menu, choose one of the following: List Layers For All Pages. Additional information can be found at this link: https://helpx.adobe.com/acrobat/using/pdf-layers.html (updated on May 4th, 2022). (PDF)</p

S1 Dataset -

(XLSX)</p