To investigate the role of protons in the cooperative mechanism of human hemoglobin, the thermodynamic linkage between stepwise oxygen binding and dimer-tetramer assembly was studied over the pH range 7.4-9.5 at 21.5 OC. At each pH, oxygen binding isotherms were measured at a series of protein concentrations. These data were analyzed for microscopic free energies of the constituent reactions according to a model-independent thermodynamic treatment [Ackers, G. K., & Halvorson, H. R. (1974) Proc. Natl. Acad. Sci. U.S.A. 74,43 12-43 161. The analysis incorporated equilibrium constants for the assembly of unliganded and fully oxygenated species that were independently determined under identical conditions [Chu, A. H., & Ackers, G. K. (1981) J. Biol. Chem. 256, 1 199-1 2051.From the pH dependencies of the derived equilibrium constants of the linkage system, we have calculated the apparent changes in proton binding that accompany all the reactions of subunit assembly and oxygen binding. The tetramer Bohr effect was also analyzed according to a thermodynamic treatment based on integral relationships between the linked functions. Principal results are as follows: (1) At x e human hemoglobin molecule changes its affinity for protons and oxygen at each successive oxygenation step. A major goal of hemoglobin research is to understand the molecular mechanism of these coupled self-regulatory processes. Since hemoglobin operates essentially as an equilibrium thermodynamic system in vivo, the problem of delineating its structure-function relationships becomes one of establishing relationships between changes in thermodynamic and structural properties that accompany the molecule's functional cycle of oxygenation-deoxygenation. Detailed knowledge of the relationships between the energetic transitions and functional events (e.g., O2 binding, proton binding) provides important constraints on all mechanistic theories of hemoglobin action.The self-regulation properties of hemoglobin are mediated through interactions at the intersubunit contacts of the tetrameric molecule. Subunit dissociation has therefore proven to be a powerful quantitative tool for probing the regulatory energy changes that accompany the molecule's functional cycle. The rationale for this approach is as follows: (1) Interactions within the tetrameric molecule which are responsible for cooperativity can be decoupled by dissociation of the tetramers into noncooperative dimers. (2) The difference between energies of dimer-tetramer assembly at any two stages of oxygenation provides a measure of how much the all pH values, a major fraction of the intersubunit interaction energy, which "pays" for cooperativity in oxygen binding, is "spent" at the first oxygenation step. Changes occur at every pH in the intersubunit contact energy between four stages of oxygenation: unliganded, singly, triply, and fully oxygenated.(2) The quaternary enhancement effect, previously found at pH 7.4 over a wide temperature range [Mills, F. C., & Ackers, G. K. (1979) J . Biol. Chem. 254,2881-...

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity.

Highlights d Crystal structures reveal binding site for Latrophilin on the Teneurin YD shell d A ternary Latrophilin-Teneurin-FLRT complex forms in vitro and in vivo d Latrophilin controls cortical migration by binding to Teneurins and FLRTs d Latrophilin elicits repulsion of cortical cell bodies/small neurites but not axons

SummaryProtein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic “lipid-altered” tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.