Reaction Cycle of Thermotoga maritima Copper ATPase and Conformational Characterization of Catalytically Deficient Mutants†

ABSTRACT: Copper transport ATPases sustain important roles in homeostasis of heavy metals and delivery of copper to metalloenzymes. The copper transport ATPase from Thermotoga maritima (CopA) provides a useful system for mechanistic studies, due to its heterologous expression and stability. Its sequence comprises 726 amino acids, including the N-terminal metal binding domain (NMBD), three catalytic domains (A, N, and P), and a copper transport domain formed by eight helices, including the transmembrane metal binding site (TMBS). We performed functional characterization and conformational analysis by proteolytic digestion of WT and mutated (NMBD deletion or mutation) T. maritima CopA, comparing it with Archaeoglobus fulgidus CopA and Ca2+ ATPase. A specific feature of T. maritima CopA is ATP utilization in the absence of copper, to form a low-turnover phosphoenzyme intermediate, with a conformation similar to that obtained by phosphorylation with Pi or phosphate analogues. On the other hand, formation of an activated state requires copper binding to both NMBD and TMBS, with consequent conformational changes involving the NMBD and A domain. Proteolytic digestion analysis demonstrates A domain movements similar to those of other P-type ATPases to place the conserved TGES motif in the optimal position for catalytic assistance.We also studied anH479Qmutation (analogous to one of human copper ATPase ATP7B in Wilson disease) that inhibits ATPase activity. We found that, in spite of the H479Q mutation within th

Suppressor mutation analysis combined with 3D modeling explains cohesin’s capacity to hold and release DNA

Cohesin is a fundamental protein complex that holds sister chromatids together. Separase protease cleaves a cohesin subunit Rad21/SCC1, causing the release of cohesin from DNA to allow chromosome segregation. To understand the functional organization of cohesin, we employed next-generation whole-genome sequencing and identified numerous extragenic suppressors that overcome either inactive separase/Cut1 or defective cohesin in the fission yeast Schizosaccharomyces pombe. Unexpectedly, Cut1 is dispensable if suppressor mutations cause disorders of interfaces among essential cohesin subunits Psm1/SMC1, Psm3/SMC3, Rad21/SCC1, and Mis4/SCC2, the crystal structures of which suggest physical and functional impairment at the interfaces of Psm1/3 hinge, Psm1 head–Rad21, or Psm3 coiled coil–Rad21. Molecular-dynamics analysis indicates that the intermolecular β-sheets in the cohesin hinge of cut1 suppressor mutants remain intact, but a large mobility change occurs at the coiled coil bound to the hinge. In contrast, suppressors of rad21-K1 occur in either the head ATPase domains or the Psm3 coiled coil that interacts with Rad21. Suppressors of mis4-G1326E reside in the head of Psm3/1 or the intragenic domain of Mis4. These may restore the binding of cohesin to DNA. Evidence is provided that the head and hinge of SMC subunits are proximal, and that they coordinate to form arched coils that can hold or release DNA by altering the angles made by the arched coiled coils. By combining molecular modeling with suppressor sequence analysis, we propose a cohesin structure designated the “hold-and-release” model, which may be considered as an alternative to the prevailing “ring” model

The tertiary structure of the human Xkr8–Basigin complex that scrambles phospholipids at plasma membranes

Xkr8–Basigin is a plasma membrane phospholipid scramblase activated by kinases or caspases. We combined cryo-EM and X-ray crystallography to investigate its structure at an overall resolution of 3.8 Å. Its membrane-spanning region carrying 22 charged amino acids adopts a cuboid-like structure stabilized by salt bridges between hydrophilic residues in transmembrane helices. Phosphatidylcholine binding was observed in a hydrophobic cleft on the surface exposed to the outer leaflet of the plasma membrane. Six charged residues placed from top to bottom inside the molecule were essential for scrambling phospholipids in inward and outward directions, apparently providing a pathway for their translocation. A tryptophan residue was present between the head group of phosphatidylcholine and the extracellular end of the path. Its mutation to alanine made the Xkr8–Basigin complex constitutively active, indicating that it plays a vital role in regulating its scramblase activity. The structure of Xkr8–Basigin provides insights into the molecular mechanisms underlying phospholipid scrambling

The Average Conformation at Micromolar [Ca2+] of Ca2+-ATPase with Bound Nucleotide Differs from That Adopted with the Transition State Analog ADP·AlFx or with AMPPCP under Crystallization Conditions at Millimolar [Ca 2+ ]: AMPPCP versus ADP.AlFx, and Ca ion occlusion in SERCA1a

International audienceCrystalline forms of detergent-solubilized sarcoplasmic reticulum Ca 2+-ATPase, obtained in the presence of either a substrate analog, AMPPCP, or a transition state complex, ADP.fluoroaluminate, were recently described to share the same general architecture despite the fact that, when studied in a test tube, these forms show different functional properties. Here, we show that the differences in the properties of the E1.AMPPCP and the E1.ADP.AlFx membraneous (or solubilized) forms are much less pronounced when these properties are examined in the presence of 10 mM Ca 2+ (the concentration prevailing in the crystallisation media) than when they are examined in the presence of the few µM Ca 2+ known to be sufficient to saturate the transport sites. This concerns various properties including ATPase susceptibility to proteolytic cleavage by Proteinase K, ATPase reactivity towards SH-directed Ellman's reagent, ATPase intrinsic fluorescence properties (here described for the E1.ADP.AlFx complex for the first time), and also the rates of 45 Ca 2+-40 Ca 2+ exchange at site "II". These results solve the above paradox at least partially, and suggest that the presence of a previously unrecognized Ca 2+ ion in the Ca 2+-ATPase.AMPPCP crystals should be re-investigated. A contrario, they emphasize the fact that the average conformation of the E1.AMPPCP complex under usual conditions in the test tube differs from that found in the crystalline form. The extended conformation of nucleotide revealed by the E1.AMPPCP crystalline form might be only indicative of the requirements for further processing of the complex, towards the transition state leading to phosphorylation and Ca 2+ occlusion