Chikashi Toyoshima

Significance: During phosphoryl transfer from ATP in P-type ion translocating ATPases, very large-scale and complex structural changes take place, resulting in occlusion of ions to be translocated. In the present report, we describe a new crystal structure of a calcium pump in the absence of Ca ²⁺ but with a bound ATP analogue. We show that the ATP analogue is delivered to the phosphorylation site with a correct geometry despite the absence of Ca ²⁺ . Yet phosphoryl transfer from ATP never occurs in the absence of Ca ²⁺ , although phosphorylation by P _i is possible as a backward reaction. We describe the structural changes that ATP binding alone elicits in the calcium pump and why Ca ²⁺ binding is absolutely necessary for phosphoryl transfer.

Ca ²⁺ -ATPase of sarcoplasmic reticulum (SERCA1a) pumps two Ca ²⁺ per ATP hydrolyzed from the cytoplasm and two or three protons in the opposite direction. In the E2 state, after transferring Ca ²⁺ into the lumen of sarcoplasmic reticulum, all of the acidic residues that coordinate Ca ²⁺ are thought to be protonated, including the gating residue Glu309. Therefore a Glu309Gln substitution is not expected to significantly perturb the structure. Here we report crystal structures of the Glu309Gln and Glu309Ala mutants of SERCA1a under E2 conditions. The Glu309Gln mutant exhibits, unexpectedly, large structural rearrangements in both the cytoplasmic and transmembrane domains, apparently uncoupling them. However, the structure definitely represents E2 and, together with the help of quantum chemical calculations, allows us to postulate a mechanism for the E2 → E1 transition triggered by deprotonation of Glu309.

Ca²⁺‐ATPase of sarcoplasmic reticulum is known to pump Mn²⁺ in addition to Ca²⁺, but whether its transport mechanism is identical to that of Ca²⁺ is ambiguous. To clarify, we examined, by atomic absorption spectroscopy, competition between Mn²⁺ and Ca²⁺ in active transport using vesicles of sarcoplasmic reticulum (SR). Here, we demonstrate that Ca²⁺‐ATPase transports Ca²⁺ and Mn²⁺ concomitantly but has a much lower affinity for Mn²⁺ (apparent K_d ~ 0.5 mm). Stoichiometries of transported ions per ATP hydrolysed, V_max values and activation energies are very similar. Altogether, Ca²⁺‐ATPase appears to use the same mechanism for transporting Mn²⁺ as that for Ca²⁺.

Na⁺,K⁺-ATPase transfers three Na⁺ from the cytoplasm into the extracellular medium and two K⁺ in the opposite direction per ATP hydrolysed. The binding and release of Na⁺ and K⁺ are all thought to occur sequentially. Here we demonstrate by X-ray crystallography of the ATPase in E2·MgF₄²⁻·2K⁺, a state analogous to E2·Pi·2K⁺, combined with isotopic measurements, that the substitution of the two K⁺ with congeners in the extracellular medium indeed occurs at different rates, substantially faster at site II. An analysis of thermal movements of protein atoms in the crystal shows that the M3–M4E helix pair opens and closes the ion pathway leading to the extracellular medium, allowing K⁺ at site II to be substituted first. Taken together, these results indicate that site I K⁺ is the first cation to bind to the empty cation-binding sites after releasing three Na⁺.

Biselyngbyasides (BLSs), macrolides from a marine cyanobacterium, are cytotoxic natural products whose target molecule is unknown. Here we report that BLSs are high affinity (K _i ∼ 10 nM) inhibitors of Ca²⁺‐pumps with a unique binding mode. The crystal structures of the Ca²⁺‐pump in complex with BLSs at 3.2–3.5 Å‐resolution show that BLSs bind to the pump near the cytoplasmic surface of the transmembrane region. The crystal structures and activity measurement of BLS analogs allow us to identify the structural features that confer high potency to BLSs as inhibitors of the pump.

Significance: Electron crystallography has the potential to analyze crystals of membrane proteins and macromolecular complexes too small or too thin for X-ray crystallography, as electrons are scattered four to five orders of magnitude more strongly than X-rays. Electron crystallography yields Coulomb potential maps, rather than electron density maps as X-rays do, providing information on charged states of amino acids and metals. Here we present such Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, of Ca ²⁺ -ATPase and catalase obtained from crystals of just a few layers thick. These maps demonstrate that it is indeed possible to build atomic models from such crystals and charge information is included, often critical in understanding protein function.