Ca 2؉ -ATPase of sarcoplasmic reticulum is an ATP-powered Ca 2؉ pump but also a H ؉ pump in the opposite direction with no demonstrated functional role. Here, we report a 2.4-Å-resolution crystal structure of the Ca 2؉ -ATPase in the absence of Ca 2؉ stabilized by two inhibitors, dibutyldihydroxybenzene, which bridges two transmembrane helices, and thapsigargin, also bound in the membrane region. Now visualized are water and several phospholipid molecules, one of which occupies a cleft between two transmembrane helices. Atomic models of the Ca 2؉ binding sites with explicit hydrogens derived by continuum electrostatic calculations show how water and protons fill the space and compensate charge imbalance created by Ca 2؉ -release. They suggest that H ؉ countertransport is a consequence of a requirement for maintaining structural integrity of the empty Ca 2؉ -binding sites. For this reason, cation countertransport is probably mandatory for all P-type ATPases and possibly accompanies transport of water as well.2ϩ -ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1a), an integral membrane protein consisting of 994 aa (1), transfers two Ca 2ϩ from the cytoplasm into the lumen of sarcoplasmic reticulum per ATP hydrolyzed and thereby establishes a Ͼ10 4 concentration gradient across the membrane (2). At the same time, Ca 2ϩ -ATPase pumps two or three H ϩ in the opposite direction (3-6) during the reaction cycle. According to the classical E1͞E2 theory (7-9), transmembrane ion-binding sites have high affinity for Ca 2ϩ and face the cytoplasm in E1, whereas they have low affinity and face the lumen of sarcoplasmic reticulum in E2. The opposite applies to H ϩ , which binds to the ATPase in E2 and dissociates in E1, presumably in exchange with Ca 2ϩ . As Na ϩ K ϩ -ATPase and gastric H ϩ K ϩ -ATPase countertransport K ϩ , instead of H ϩ , countertransport of monovalent cations may be a common feature of the P-type ATPase superfamily (2, 10), of which SERCA1a is the best-studied member (11,12). However, the sarcoplasmic reticulum membrane is leaky to monovalent cations including H ϩ (13). As has been pointed out before (14), the membrane potential and pH gradient must be minimized to achieve such a large concentration gradient of Ca 2ϩ . Therefore, the physiological role of H ϩ countertransport has been a puzzle. Although protonation of carboxyls in the Ca 2ϩ -binding site has been suggested from biochemical (15, 16) and mutagenesis studies (17, 18), crystal structures of SERCA1a in various states (19-24) did not clarify the role of countertransport or identify protonation sites.These questions, however, can be addressed computationally by calculating stabilization energy provided by H ϩ binding (25)(26)(27). Protonation probability of a particular residue is related directly to the free energy difference between protonated and unprotonated forms. Such calculations, therefore, should be able to identify residues likely to be protonated in crystal structures and were indeed successful (28) for a Ca 2ϩ -bound form [E1⅐2C...

Aggregation Amyloid β (Aβ) peptide has been linked to the neurodegenerative Alzheimer's Disease and implicated in other amyloid diseases including cerebral amyloid angiopathy. Aβ peptide is generated by cleavage of the amyloid precursor protein (APP) by transmembrane proteases. It is crucial to determine the structures of β-amyloid peptides in a membrane to provide a molecular basis for the cleavage mechanism. We report the structures of amyloid β peptide (Aβ 1-40 and Aβ 1-42 ) as well as the 672-726 fragment of APP (referred to as Aβ 1-55 ) in a membrane environment determined by replica-exchange molecular dynamics simulation. Aβ 1-40 is found to have two helical domains A (13-22) and B(30-35) and a type I β turn at 23-27. The peptide is localized at the interface between membrane and solvent. Substantial fluctuations in domain A are observed. The dominant simulated tertiary structure of Aβ 1-40 is observed to be similar to the simulated Aβ 1-42 structure. However, there are differences observed in the overall conformational ensemble as characterized by the twodimensional free energy surfaces. The fragment of APP (Aβ 1-55 ) is observed to have a long transmembrane helix. The position of the transmembrane region and ensemble of membrane structures are elucidated. The conformational transition between the transmembrane Aβ 1-55 structure, prior to cleavage, and the Aβ 1-40 structure, following cleavage, is proposed.