P-type ion transporting ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes. Transfer of bound cations to the lumenal or extracellular side occurs while the ATPase is phosphorylated. Here we report at 2.3 A resolution the structure of the calcium-ATPase of skeletal muscle sarcoplasmic reticulum, a representative P-type ATPase that is crystallized in the absence of Ca2+ but in the presence of magnesium fluoride, a stable phosphate analogue. This and other crystal structures determined previously provide atomic models for all four principal states in the reaction cycle. These structures show that the three cytoplasmic domains rearrange to move six out of ten transmembrane helices, thereby changing the affinity of the Ca2+-binding sites and the gating of the ion pathway. Release of ADP triggers the opening of the lumenal gate and release of phosphate its closure, effected mainly through movement of the A-domain, the actuator of transmembrane gates.

The inhibitory interaction of phospholamban (PLN) with the sarco(endo)plasmic reticulum Ca 2؉ ATPase isoform 1 (SERCA1a) was modeled on the basis of several constraints which included (i) spontaneous formation of SS-bridges between mutants L321C in transmembrane helix 4 (M4) of SERCA1a and N27C in PLN and between V89C (M4) and V49C (PLN); (ii) definition of the face of the PLN transmembrane helix that interacts with SERCA; (iii) crosslinking between Lys-3 of PLN and Lys-397 and Lys-400 of SERCA2a. The crystal structure of SERCA1a in the absence of Ca 2؉ , which binds PLN, was used as the structure into which an atomic model of PLN was built. PLN can fit into a transmembrane groove formed by the juxtaposition of M2, the upper part of M4, M6, and M9. In the SERCA1a structure with bound Ca 2؉ , this groove is closed, accounting for the ability of Ca 2؉ to disrupt PLN-SERCA interactions. Near the cytoplasmic surface of the bilayer, the PLN helix is disrupted to prevent its collision with M4. The model can be extended into the cytoplasmic domain so that Lys-3 in PLN can be cross-linked with Lys-397 and Lys-400 in SERCA1a with little unwinding of the N-terminal helix of PLN. S arco(endo)plasmic reticulum Ca 2ϩ ATPases (SERCAs) have proven to be exceptionally useful models for the investigation of structure-function relationships and the mechanism of action of P-type cation pumps (1-4). SERCA1a is also the first P-type ATPase for which high-resolution crystal structures have been presented in both E1 (5) and E2 (6) conformations. The high-resolution structures have not only defined the gross movements of the major actuator (A), nucleotide binding (N), phosphorylation (P), and Ca 2ϩ binding domains, but have provided precise information on the relationship among individual amino acids and helices.The activity of SERCA2a is regulated by phospholamban (PLN) (7,8), a member of a family of low-molecular-mass transmembrane proteins that includes sarcolipin (SLN) (9-11). Because PLN has been demonstrated to be a major regulator of the kinetics of cardiac contractility through its effects on SERCA2a in the heart (12, 13), it is a potential drug target for management of cardiac function. Accordingly, it has been of great interest to determine the sites of interaction between PLN and SERCA2a or other SERCA isoforms.James et al. (14) cross-linked PLN to SERCA2a by using a cross-linking agent (the Denny-Jaffe reagent) to show that Lys-3 of PLN lies within 15 Å of both Lys-397 and Lys-400 in the sequence Lys-Asp-Asp-Lys-400 in what is now recognized as the cytosolic N domain of SERCA2a. Studies using chimera formation and mutagenesis confirmed that the sequence, LysAsp-Asp-Lys-Pro-Val-402 is involved in PLN-SERCA2a interactions (15). Further studies then showed that a series of charged and hydrophobic amino acids between Glu-2 and Ile-18 in PLN were also mutation-sensitive in the assay used for measurement of Ca 2ϩ affinity of SERCA2a (16). The C-terminal region of PLN, involving either Leu-28-Leu-52 or Asn-30-Leu-52 fused to a var...

For the p53 Special IssueOf the various genetic alterations in lung cancer, the abnormalities of the TP53 gene (p53) are among the most frequent and important events. Because of its importance, many aspects of TP53 have been studied, including preneoplastic lesions and TP53 as a marker for early detection and prognosis and as a therapeutic option. We summarize recent knowledge of TP53 in lung cancer with a special emphasis on the relationship between smoking exposure (e.g, cigarette, etc.) and specific mutational pattern of TP53 by analyzing the latest version of the International Agency for Research on Cancer (IARC) database on TP53 mutations in human cancer. Our analysis confirmed several other studies showing significant differences in the frequencies of G:C to T:A transversions between ever-smokers and never-smokers. Furthermore, when comparing the mutational spectrum by gender, important differences were noted between male and female never-smokers. We concluded that the previously noted G:C to T:A transversions were mainly due to female smokers having a high frequency of these changes compared to female never-smokers. There was no relationship between adenocarcinomas and squamous cell carcinomas independent of gender. We also examined the seven codons which have been previously identified as hot spots, that is, the sites of frequent G:C to T:A transversions in smoking-related lung cancers. However, there was no specific codon which was strongly related to smoke exposure despite a moderate relationship. We considered the term ''warmspot'' may be more appropriate. While mutations of TP53 are frequent in lung cancers, further investigation is necessary to understand their role for lung carcinogenesis, especially as they relate to gender differences, and to translate our laboratory knowledge to clinical applications. Hum Mutat 21:229-239,

Ca 2؉ -ATPase of skeletal muscle sarcoplasmic reticulum is the beststudied member of the P-type or E1/E2 type ion transporting ATPases. It has been crystallized in seven different states that cover nearly the entire reaction cycle. crystal structure ͉ phosphorylation ͉ P-type ATPase C a 2ϩ -ATPase from skeletal muscle sarcoplasmic reticulum (SERCA1a) is an ATP-powered calcium pump that transfers Ca 2ϩ from the cytoplasm to the lumen of sarcoplasmic reticulum against a Ͼ10 4 concentration gradient. It is an integral membrane protein of M r 110K (1), and it pumps ions by alternating the affinity of the transmembrane (TM) binding sites and synchronizing opening and closing of the cytoplasmic and luminal gates. According to the conventional E1-E2 theory (2-4), E1 and E2 respectively refer to high-affinity and low-affinity states to Ca 2ϩ . Gating of the ion pathway is coupled to autophosphorylation and dephosphorylation of the ATPase. Phosphoryl transfer from ATP to an Asp in the cytoplasmic domain (i.e., E1⅐2Ca 2ϩ 3 E1P; here P stands for phosphorylated state) closes the cytoplasmic gate, and the release of ADP triggers a change in affinity of the Ca 2ϩ binding sites (i.e., E1P 3 E2P) and opening of the luminal gate. Hydrolysis of the aspartylphosphate (E2P 3 E2) closes the gate.