Structural aspects of ion pumping by Ca2+-ATPase of sarcoplasmic reticulum

Ca²⁺-ATPase of muscle sarcoplasmic reticulum is an ATP-powered Ca²⁺-pump that establishes a >10,000-fold concentration gradient across the membrane. Its crystal structures have been determined for nine different states that cover nearly the entire reaction cycle. Presented here is a brief structural account of the ion pumping process, which is achieved by a series of very large domain rearrangements.     

Intermediate phosphorylation reactions in the mechanism of ATP utilization by the copper ATPase (CopA) of Thermotoga maritima

Recombinant and purified Thermotoga maritima CopA sustains ATPase velocity of 1.78-2.73 micromol/mg/min in the presence of Cu+ (pH 6, 60 degrees C) and 0.03-0.08 micromol/mg/min in the absence of Cu+. High levels of enzyme phosphorylation are obtained by utilization of [gamma-32P]ATP in the absence of Cu+. This phosphoenzyme decays at a much slower rate than observed with Cu.E1 approximately P. In fact, the phosphoenzyme is reduced to much lower steady state levels upon addition of Cu+, due to rapid hydrolytic cleavage. Negligible ATPase turnover is sustained by CopA following deletion of its N-metal binding domain (DeltaNMBD) or mutation of NMBD cysteines (CXXC). Nevertheless, high levels of phosphoenzyme are obtained by utilization of [gamma-3)P]ATP by the DeltaNMBD and CXXC mutants, with no effect of Cu+ either on its formation or hydrolytic cleavage. Phosphoenzyme formation (E2P) can also be obtained by utilization of Pi, and this reaction is inhibited by Cu+ (E2 to E1 transition) even in the DeltaNMBD mutant, evidently due to Cu+ binding at a (transport) site other than the NMBD. E2P undergoes hydrolytic cleavage faster in DeltaNMBD and slower in CXXC mutant. We propose that Cu+ binding to the NMBD is required to produce an "active" conformation of CopA, whereby additional Cu+ bound to an alternate (transmembrane transport) site initiates faster cycles including formation of Cu.E1 approximately P, followed by the E1 approximately P to E2-P conformational transition and hydrolytic cleavage of phosphate. An H479Q mutation (analogous to one found in Wilson disease) renders CopA unable to utilize ATP, whereas phosphorylation by Pi is retained.     

Critical role of Val-304 in conformational transitions that allow Ca2+ occlusion and phosphoenzyme turnover in the Ca2+ transport ATPase

Site-directed mutations were produced in the distal segments of the Ca(2+)-ATPase (SERCA) transmembrane region. Mutations of Arg-290 (M3-M4 loop), Lys-958, and Thr-960 (M9 - M10 loop) had minor effects on ATPase activity and Ca(2+) transport. On the other hand, Val-304 (M4) mutations to Ile, Thr, Lys, Ala, or Glu inhibited transport by 90-95% while reducing ATP hydrolysis by 83% (Ile, Thr, and Lys), 56% (Ala), or 45% (Glu). Val-304 participates in Ca(2+) coordination with its main-chain carbonyl oxygen, and this function is not expected to be altered by mutations of its side chain. In fact, despite turnover inhibition, the Ca(2+) concentration dependence of residual ATPase activity remained unchanged in Val-304 mutants. However, the rates (but not the final levels) of phosphoenzyme formation, as well the rates of its hydrolytic cleavage, were reduced in proportion to the ATPase activity. Furthermore, with the Val-304 --> Glu mutant, which retained the highest residual ATPase activity, it was possible to show that occlusion of bound Ca(2+) was also impaired, thereby explaining the stronger inhibition of Ca(2+) transport relative to ATPase activity. The effects of Val-304 mutations on phosphoenzyme turnover are attributed to interference with mechanical links that couple movements of transmembrane segments and headpiece domains. The effects of thermal activation energy on reaction rates are thereby reduced. Furthermore, inadequate occlusion of bound Ca(2+) following utilization of ATP in Val-304 side-chain mutations is attributed to inadequate stabilization of the Glu-309 side chain and consequent defect of its gating function.     

Detection of porphyrin using a short peptide immobilized on a surface plasmon resonance sensor chip

In this paper the development and feasibility of a novel detection system for a low molecular weight chemical, in which a peptide was utilized as a binding molecule, are described. Surface plasmon resonance (SPR) apparatus was used as a transducer. The porphyrin binding peptide, PSP2, was used as a model peptide ligand, while a porphyrin derivative, H₂TMpyP, was used as a model low-molecular-weight chemical. PSP2 was covalently immobilized onto the SPR sensor chip and SPR measurement using the PSP2-immobilized chip for various concentrations of porphyrin was carried out. H₂TMpyP was detectable in the range from 100 ng ml⁻¹ to 10 μg ml⁻¹ with a linear correlation and good precision and the PSP2-immobilized chip could be regenerated within 1 min after measurement in this system. From comparison of the detection manners of three porphyrin derivatives, the ability of a short peptide to discriminate between differences in molecular structure was demonstrated. Moreover, the self-assembled monolayer (SAM) of PSP2 was successfully prepared on the gold substrate and H₂TMpyP could be detected using the PSP2-SAM chip.     

Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry

DNA replication is a key step in initiating cell proliferation. Loading hexameric complexes of minichromosome maintenance (MCM) helicase onto DNA replication origins during the G1 phase is essential for initiating DNA replication. Here, we examined MCM hexamer states during the cell cycle in human hTERT-RPE1 cells using multicolor immunofluorescence-based, single-cell plot analysis, and biochemical size fractionation. Experiments involving cell-cycle arrest at the G1 phase and release from the arrest revealed that a double MCM hexamer was formed via a single hexamer during G1 progression. A single MCM hexamer was recruited to chromatin in the early G1 phase. Another single hexamer was recruited to form a double hexamer in the late G1 phase. We further examined relationship between the MCM hexamer states and the methylation levels at lysine 20 of histone H4 (H4K20) and found that the double MCM hexamer state was correlated with di/trimethyl-H4K20 (H4K20me2/3). Inhibiting the conversion from monomethyl-H4K20 (H4K20me1) to H4K20me2/3 retained the cells in the single MCM hexamer state. Non-proliferative cells, including confluent cells or Cdk4/6 inhibitor-treated cells, also remained halted in the single MCM hexamer state. We propose that the single MCM hexamer state is a halting step in the determination of cell cycle progression.     

Fission Yeast Ste9, a Homolog of Hct1/Cdh1 and Fizzy-related, Is a Novel Negative Regulator of Cell Cycle Progression during G1-Phase

When proliferating fission yeast cells are exposed to nitrogen starvation, they initiate conjugation and differentiate into ascospores. Cell cycle arrest in the G₁-phase is one of the prerequisites for cell differentiation, because conjugation occurs only in the pre-Start G₁-phase. The role of ste9⁺ in the cell cycle progression was investigated. Ste9 is a WD-repeat protein that is highly homologous to Hct1/Cdh1 and Fizzy-related. The ste9 mutants were sterile because they were defective in cell cycle arrest in the G₁-phase upon starvation. Sterility was partially suppressed by the mutation in cig2 that encoded the major G₁/S cyclin. Although cells lacking Ste9 function grow normally, the ste9 mutation was synthetically lethal with the wee1 mutation. In the double mutants of ste9 cdc10^ts, cells arrested in G₁-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) did not decrease. In these cells, abortive mitosis occurred from the pre-Start G₁-phase. Overexpression of Ste9 decreased the Cdc13 protein level and the H1-histone kinase activity. In these cells, mitosis was inhibited and an extra round of DNA replication occurred. Ste9 regulates G₁ progression possibly by controlling the amount of the mitotic cyclin in the G₁-phase.     

Stabilities of Enzymes in Polyhydric Alcohols

To keep an enzyme stable in solution, the stabilities of the enzyme in various polyhydric alcohols were investigated, and it was found that the enzyme in 70% aqueous sorbitol solution was strikingly stable against both storage at 33°C and heating at 80°C. It seems reasonable to ascribe this stability to such high osmotic pressure of the sorbitol concentration that decreased the viable counts of microorganisms. The reason why the sorbitol is the most effective among various polyhydric alcohols is now being investigated. This stable enzyme solution will be of good advantage as food improver, medicines or so on.     

Structural changes in the cytoplasmic domain of phospholamban by phosphorylation at Ser16: a molecular dynamics study

Phospholamban is a 52-residue integral membrane protein that regulates the activity of the sarcoplasmic reticulum calcium pump in cardiac muscle. Its inhibitory action is relieved when phospholamban is phosphorylated at Ser16 by cAMP-dependent protein kinase. To computationally explore all possible conformations of the phosphorylated form, and thereby to understand the structural effects of phosphorylation, replica-exchange molecular dynamics (REMD) was applied to the cytoplasmic domain that includes Ser16. The simulations showed that (i) without phosphorylation, the region from Lys3 to Ser16 takes all alpha-helical conformations; (ii) when phosphorylated, the alpha-helix is partially unwound in the C-terminal part (from Ser10 to Ala15) resulting in less extended conformations; (iii) the phosphate at Ser16 forms salt bridges with Arg9, Arg13, and/or Arg14; and (iv) the salt bridges with Arg13 and Arg14 distort the alpha-helix and induce unwinding of the C-terminal part. We then applied conventional all-atom molecular dynamics simulations to the full-length phospholamban in the phospholipid bilayer. The results were consistent with those obtained with REMD simulations, suggesting that the transmembrane part of phospholamban and the lipid bilayer itself have only minor effects on the conformational changes in the cytoplasmic domain. The distortions caused by the salt bridges involving the phosphate at Ser16 readily explain the relief of the inhibitory effect of phospholamban by phosphorylation, as they will substantially reduce the population of all helical conformations, which are presumably required for the binding to the calcium pump. This will also be the mechanism for releasing the phosphorylated phospholamban from kinase.     

Concerted conformational effects of Ca2+ and ATP are required for activation of sequential reactions in the Ca2+ ATPase (SERCA) catalytic cycle

We relate solution behavior to the crystal structure of the Ca2+ ATPase (SERCA). We find that nucleotide binding occurs with high affinity through interaction of the adenosine moiety with the N domain, even in the absence of Ca2+ and Mg2+, or to the closed conformation stabilized by thapsigargin (TG). Why then is Ca2+ crucial for ATP utilization? The influence of adenosine 5'-(beta,gamma-methylene) triphosphate (AMPPCP), Ca2+, and Mg2+ on proteolytic digestion patterns, interpreted in the light of known crystal structures, indicates that a Ca2+-dependent conformation of the ATPase headpiece is required for a further transition induced by nucleotide binding. This includes opening of the headpiece, which in turn allows inclination of the "A" domain and bending of the "P" domain. Thereby, the phosphate chain of bound ATP acquires an extended configuration allowing the gamma-phosphate to reach Asp351 to form a complex including Mg2+. We demonstrate by Asp351 mutation that this "productive" conformation of the substrate-enzyme complex is unstable because of electrostatic repulsion at the phosphorylation site. However, this conformation is subsequently stabilized by covalent engagement of the -phosphate yielding the phosphoenzyme intermediate. We also demonstrate that the ADP product remains bound with high affinity to the transition state complex but dissociates with lower affinity as the phosphoenzyme undergoes a further conformational change (i.e., E1-P to E2-P transition). Finally, we measured low-affinity ATP binding to stable phosphoenzyme analogues, demonstrating that the E1-P to E2-P transition and the enzyme turnover are accelerated by ATP binding to the phosphoenzyme in exchange for ADP.