Mutagenesis of Segment 487Phe-Ser-Arg-Asp-Arg-Lys492 of Sarcoplasmic Reticulum Ca2+-ATPase Produces Pumps Defective in ATP Binding

.5 ) and the apparent K m for Mg 2؉ , Na ؉ , K ؉ , and vanadate in Na/K-ATPase were also estimated. For all the mutants, the value for ATP was ϳ2-10-fold larger than that of the wild type. While the turnover number for Na/K-ATPase activity were unaffected or reduced by 20ϳ50% in mutants G442(A/P) and D443A. Although both affinities for ATP effects were reduced as a result of the mutations, the ratio, K m l /K m h , for each mutant was 1.3ϳ3.7, indicating that these mutations had a greater impact on the low affinity ATP effect than on the high affinity effect. Each K m P value with the turnover number suggests that these mutations favor the binding of pNPP over that of ATP. These data and others indicate that the sequence 442 GDASE 446 in the ATP binding pocket is an important motif that it is involved in both the high and low affinity ATP effects rather than in free Mg 2؉ , Na ؉ , and K ؉ effects.

mentioning

confidence: 73%

The Amino Acid Sequence 442GDASE446 in Na/K-ATPase Is an Important Motif in Forming the High and Low Affinity ATP Binding Pockets

Imagawa

Kaya

Taniguchi

2003

“…If an interaction between nucleotide and ATPase had only local effects on the protein structure, a weakened interaction would selectively reduce the amplitude of difference bands associated with that conformational change, but not of all of the bands as observed here. Particularly interesting is that functional groups of ATP, which interact with different domains of the protein, produce the same type of conformational change: the amino function is thought to interact with the N-domain (19,24,46) and the ␥-phosphate with the P-domain (25,26,46). Despite that, the absence of the ␥-phosphate in ADP (28) or of the adenine amino group in ITP both reduce the amplitude of the same bands.…”

Section: Discussionmentioning

confidence: 99%

Mapping Interactions between the Ca2+-ATPase and Its Substrate ATP with Infrared Spectroscopy

Liu

Barth

2003

Infrared spectroscopy has been used to map substrate-protein interactions: the conformational changes of the sarcoplasmic reticulum Ca 2؉ -ATPase upon nucleotide binding and ATPase phosphorylation were monitored using the substrate ATP and ATP analogues (2 -deoxy-ATP, 3 -deoxy-ATP, and inosine 5 -triphosphate), which were modified at specific functional groups of the substrate. Modifications to the 2 -OH, the 3 -OH, and the amino group of adenine reduce the extent of bindinginduced conformational change of the ATPase, with particularly strong effects observed for the latter two. This demonstrates the structural sensitivity of the nucleotide-ATPase complex to individual interactions between nucleotide and ATPase. All groups studied are important for binding and interactions of a given ligand group with the ATPase depend on interactions of other ligand groups.Phosphorylation of the ATPase was observed for ITP and 2 -deoxy-ATP, but not for 3 -deoxy-ATP. There is no direct link between the extent of conformational change upon nucleotide binding and the rate of phosphorylation showing that the full extent of the ATP-induced conformational change is not mandatory for phosphorylation. As observed for the nucleotide-ATPase complex, the conformation of the first phosphorylated ATPase intermediate E1PCa 2 also depends on the nucleotide, indicating that ATPase states have a less uniform conformation than previously anticipated.Ligand binding to proteins controls vast numbers of cellular processes and has attracted great scientific and economic interest. Protein and ligand flexibility are important determinants of the interaction and often lead to ligand binding modes that are not anticipated from structures obtained with other ligands. To these "failure(s) of the rigid receptor hypothesis" (1) is added here an impressive example: induced-fit binding of nucleotides to the Ca 2ϩ -ATPase. This finding stems from a systematic mapping of substrate-protein interactions with infrared (IR) 1 spectroscopy. New approaches like this are welcome in the field of ligand-protein recognition, since the most informative techniques, NMR and x-ray crystallography, are laborious and problematic for some systems. Methods like fluorescence and luminescence that require less expenditure also provide less molecular information. We expect that this technology gap will be bridged by IR spectroscopy.IR spectroscopy, one of the methods of vibrational spectroscopy, provides direct information on the molecular level, is cost-effective, and can be universally applied from small soluble proteins to large membrane proteins under near-physiological conditions. Work summarized in recent reviews (2-5) has shown that the vibrational spectrum changes characteristically when a ligand binds to a protein. This provides a direct observation of ligand binding: no marker compound has to be introduced to report the binding process, as with many other methods. Previous work has mostly focused on individual interactions between a ligand and a protein by monitoring the ...

“…Calculations and Data Analysis-The phosphorylation data and the [␥-32 P]TNP-8N 3 -ATP labeling data were analyzed as detailed previously (14,16). The "true" dissociation constant for ATP and MgATP binding was calculated using the validated equation for competitive inhibition of the [␥-32 P]TNP-8N 3 -ATP labeling (16).…”

Section: Methodsmentioning

confidence: 99%

“…Assays for Nucleotide Binding-The synthesis of [␥-32 P]TNP-8N 3 -ATP, the photolabeling of COS-1 cell microsomes containing wild-type or mutant Ca 2ϩ -ATPase (at room temperature), the inhibition by ATP, and the quantification of labeled bands by radio-imaging following SDS-polyacrylamide gel electrophoresis were carried out as described previously (16). The concentration of [␥-32 P]TNP-8N 3 -ATP was 3 ϫ K 0.5 in the inhibition experiments with ATP.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Roles of Conserved P Domain Residues and Mg2+ in ATP Binding in the Ground and Ca2+-activated States of Sarcoplasmic Reticulum Ca2+-ATPase

McIntosh

Clausen

Woolley

et al. 2004

Self Cite

Residues in conserved motifs 625 The sarcoplasmic reticulum Ca 2ϩ -ATPase 1 from rabbit muscle couples the hydrolysis of ATP to the transport of Ca 2ϩ to the reticular lumen, and the lowering of the sarcoplasmic Ca 2ϩ concentration leads to muscle relaxation. This ion pump is a well studied example of P-type ATPases, the catalytic cycle of which includes a phosphorylated aspartyl intermediate and progression through E1 and E2 states. ATP-dependent phosphorylation takes place upon Ca 2ϩ binding in the E1 form, and hydrolysis of the aspartyl phosphoryl bond occurs in the E2P phosphoenzyme conformation following Ca 2ϩ translocation across the membrane. Two atomic structures of the protein have been elucidated by x-ray crystallography, one in the presence of Ca 2ϩ (designated E1(Ca 2 )) and another in the absence of Ca 2ϩ and presence of the specific inhibitor thapsigargin (E2(TG)) (1, 2). They reveal 10 membrane-spanning helices, connected through a stalk section to the phosphorylation (P) domain in association with nucleotide (N) and actuator (A) domains. The three head domains are loosely attached in E1(Ca 2 ) and closer together, forming a more compact entity, in E2(TG). For ATP-dependent phosphorylation of Asp 351 to occur in the Ca 2ϩ bound E1 state, domain N must close over the phosphorylation site, such that the nucleotide site is brought close to Asp 351 . ATP likely straddles both domains, the nucleoside portion anchored in domain N and the phosphates stretching into domain P (cf. Fig. 1A). Domain P contains the highly conserved segments 625 TGD, 676 FARXXPXXK, and 701 TGDGVND that surround the phosphorylation loop 351 DKTGTLT. Many of these residues have been implicated in ATP binding and catalysis on the basis of chemical labeling and cross-linking experiments, and mutagenesis of Ca 2ϩ -ATPase and related pumps (for review, see Ref.3). Most of them recur at the active site of a large class of soluble phosphohydrolases and phosphotransferases believed to have a similar reaction mechanism (4).