Comparing the structure and dynamics of phospholamban pentamer in its unphosphorylated and pseudo‐phosphorylated states

Oxenoid, Kirill; Rice, Amanda J.; Chou, James J.

doi:10.1110/ps.072975107

Cited by 25 publications

(27 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The PLB-PLB FRET max parameter, a measure of the probe separation distance, did not change with pseudo-phosphorylation indicating the quaternary structure was unaffected. These results are consistent with a recent NMR study that observed no significant changes to short-range distance constraints with the S16E mutation (19). According to a computational model of intrapentameric energy transfer based on ring-oligomer FRET theory (29), the average nearest-neigh-…”

Section: Discussionsupporting

confidence: 92%

“…To test whether phosphorylation stabilizes a compact pentamer conformation, we obtained distance constraints by measuring fluorescence resonance energy transfer (FRET) between N-terminal fluorescent protein fusion tags. In this study, we mimicked phosphorylation of PLB by PKA and CaMKII with glutamate substitutions at positions 16 and 17 (18,19). The extent to which glutamate substitutions recapitulate phosphorylation of PLB is not known.…”

Section: Phospholamban (Plb)mentioning

confidence: 99%

See 1 more Smart Citation

Phosphomimetic Mutations Increase Phospholamban Oligomerization and Alter the Structure of Its Regulatory Complex

Hou

Kelly

Robia

2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

To investigate the effect of phosphorylation on the interactions of phospholamban (PLB) with itself and its regulatory target, SERCA, we measured FRET from CFP-SERCA or CFP-PLB to YFP-PLB in live AAV-293 cells. Phosphorylation of PLB was mimicked by mutations S16E (PKA site) or S16E/T17E (PKA؉CaMKII sites). FRET increased with protein concentration up to a maximum (FRET max ) that was taken to represent the intrinsic FRET of the bound complex. The concentration dependence of FRET yielded dissociation constants (K D ) for the PLB-PLB and PLB-SERCA interactions. PLB-PLB FRET data suggest pseudo-phosphorylation of PLB increased oligomerization of PLB but did not alter PLB pentamer quaternary structure. PLB-SERCA FRET experiments showed an apparent decrease in binding of PLB to SERCA and an increase in the apparent PLB-SERCA binding cooperativity. It is likely that these changes are secondary effects of increased oligomerization of PLB; a change in the inherent affinity of monomeric PLB for SERCA was not detected. In addition, PLB-SERCA complex FRET max was reduced by phosphomimetic mutations, suggesting the conformation of the regulatory complex is significantly altered by PLB phosphorylation.Phospholamban (PLB) 2 is a 52-amino acid peptide localized primarily to the sarcoplasmic reticulum of cardiac muscle cells. The peptide comprises two ␣-helical domains connected by a flexible linker. NMR studies suggest an L-shaped tertiary structure; the PLB cytoplasmic domain N-terminal ␣-helix (domain IA) is significantly parallel to the bilayer surface, while the transmembrane helix (domain II) is roughly normal to the membrane (1, 2). The monomeric form of PLB binds and inhibits the sarco(endo)plasmic reticulum calcium ATPase (SERCA) (3). Inhibition is partially relieved by phosphorylation of PLB by PKA (4) and CaMKII (5), and PLB is a major effector of adrenergic signaling (6). PLB also binds to itself, forming pentamers (7). Most evidence suggests that the pentamer is unable to bind or regulate SERCA (8, 9). NMR and EPR (1) and FRET (10, 11) data suggest the pentamer is a "pinwheel" quaternary complex of L-shaped subunits, however, other diverse structures have been proposed (12)(13)(14).Phosphorylation has been proposed to alter the architecture of PLB. Wegener and Jones (7) observed a significant shift in the PLB pentamer electrophoretic mobility in SDS gels, and postulated that mobility was changed as a result of altered quaternary conformation of the pentamer. Specifically, they envisioned PLB cytoplasmic domains assuming a more compact conformation after phosphorylation (15). Consistent with such a structural transition is a recent NMR study showing that phosphorylation decreases the membrane association of the phospholamban cytoplasmic domain (16). Furthermore, Karim et al. (17) found that phosphorylation influenced the proportion of a structurally dynamic form of PLB. To test whether phosphorylation stabilizes a compact pentamer conformation, we obtained distance constraints by measuring fluorescence resonance ...

show abstract

Section: Discussionsupporting

confidence: 92%

Section: Phospholamban (Plb)mentioning

confidence: 99%

Phosphomimetic Mutations Increase Phospholamban Oligomerization and Alter the Structure of Its Regulatory Complex

Hou

Kelly

Robia

2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…If the subunits in a complex are identical, then the complex is called homo-oligomer; otherwise hetero-oligomer. For example, the sodium channel is formed by a monomer [192] while the potassium channel by a homo-tetramer [88]; the phospholamban is formed by homo-pentamer [93,193] while the Gamma-aminobutyric acid type A (GABAA) receptor by a hetero-pentamer [84,194]; the M2 proton channel is formed by a homo-tetramer [87] while hemoglobin by a hetero-tetramer [195].…”

Section: Quatidentmentioning

confidence: 99%

REVIEW : Recent advances in developing web-servers for predicting protein attributes

Chou¹,

Shen²

2009

277

173

View full text Add to dashboard Cite

Recent advance in large-scale genome sequencing has generated a huge volume of protein sequences. In order to timely utilize the information hidden in these newly discovered sequences, it is highly desired to develop computational methods for efficiently identifying their various attributes because the information thus obtained will be very useful for both basic research and drug development. Particularly, it would be even more useful and welcome if a user-friendly web-server could be provided for each of these methods. In this minireview, a systematic introduction is presented to highlight the development of these web-servers by our group during the last three years.

show abstract

“…Therefore, the final predicted result should be determined by a fusion approach through the following voting mechanism. According to eq 11, the voting score for the query protein P belonging to the ith class is given by 2,3,4,5,6,7,8,10,12) (12) where i ) 1 is for monomer, i ) 2 for dimer and so forth, w K 1 and w K, 2 are the weight factors and were set at 1 for simplicity, thus the query protein P is predicted belonging to the class or subset for which the score of eq 12 is the highest; i.e., 2,3,4,5,6,7,8,10,12) …”

Section: Optimized Evidence-theoretic K Nearest Neighbormentioning

confidence: 99%

“…If there is a tie among two or more classes, then the final predicted result will be randomly assigned to one of their corresponding classes although this kind of tie case rarely happens and actually was not observed in the current study. By changing (i ) 1, 2, 3,4,5,6,7,8,10,12) to (i ) homo, hetero) and working on the subclass benchmark data set (cf. eq 2 and the Supporting Information B), eqs 11 and 13 can be automatically used to solve the second-level problem, that is, identify the half-query protein P (which has already been identified as an oligoprotein in the first-layer prediction) belonging to a homo-oligomer or hetero-oligomer.…”

Section: Optimized Evidence-theoretic K Nearest Neighbormentioning

confidence: 99%

QuatIdent: A Web Server for Identifying Protein Quaternary Structural Attribute by Fusing Functional Domain and Sequential Evolution Information

Shen

Chou

2009

J. Proteome Res.

View full text Add to dashboard Cite

Many proteins exist in vivo as oligomers with various different quaternary structural attributes rather than as single individual chains. They are the structural bases of various marvelous biological functions such as cooperative effects, allosteric mechanism, and ion-channel gating. Therefore, with the avalanche of protein sequences generated in the postgenomic era, it is very important for both basic research and drug discovery to identify their quaternary structural attributes in a timely manner. In view of this, a powerful ensemble identifier, called QuatIdent, is developed by fusing the functional domain and sequential evolution information. QuatIdent is a 2-layer predictor. The 1st layer is for identifying a query protein as belonging to which one of the following 10 main quaternary structural attributes: (1) monomer, (2) dimer, (3) trimer, (4) tetramer, (5) pentamer, (6) hexamer, (7) heptamer, (8) octamer, (9) decamer, and (10) dodecamer. If the result thus obtained turns out to be anything but monomer, the process will be automatically continued to further identify it as belonging to a homo-oligomer or hetero-oligomer. The overall success rate by QuatIdent for the 1st layer identification was 71.1% and that for the 2nd layer ranged from 84 to 96%. These rates were derived by the jackknife cross-validation tests on the stringent benchmark data sets where none of proteins has > or =60% pairwise sequence identity to any other in a same subset. QuatIdent is freely accessible to the public as a web server via the site at http://www.csbio.sjtu.edu.cn/bioinf/Quaternary/ , by which one can get the desired 2-level results for a query protein sequence in around 25 seconds. The longer the sequence is, the more time that is needed.

show abstract

Comparing the structure and dynamics of phospholamban pentamer in its unphosphorylated and pseudo‐phosphorylated states

Cited by 25 publications

References 22 publications

Phosphomimetic Mutations Increase Phospholamban Oligomerization and Alter the Structure of Its Regulatory Complex

Phosphomimetic Mutations Increase Phospholamban Oligomerization and Alter the Structure of Its Regulatory Complex

REVIEW : Recent advances in developing web-servers for predicting protein attributes

QuatIdent: A Web Server for Identifying Protein Quaternary Structural Attribute by Fusing Functional Domain and Sequential Evolution Information

Contact Info

Product

Resources

About