REVIEW 4 major objections 5 minor 19 references
Investigation of the impact of PTMs on the protein backbone conformation
T0 review · 4 major / 5 minor · reviewed 2026-08-14 · deepseek-v4-flash
Pith's one-line read A structural survey argues that post-translational modifications alter backbone flexibility in type-specific ways, with CDK2's Thr160 phosphorylation stiffening its activation loop and increasing flexibility at two other regulatory…
desk verdict A workmanlike PB/Neq study of PTM backbone effects; the CDK2 allosteric observation is plausible but not yet secured by the species/ligand-matched comparison. read the letter →
The pith
A machine-rendered reading of the paper's core claim, the machinery that carries it, and where it could break.
The reading
What carries the argument
Protein Blocks (PBs) are a 16-letter structural alphabet for the backbone: each letter represents the local conformation of a five-residue fragment through eight backbone dihedral angles, so every crystal structure becomes a one-dimensional string of letters. The central measure is $N_{eq}$, defined as $N_{eq} = \exp(-\sum_x f_x \ln f_x)$ over the 16 PB frequencies at a position, ranging from 1 (always the same PB) to 16 (all conformations equally frequent). $N_{eq}$ quantifies local backbone deformability across aligned copies of the same protein, and it is compared between PTM regions and the rest of the protein, and between modified and unmodified structure sets, using normalized B-factors and a nonparametric rank test.
What would settle it
Find a matched set of CDK2 crystal structures that use the same cyclin A2 species, similar ligands, and comparable crystal forms and differ only in whether Thr160 is phosphorylated, then recompute $N_{eq}$ at position +1, positions 8-18, and around Thr39. If the rigidification at +1 or the flexibility increase at the two distant regions disappears in the matched comparison, the paper's central allostery claim is not supported.
Extended reading notes
Core claim
The paper's central discovery is that PTMs affect backbone diversity in a type- and position-dependent way. In a non-redundant screen, neither N-glycosylation nor phosphorylation sites have a single privileged local conformation, although Ser/Thr phosphorylation prefers the beta-strand core Protein Block d in about 40% of cases. In the four case studies, only phosphorylation produces a statistically significant difference between the PTM region and the rest of the protein: CDK2's Thr160 phosphorylation lowers $N_{eq}$ at position +1 from 5.27 to 2.40 and lowers normalized B-factors, rigidifying the activation loop, while raising $N_{eq}$ around positions 8-18 (near Thr14/Tyr15) and near Thr39, two other regulatory phosphorylation sites. The paper interprets this as a coupled stiffening-flexibility pattern indicative of underlying allostery. N-glycosylation in renin endopeptidase and liver carboxylesterase changes no $N_{eq}$ profile, while actin H75 methylation increases local $N_{eq}$ around the site but cannot sustain a global claim because of ligand heterogeneity.
Load-bearing premise
The load-bearing premise is that the modified and unmodified crystal-structure sets differ only in the PTM; ligand variation, crystal contacts, additional modifications, and construct boundaries are not fully controlled, and for actin the authors concede this heterogeneity precludes a global conclusion.
Editorial extensions
If this is right
- The paper's results imply that a single PTM can have opposite structural effects inside one protein: phosphorylation of CDK2's Thr160 both rigidifies the activation loop and increases backbone diversity at two distant regulatory regions.
- The non-redundant survey implies that N-glycosylation and phosphorylation do not require a particular backbone context, so PTM sites should not be assumed to sit in special or conserved local structures.
- The two N-glycosylation case studies imply that some glycans, particularly those solvent-exposed on disulfide-stabilized proteins, can be structurally silent, so detecting a PTM in a crystal does not by itself indicate a conformational role.
- The local and distant pattern in CDK2, together with the known opposing roles of Thr160 and Thr14/Tyr15 phosphorylation, implies a structural route for PTM crosstalk in cell-cycle regulation.
Reading between the lines
- A testable extension would replace Thr160 of CDK2 with a phosphomimetic aspartate or a nonphosphorylatable alanine and compare crystal structures; if the two distant regions do not become more flexible, the observed coupling is specific to the phosphate group or to crystallographic conditions rather than a general phosphoregulatory mechanism.
- The same $N_{eq}$ strategy could be applied to other kinases with a single well-resolved phosphorylation site to see whether 'stiffen one's own loop, loosen other regulatory loops' is a recurrent motif of kinase activation rather than a CDK2 peculiarity.
- Because the actin conclusion is ligand-confounded, a cleaner test would compare methylated and unmethylated actin structures holding the bound nucleotide and drug constant; local differences near His75 would then be attributable to methylation with higher confidence.
Signed reviews
Editorial analysis
A structured set of objections, weighed in public.
Referee Report
Summary. This manuscript investigates how post-translational modifications (PTMs) affect protein backbone conformation using the PTM-SD structural database and the Protein Blocks (PB) structural alphabet. The authors first analyze local backbone diversity around N-glycosylation and phosphorylation sites in non-redundant datasets, and then focus on four proteins: renin endopeptidase (N-glycosylation), liver carboxylesterase (N-glycosylation), cyclin-dependent kinase 2 (CDK2, phosphorylation of Thr160), and actin (methylation of His75). They compute Neq, an entropy-like measure of local backbone diversity, from ensembles of X-ray structures, and compare Neq profiles between modified and unmodified proteins, using Mann-Whitney-Wilcoxon tests. The central reported finding is that CDK2 Thr160 phosphorylation rigidifies the activation loop locally while increasing backbone flexibility near Thr14/Tyr15 and Thr39, leading the authors to propose a coupled stiffening-flexibility allosteric mechanism. The manuscript also reports a local destabilizing effect of actin His75 methylation and concludes that N-glycosylation does not alter backbone flexibility in the two examples studied.
Significance. If the CDK2 allosteric claim is correct, it would be a valuable structural observation linking a single phosphorylation event to a specific distal regulatory response in a medically important kinase. The study uses a curated database (PTM-SD) and an established conformational alphabet (PBxplore), and it provides the list of PDB entries used, which supports reproducibility of the dataset. The comparative design, measuring Neq profiles in modified versus unmodified structures, does not fit parameters to the target conclusion, so circularity is not a concern. However, the causal interpretation of the CDK2 and actin comparisons is currently undermined by uncontrolled covariates and by the statistical treatment of the data.
major comments (4)
- [Results: 'Local and global backbone diversity compared between modified and unmodified proteins'; Table 2] The CDK2 comparison is confounded by differences in the cyclin A2 partner species and by ligand chemistry. As the authors correctly note, all chains are cyclin A2 complexes, but Table 2 shows that the pT160 set contains 70 human, 24 bovine, and 4 mouse cyclin A2 chains, whereas the non-pT160 set contains only human cyclin A2. In addition, the manuscript does not report whether the ligand types (ATP, ADP, ATP analogs, inhibitors) are matched between the two sets; Supplementary Figure S12 only states that no visible change in binding pattern is observed, which cannot exclude systematic differences in ligand chemistry or crystal contacts. Because Neq is a per-position ensemble statistic, any covariate correlated with phosphorylation state could produce the reported local rigidification at position 160 and the increased flexibility near positions 14-15 and 39. The authors should perform a human-only (or species-matched) sensitivity analysis and, if feasible, a ligand-matched analysis to establish that the effect is attributable to Thr160 phosphorylation.
- [Table 1 and 'Various analyses' in Methods] The Mann-Whitney-Wilcoxon tests in Table 1 treat individual sequence positions as independent samples. The PTM-region test uses n=21 positions and the whole-protein test uses n≈300 positions, but neighboring Neq values along a polypeptide are spatially correlated rather than statistically independent. Moreover, the 'Neq 0 PTM vs Neq 1 PTM' comparisons are, for a given protein, paired by residue position, yet an unpaired Mann-Whitney-Wilcoxon test is applied; a paired Wilcoxon signed-rank test or a permutation test respecting the spatial structure would be more appropriate. No correction for multiple testing is made across the eight MWW comparisons reported in Table 1 and Supplementary Table S4. These issues mean the reported p-values (e.g., CDK2 p=0.0132, actin p=0.0007) do not provide reliable statistical evidence as presented. The authors should re-analyze the data with an appropriate paired or block-resampling procedure and report effect sizes or at least the unadjusted versus adjusted results.
- [Results, N-glycosylation section; Discussion, 'For the two examples of N-glycosylation'] The manuscript over-interprets non-significant results as evidence of absence when concluding that N-glycosylation 'does not impact the intrinsic flexibility' of renin endopeptidase and liver carboxylesterase. The local comparison is based on only 21 positions in the PTM region, and no power analysis or equivalence testing is provided. A non-significant Mann-Whitney-Wilcoxon test with such a small sample cannot distinguish a true null effect from an underpowered test. Because this null conclusion is part of the paper's general claim that PTM effects depend on PTM type, the authors should either adopt an equivalence testing framework or explicitly describe the conclusion as limited by low statistical power.
- [Methods and Table 1] The definition of the PTM region is not specified. Table 1 consistently shows n=21 for the PTM-region sample, and the text refers to 'the PTM site and its neighboring positions,' but the window size, the exact set of positions, and how the Neq values are pooled across chains or structures are not described in Methods. Without this information, the local MWW test is not reproducible, and it is unclear whether the window is the same for all four proteins. Please provide the precise construction of the PTM-region sample, including the flanking window length and any residue-position exclusion criteria.
minor comments (5)
- [Methods, 'Dataset'] The Methods state '92 phosphorylations on 76 structures', while the Results and Supplementary Table S1 report 75 chains; please reconcile this inconsistency.
- [Equation (2) in Methods] Equation (2) is malformed as printed: 'Neq=exp(−fxx=116∑lnfx)' should read Neq = exp(−Σ_{x=1}^{16} f_x ln f_x). Please correct the typesetting.
- [Equation (1) in Methods] The RMSDA formula in Equation (1) is garbled in the manuscript; it should be presented as a sum over the eight dihedral-angle differences, properly formatted.
- [Figure 2 legend] The legend for Figure 2 indicates the red line as 'the amount of data' but does not define what 'data' means; the figure legend should explicitly state that the red curve is the percentage of ordered residues used to compute each Neq value.
- [Table 1 caption] The table caption would be clearer if it explicitly stated that the sample sizes n are the number of sequence positions included in each Neq comparison.
Circularity Check
No significant circularity: the PTM-vs-no-PTM Neq comparison is an external PDB-based measurement; self-cited tools are infrastructure, not load-bearing results.
full rationale
The paper's derivation chain is: (1) obtain PTM-annotated and unannotated X-ray chains from PTM-SD; (2) assign Protein Blocks with PBxplore; (3) compute Neq per position; (4) compare Neq profiles between modified and unmodified sets with Mann-Whitney-Wilcoxon; (5) interpret significant differences as local or distal flexibility changes. No step fits a parameter to the conclusion or defines the outcome into the input. The PTM/non-PTM group labels come from structural annotation, while Neq is computed independently from backbone dihedrals; the CDK2 local rigidification and distal flexibility changes are measured, not assumed. Self-citations (PTM-SD, PBxplore, de Brevern et al. 2000) provide software and a metric, not the biological conclusion; they are not uniqueness theorems and no result is imported as a forced alternative. The actin ligand heterogeneity and the unmatched cyclin species/constructs in the CDK2 comparison are confounding or validity limitations, explicitly acknowledged by the authors, but confounding is not circularity. No equation in the paper reduces to another by construction, and no fitted input is renamed as a prediction. Thus there is no specific circular step to quote.
Assumptions & free parameters
free parameters (2)
- Redundancy threshold (25% sequence identity) =
25%
- PTM flanking window size (about 21 positions) =
-10 to +10 residues
assumptions (5)
- domain assumption Protein Blocks (16 prototypes) adequately represent local backbone conformation
- domain assumption Neq computed from a set of X-ray structures measures local backbone deformability
- domain assumption PTM annotations in PTM-SD (from dbPTM and PTMCuration) are correct
- domain assumption Sequence positions are independent samples in the Mann-Whitney-Wilcoxon tests
- domain assumption Structure sets with and without the PTM differ only in the PTM
Cite this review
Pith. "Pith review of Investigation of the impact of PTMs on the protein backbone conformation." pith.science (2026). https://pith.science/paper/JI3U7NSP
@misc{pith2026190805122,
author = {Pith},
title = {Pith review of: Investigation of the impact of PTMs on the protein backbone conformation},
year = {2026},
howpublished = {\url{https://pith.science/paper/JI3U7NSP}},
note = {Machine review of arXiv:1908.05122}
}
read the original abstract
Post-Translational Modifications (PTMs) are known to play a critical role in the regulation of the protein functions. Their impact on protein structures, and their link to disorder regions have already been spotted on the past decade. Nonetheless, the high diversity of PTMs types, and the multiple schemes of protein modifications (multiple PTMs, of different types, at different time, etc) make difficult the direct confrontation of PTM annotations and protein structures data.We so analyzed the impact of the residue modifications on the protein structures at local level. Thanks to a dedicated structure database, namely PTM-SD, a large screen of PTMs have been done and analyze at a local protein conformation levels using the structural alphabet Protein Blocks (PBs). We investigated the relation between PTMs and the backbone conformation of modified residues, of their local environment, and at the level of the complete protein structure. The two main PTM types (N-glycosylation and phosphorylation) have been studied in non-redundant datasets, and then, 4 different proteins were focused, covering 3 types of PTMs: N-glycosylation in renin endopeptidase and liver carboxylesterase, phosphorylation in cyclin-dependent kinase 2 (CDK2), and methylation in actin. We observed that PTMs could either stabilize or destabilize the backbone structure, at a local and global scale, and that these effects depend on the PTM types.
Figures
Reference graph
Works this paper leans on
-
[1]
Pierrick Craveur1,2,3,4,5,#, Tarun J
Flexibility & PTMs 1 Investigate the impact of PTMs on the protein backbone conformation. Pierrick Craveur1,2,3,4,5,#, Tarun J. Narwani1,2,3,4,#, Joseph Rebehmed1,2,3,4,6,+ & Alexandre G. de Brevern 1,2,3,4,+,* 1 INSERM, U 1134, DSIMB, F-75739 Paris, France. 2 Univ Paris, Univ de la Réunion, Univ des Antilles, UMR_S 1134, F-75739 Paris, France. 3 Institut...
work page 2010
-
[2]
Cellular components, domains, technical terms, developmental processes, and coding sequence diversities correlated with long disordered regions J Proteome Res 6:1899-1916 doi:10.1021/pr060393m Welburn JP et al. (2007) How tyrosine 15 phosphorylation inhibits the activity of cyclin-dependent kinase 2-cyclin A J Biol Chem 282:3173-3181 doi:10.1074/jbc.M6091...
-
[3]
Ligands, post-translational modifications, and diseases associated with intrinsically disordered proteins J Proteome Res 6:1917-1932 doi:10.1021/pr060394e Xie H, Vucetic S, Iakoucheva LM, Oldfield CJ, Dunker AK, Uversky VN, Obradovic Z (2007b) Functional anthology of intrinsic disorder
-
[4]
suggest that disorder-to-order transition could be induced by the modifications of phospho-serine/-threonine, various types of methyllysines, sulfotyrosine, 4-carboxyglutamate, and potentially 4-hydroxyproline. Disorder regions are mainly defined as series of missing residues in X-ray structures taken from the PDB, they are highly frequent (more than 80% ...
work page 2015
-
[12]
by expanding their partnered interactions with other proteins, collectively termed as protein-protein interactions (PPI). The selectivity of interacting partners and order-disorder transition of the protein structures is regulated by PTMs, and most of the times by phosphorylations. (Hsu et al. 2013). During our analysis of CDK2, we also found identical st...
work page 2013
-
[13]
We may suggest that the number of phosphorylations, in and around the catalytic domain, may also impact this selectivity of interacting partners for Flexibility & PTMs 20 CDK2. The example of actin methylation shows an opposite effect of the modification compared to the example of CDK2. The methylation induces a local increase of the backbone diversity at...
work page 2012
-
[15]
Trapping of ultracold polar molecules with a Thin Wire Electrostatic Trap
Phosphorylation on Thr160 of human cyclin-dependent kinase 2 (P24941) The Neq profiles are given at a local scale (A), for the surrounding positions of the PTM site (coloured in green), and at a global scale (B), for all sequence positions. (C) The 96 structures used for the computation were aligned on the backbone, and represented as cartoon. The phospho...
work page Pith review arXiv 2017
-
[18]
Biological processes and functions of proteins with long disordered regions J Proteome Res 6:1882-1898 doi:10.1021/pr060392u Xin F, Radivojac P (2012) Post-translational modifications induce significant yet not extreme Flexibility & PTMs 30 changes to protein structure Bioinformatics 28:2905-2913 doi:10.1093/bioinformatics/bts541 Yao Q, Xu D (2017) Bioinf...
Show all 19 references
-
[79]
Figure S5
The blue spheres mark the disulphide bridges. Figure S5 . S t r u c t u r a l A n a l y s i s o f A c t i n. A ) T h e Neq p r o f i l e o f t h e m e t h y l a t i o n o f H i s t i d i n e residue 75 and its neighbouring positions. B) The entire Neq profile of the actin chai...
-
[92]
are close to the protein extremities. A more precise analysis of the distribution of each type of PBs (see Supplementary Figure S1) underlines that N-glycosylation and phosphorylation sites are observed for all types of local conformations, almost any kind of PBs (except PBs g...
2007
-
[160]
In the absence of the phosphorylation (see Supplementary Figure S10), this area corresponds to a wide flexible loop, sometimes not even ordered in few structures (see red line)
and its surrounding positions. In the absence of the phosphorylation (see Supplementary Figure S10), this area corresponds to a wide flexible loop, sometimes not even ordered in few structures (see red line). Nonetheless, in the presence of the phosphorylation, the Neq value i...
1992
-
[1989]
Briefly described, PBs m and d are prototypes for the central region of α-helix and β-strand, respectively
obtained them. Briefly described, PBs m and d are prototypes for the central region of α-helix and β-strand, respectively. PBs a to c primarily represent the N-cap of β-strand while e and f correspond to C-caps; PBs g to j are specific to coils, PBs k and l correspond to N cap...
2005
-
[2008]
of protein binding sites, and structural analysis of β-bulges (Craveur et al. 2013). Protein Blocks assignment. The assignment translates a 3D structure to 1D sequence of PBs. In our study input structures come from PDB files. The algorithm uses 5 residues long window for each...
2013
-
[2009]
Flexibility & PTMs 10 B-factor normalization
for similar analyses) is to provide a simple measure that quantifies locally the divergence. Flexibility & PTMs 10 B-factor normalization. B-factor values are partly dependent on the resolution of the crystal and of the refinement process (Hinsen 2008; Linding et al. 2003; Sch...
2008
-
[2010]
2009; Zimmermann and Hansmann
or prediction (Rangwala et al. 2009; Zimmermann and Hansmann
2009
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[2012]
2011), or the co-evolution of different PTMs sites within the same protein (Minguez and Bork 2017; Minguez et al
their competition for the same residue (Danielsen et al. 2011), or the co-evolution of different PTMs sites within the same protein (Minguez and Bork 2017; Minguez et al. 2013; Minguez et al. 2012). Many databases and prediction tools have been developed to enhance the underst...
2011
-
[2015]
2016; Imberty and Perez 1995), tagging proteins for degradation by ubiquitination (Zhou and Zeng 2016), and regulation of kinase activity with phosphorylation (Krupa et al
or glycosylation (Dewald et al. 2016; Imberty and Perez 1995), tagging proteins for degradation by ubiquitination (Zhou and Zeng 2016), and regulation of kinase activity with phosphorylation (Krupa et al. 2004). PTMs are also associated with major human diseases such as cancer...
2016
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[2016]
and PTMCuration (Khoury et al. 2011). PTM-SD uses diverse set of rules to underline the discrepancies between annotation in the structure and the sequences owing to different sources. Also, PTM-SD allows the user to create customized PTMs queries and perform different analyses...
2011
-
[2017]
on the stability of specific proteins, but the growing success of these kind of simulations also rely upon the growing number of experimental data, for the development of accurate PTM force field parameters. Acknowledgments This work was supported by grants from the Ministry o...
2010
Reviewed August 14, 2026 · model on record in the stance chip above.
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