A systematic comparative and structural analysis of protein phosphorylation sites based on the mtcPTM database

Abstract

mtcPTM is an online repository of human and mouse phosphosites in which data are hierarchically organized to preserve biologically relevant experimental information, thus allowing straightforward comparisons of phosphorylation patterns found under different conditions. The database also contains the largest available collection of atomic models of phosphorylatable proteins. Detailed analysis of this structural dataset reveals that phosphorylation sites are found in a heterogeneous range of structural and sequence contexts. mtcPTM is available on the web http://www.mitocheck.org/cgi-bin/mtcPTM/search.

Figure 1

Gene view: display of genomic peptide matches. The figure depicts an example of how genomic matches of peptides from a single experiment are dealt with. Gene ENSG00000171467 (top), which has three possible transcripts/proteins (middle), was matched by several peptides obtained from an experiment. Of all the three transcripts, ENSP00000354964 was the one containing the highest number of peptides, even though none of them was unique for this protein. Therefore, ENSP00000354964 was considered to be part of the minimal list (peptides highlighted in red). However, it may be that the peptide patterns could be explained by the presence of the other two transcripts that are not included in the minimal list (peptides in green). However, even though more information would be needed to confirm either scenario, the raw data are kept for the users to draw their own conclusions. Peptides matching to proteins from other genes are shown at the bottom of the figure. Some of these protein/genes matched additional peptides and therefore they were included in the minimal list (red) whereas others did not (green). The latter assignments could thus be considered spurious.

Figure 2

Protein view: graphical comparison of experiments. The figure shows an example of the graphical display used to present all the phosphosites stored for a given protein entry. The protein is represented by a horizontal bar, with the positions of known domains and phosphosites indicated by colored boxes and vertical lines, respectively. The top panel depicts a complete summary of all modifications, in which phosphosites are color coded according to whether they were fully resolved by the experiment, because sometimes the position of a phosphosite cannot be unambiguously determined by mass spectrometry. Thus, confidently determined positions are shown in red, uncertain positions in orange, and positions that have been retrieved from literature or other sources and are still awaiting manual curation to confirm their status in gray. The peptide maps for each experiment are then shown underneath, in which related experiments are grouped together to allow easy comparison. The color coding is the same as above with the exception that gray is now used to highlight residues that have been seen phosphorylated but not in that particular experiment. Further information about individual peptides can be retrieved via links from the lines representing them. These peptide pages include details about the sequence of the peptide, experimental data (such as protease and software used for their identification), whether the peptide is unique for a gene/protein, its position in the full-length protein, and whether there exist sequence variations with respect to the Ensembl sequence.

Figure 3

Phosphosite location relative to the structured domains. The plots show the distributions with the frequencies of occurrences of potential (yellow) and known (cyan and red) phosphosites along the length of the structures. The positions correspond to all nonoverlapping and equally long tenths in which the sequences can be split, from the amino- (left) to the carboxyl-termini (right). The distributions are shown separately for (a) serine/threonine and (b) tyrosine residues. As explained in the main text, the occurrences of known phosphosites were calculated in two different ways: directly from the full-length structure (cyan) or from trimmed versions of the domains in which disordered and exposed termini had been removed (red).

Figure 4

Phosphosites at unstructured termini. (a) Structure of the Rho GDP-dissociation inhibitor 2 in complex with RAC [81] (Protein Data Bank [PDB]: 1ds6). (b) Structure of the orphan nuclear receptor NR4A1 bound to DNA [82] (PDB: 1cit). In both panels the phosphosite-containing domains are colored in cyan and their interacting partners in light yellow. The modified sites are shown in space-filled representation. (c,d) Two examples of phosphorylations found in short linkers between domains within the human Zinc finger protein 174 and the mouse discs large homolog 4, respectively. Notice that, for the latter, the displayed boundaries of the PDZ domain correspond to those from the structural assignment and not to those defined by Pfam, because the latter did not include the carboxyl-terminus. A list with additional details on the examples, including links to the appropriate mtcPTM entries, can be found in Additional data file 1.

Figure 5

Solvent accessibility of phosphorylatable residues. The plots show the distributions of the percentage of solvent accessibility of the (a) serine/threonine and (b) tyrosine side chains in the structures, as calculated by NACCESS [44]. The cyan and red columns correspond to the distributions for all potential and known phosphorylated residues, respectively, whereas the yellow columns are controls summarizing the solvent accessibility of all amino acids. Exposed terminal regions were not included in the calculations. These distributions were identical to that calculated from the templates or from models sharing at least 80% identity to the templates, indicating that, overall, the modeled conformations of the residues holding the phosphosites are expected to be accurate.

Figure 6

Distribution of phosphosites with respect to secondary structure elements. The plots represent the frequency of occurrences of phosphorylated (a) serine/threonine and (b) tyrosine residues in the elements of secondary structure of the models as defined by Dictionary of Protein Secondary Structure (DSSP) [43]. The three sets shown as well as their color coding are identical to those from Figure 5.

Figure 7

Evolutionary conservation of phosphorylated sites. The plots show the distribution of the percentage of known (red) or potential (cyan) phosphosites presenting a given degree of conservation (between 0 and 20, 20 and 40, and so on) in four sets of multiple alignments. These four sets of multiple alignments, which contain different sequence diversity, comprise sequences sharing at least (a) 30%, (b) 40%, (c) 50%, or (d) 60% identity with respect to the human or mouse queries.

Figure 8

Phosphorylatable buried residues at or close to functional sites. The figure shows examples of phosphorylatable buried residues that are found at or in the vicinity of binding sites for small or large molecules. All the structures are shown differentially colored from their amino- (cold colors) to their carboxyl-termini (hot colors), with the phosphosites in white space-filled representation and their bound substrate in dark gray unless stated otherwise. (a) Structure of human 5'-deoxy-5'-methylthioadenosine phosphorylase [83] (Protein Data Bank [PDB]: 1cb0) with an adenine molecule. (b) Structure of homodimeric human malate dehydrogenase type 2 bound to NAD co-factor (unpublished data; PDB: 2dfd). (c) Structure of the carboxyl-terminal C2 domain of human tricalbin modeled onto the C2 domain of phospatidylinositide 3-kinase C2 α [55] (PDB: 2b3r). (d) Structure of a histone dimer (red and light yellow) bound to DNA (green and cyan) [84] (PDB: 1kx5). (e) Structure of the homodimer formed by the zinc-finger domains of the estrogen receptor in complex with DNA (cyan and blue) [85] (PDB: 1hcq). A list with additional details of the examples, including links to the appropriate mtcPTM entries, can be found in Additional data file 2.

Figure 9

Phosphorylatable residues buried between domains. The figure shows examples of phosphorylated buried residues found in hinge regions or at the interface between domains from the same protein. All of the structures are shown differentially colored from their amino- (cold colors) to their carboxyl-termini (hot colors) and the phosphosites in white space-filled representations unless stated otherwise. (a) Structure of human thioredoxin reductase 1 (unpublished data; Protein Data Bank [PDB]: 2cfy). The residue that can be phosphorylated, Y422, participates in the interface between the two domains of the protein, namely the pyridine nucleotide-disulphide oxidoreductase (cyan) and the dimerization (red) domains. The substrate for the enzyme is shown in yellow. (b) Structure of the α isoform of the guanine nucleotide dissociation inhibitor [86] (PDB: 1gnd). (c) Structure of mouse Sec1 complexed with syntaxin-1 (light gray) [58] (PDB: 1dn1). The phosphorylatable residues are shown in the same color as the protein backbone to facilitate their localization along the structure. A list with additional details of the examples, including links to the appropriate mtcPTM entries, can be found in Additional data file 2.

Figure 10

Buried residues whose phosphorylation state could affect local structural conformation. The figure shows several examples of buried residues whose phosphorylation may result in conformational rearrangements, including detachment of secondary structure elements from the protein domain. Unless stated otherwise, all the structures are shown differentially colored from their amino- (cold colors) to their carboxyl-termini (hot colors), with the regions whose conformation is predicted to be affected in gray, and the phosphosites in white space-filled representation. (a) Structure of the human 7508A NBD1 domain [60] (Protein Data Bank [PDB]: 1xmi). (b) Structure of the autoinhibited p47^phox [62] (PDB: 1ng2). (c) Structure of annexin-1 [64] (PDB: 1hm6). Other residues, namely T24, S27 and S28, that can also be phosphorylated, although they are not buried, are shown in gray. The region encircled is likely to be affected by phosphorylation of the enclosed amino acids, as described in the main text. (d) Structure of the regulator of G-protein signaling 16 (unpublished data; PDB: 2bt2). (e) Structure of the serine/threonine protein phosphatase PP1-β catalytic subunit [67] (PDB: 1s70). A PEG molecule is shown in red and the 130 kDa myosin-binding subunit of smooth muscle myosin phosphatase in white. (f) Structure of DJ-1, a protein related to male fertility and Parkinson's disease [69] (PDB: 1ps4). (g) Structure of the 60S ribosomal protein L7-A [70] (PDB: 1s1i). (h) Structure of the elongation factor EEF1A [87] (PDB: 1f60), in which their individual domains are shown in blue (elongation factor Tu GTP-binding domain), cyan (elongation factor Tu domain 2), and green (elongation factor Tu carboxyl-terminal domain). The catalytic carboxyl-terminal domain of EEF1BA is shown in yellow. A list with additional details of the examples, including links to mtcPTM entries, can be found in Additional data file 2.