Interface Analysis of the Complex between ERK2 and PTP-SL

The activity of ERK2, an essential component of MAP-kinase pathway, is under the strict control of various effector proteins. Despite numerous efforts, no crystal structure of ERK2 complexed with such partners has been obtained so far. PTP-SL is a major regulator of ERK2 activity. To investigate the ERK2–PTP-SL complex we used a combined method based on cross-linking, MALDI-TOF analysis, isothermal titration calorimetry, molecular modeling and docking. Hence, new insights into the stoichiometry, thermodynamics and interacting regions of the complex are obtained and a structural model of ERK2-PTP-SL complex in a state consistent with PTP-SL phosphatase activity is developed incorporating all the experimental constraints available at hand to date. According to this model, part of the N-terminal region of PTP-SL has propensity for intrinsic disorder and becomes structured within the complex with ERK2. The proposed model accounts for the structural basis of several experimental findings such as the complex-dissociating effect of ATP, or PTP-SL blocking effect on the ERK2 export to the nucleus. A general observation emerging from this model is that regions involved in substrate binding in PTP-SL and ERK2, respectively are interacting within the interface of the complex

MALDI-TOF spectrum of non-digested and trypsin digested, cross-linked ERK2−PTP-SL complex.

<p>(A)The BS³ cross-linked complex was purified by size exclusion chromatography, concentrated, SDS removed and finally analysed in presence of sinapinic acid matrix. Representative data for the digested cross-linked (B) and digested non-crosslinked (C) complex are shown. The magnified images a, b, c, d show the four identified tryptic fragments which contain Lys-Lys cross-linked peptides.</p

Potential networks of cross-linked lysine residues within the complex between ERK2 and PTP-SL.

<p>Processing the MALDI-TOF data resulted from trypsinolysis of the cross-linked complex, three possible networks are revealed (denoted A,B and C). Arrows connect cross-linked lysine residues from PTP-SL and ERK2. Dashed line arrow stands for: (i) an alternative involvement of K400 instead of K392 from PTP-SL in cross-linking K201 from ERK2 and (ii) for a potential cross-link between K155 to K310 of PTP-SL, supporting the signal at <i>m/z</i> value of 2221.75. Most probable network (see the text) of cross-links is illustrated in D.</p

MD simulation RMSD and RMSF profiles.

<p>Over a 10 ns span the RMSD remains very low, below 0.75 Å for trace alpha carbons of the PECr complex. The RMSF profile over the sequence, shown in the inset, indicates that the RMSD is mainly due to the loop flexibility.</p

Intrinsic disorder propensity (IDP) of the PTP-SL sequence.

<p>IDP was calculated using DISOPRED (A) and IUPRED (B) methods.</p

Purification of major cross-linking product of the ERK2−PTP-SL complex.

<p>(A) Separation of the reaction products formed by BS³ cross-linking of the ERK2−PTP-SL complex was performed by size exclusion cromatography. (B) Fractions obtained through the chromatographic separation of the cross-linking reaction mixture (lanes 1–2, 4–6) were analysed by SDS-PAGE (12,5%). The non-fractionated reaction mixture of BS³ cross-linked ERK2−PTP-SL complex was applied on lane 3. Staining was performed with Zn-imidazole reagent.</p

The PECf Model of the overall structure of ERK2−PTP-SL complex in a conformation consistent with phosphatase activity.

<p>(A) Network of crosslinks (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005432#pone-0005432-g004" target="_blank">FIG. 4</a>) within the complex of ERK2 (orange) with PTP-SL (blue). The Lys^PTP-SL(yellow)-Lys^ERK2(green) pairs are connected by orange links; (B) Representation of multiple interactions between the KIM containing segment from PTP-SL and ERK2. Cartoon representation: PTP-SL main chain; surface representation: ERK2. The hydrophobic cluster is represented in white; the salt bridges are represented in magenta-yellow pairs. The blue labels designate PTP-SL residues; the red labels designate ERK2 residues. (C) Spacefilling representation of the model of ERK2−PTP-SL complex with and without the segment 147–253^PTP-SL. Color coding: residues 254–549^PTP-SL in blue; residues 147–253^PTP-SL in dark magenta; residues of ERK2 involved in ATP binding are colored in red whereas all other residues of the ERK2 chain are in orange.</p

Experimental and theoretical MWs of various PTP-SL constructs and of their complexes with ERK2.

<p>Experimental MWs were calculated based on gel filtration experiments performed on a Superdex 200 HR analytical column run with mentioned protein preparations under similar experimental conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005432#s4" target="_blank">Materials and Methods</a>).</p

Potential assignments for peptides obtained by tryptic digestion of cross-linked complex between PTP-SL and ERK2.

<p>Sequence data corresponding to peptides formed by intramolecular cross-linking of PTP-SL are written in italics. Lysine residues potentially involved in cross-linking through BS³ are underlined (numbering is mentioned as superscript). “<u>Wox</u> ”denotes the oxidized form of tryptophan. “1” and “2” between parantheses refer to the number of predicted missed cleavage sites.</p

BS³ cross-linking reaction specificity.

<p>To compare reaction specificity the cross-linking reaction with BS³ was performed in parallel with equimolar quantities of ERK2 (lane 1), PTP-SL (lane 2) and ERK2−PTP-SL complex (lane 3). Reaction mixtures were analysed by SDS-PAGE (12,5%).</p