Weak oligomerization of low-molecular-weight protein tyrosine phosphatase is conserved from mammals to bacteria Jascha Blobel 1 , Pau Bernado ´ 1 , Huimin Xu 2 , Changwen Jin 2 and Miquel Pons 1,3 1 Laboratory of Biomolecular NMR, Institute for Research in Biomedicine, Barcelona, Spain 2 Beijing Nuclear Magnetic Resonance Center, College of Life Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China 3 Departament de Quı ´ mica Orga ` nica, Universitat de Barcelona, Spain Introduction Low-molecular-weight protein tyrosine phosphatases (lmwPTPs) constitute one of the four families of protein tyrosine phosphatases [1]. In eukaryotic cells, lmwPTPs participate in platelet-derived growth factor (PDGF)- induced mitogenesis [2] and insulin-mediated mitotic signaling [3]. Dephosphorylation of different substrates, such as the ephrin [4] or fibroblast growth factor receptor, resulting in cell proliferation [5], has also been Keywords low-molecular-weight protein tyrosine phosphatase (lmwPTP); phosphatase regulation; protein oligomerization; signaling pathways; supramolecular proenzyme Correspondence M. Pons, Laboratory of Biomolecular NMR, Institute for Research in Biomedicine, Parc Cientı ´ fic de Barcelona, Baldiri Reixac, 10, 08028 Barcelona, Spain Fax: +34934039976 Tel: +34934034683 E-mail: mpons@ub.edu (Received 13 April 2009, revised 5 June 2009, accepted 9 June 2009) doi:10.1111/j.1742-4658.2009.07139.x The well-characterized self-association of a mammalian low-molecular- weight protein tyrosine phosphatase (lmwPTP) produces inactive oligomers that are in equilibrium with active monomers. A role of the inactive oligo- mers as supramolecular proenzymes has been suggested. The oligomeriza- tion equilibrium of YwlE, a lmwPTP from Bacillus subtilis, was studied by NMR. Chemical shift data and NMR relaxation confirm that dimerization takes place through the enzyme’s active site, and is fully equivalent to the dimerization previously characterized in a eukaryotic low-molecular-weight phosphatase, with similarly large dissociation constants. The similarity between the oligomerization of prokaryotic and eukaryotic phosphatases extends beyond the dimer and involves higher order oligomers detected by NMR relaxation analysis at high protein concentrations. The conservation across different kingdoms of life suggests a physiological role for lmwPTP oligomerization in spite of the weak association observed in vitro. Struc- tural data suggest that substrate modulation of the oligomerization equilib- rium could be a regulatory mechanism leading to the generation of signaling pulses. The presence of a phenylalanine residue in the dimeriza- tion site of YwlE, replacing a tyrosine residue conserved in all eukaryotic lmwPTPs, demonstrates that lmwPTP regulation by oligomerization can be independent from tyrosine phosphorylation. Structured digital abstract l MINT-7148507: ywle (uniprotkb:P39155) and YwlE (uniprotkb:P39155) bind (MI:0407)by nuclear magnetic resonance ( MI:0077) Abbreviations AIR, ambiguous interaction restraints; BPTP, Bos taurus protein tyrosine phosphatase; BR, best representative; EM, energy minimization; HSQC, heteronuclear single-quantum correlation; lmwPTP, low-molecular-weight protein tyrosine phosphatase; PDGF, platelet-derived growth factor; SAXS, small-angle X-ray scattering; YwlE, Bacillus subtilis protein tyrosine phosphatase. 4346 FEBS Journal 276 (2009) 4346–4357 ª 2009 The Authors Journal compilation ª 2009 FEBS suggested. The regulation of lmwPTPs is not completely understood and different mechanisms have been described [6]. Reversible inactivation by oxidation of the catalytic cysteine of phosphatases has been shown to provide a regulation mechanism [7]. In the case of lmwPTP, the presence of an additional cysteine results in the reversible formation of a disulfide bond [8]. Phosphorylation of a tandem repeat of tyrosine residues (YY-loop) lining the active site [9] has also been suggested as a possible regulation mechanism leading to either activation or inactivation, depending on the substrate involved [9–12]. Dimerization of Bos taurus lmwPTP (BPTP) has been observed in crystals [13] and has also been shown to take place in solution [14], and the protein evolves further into higher molecular weight species which stand in fast equilibrium with the mono- mer and dimer [15]. Dimerization involves the tyrosines of the YY-loop and residues of the active site, leading to an intrinsically inactive species. It has been suggested that lmwPTP oligomerization could be an additional regulation mechanism, although its physiological relevance remains unproven [13]. A major objection is the high dissociation constant of the lmwPTP dimer in vitro, although crowding conditions inside the cytoplasm may enhance oligomerization [16,17]. Prokaryotic lmwPTPs have recently been identified and have been far less studied than eukaryotic lmwPTPs [18]. Some prokaryotic lmwPTPs are viru- lence factors that mimic eukaryotic phosphatases and dephosphorylate eukaryotic proteins, thereby interfer- ing with the host defense response. An example is a phosphatase from Mycobacterium tuberculosis released into the extracellular medium, from which it is proba- bly translocated into macrophages, interfering with the host signaling pathways [19]. Endogenous prokaryotic lmwPTPs participate in the regulation of bacterial metabolism [20]. They can be divided into two types on the basis of their sequence and biological characteristics [21]. The first type binds and dephosphorylates endogenous kinases (BY-kinases), with which they often appear to be co-regulated in a single operon [22–24]. They control the biosynthesis and transport of virulence factors, such as exo- and capsular polysaccharides [25–28]. One member is Wzb from Escherichia coli, which acts with the kinase Wzc in the regulation of colanic acid production [29–31]. The second type has been found in Gram-positive bacteria [32]. Although little is known about their function to date, one representative, YwlE from Bacil- lus subtilis, has been identified as the cognate phosp- hotyrosine-phosphatase of McsB, McsA and CtsR [33]. It has been suggested that they are essential in certain processes, such as bacterial stress resistance. Comparison of lmwPTPs from phylogenetically dis- tant species (endogenous prokaryotic and eukaryotic forms) is expected to identify conserved features that may shed light on the intrinsic regulation mechanisms of this class of phosphatases. Prokaryotic and eukaryotic lmwPTPs show low sequence homology (less than 30% sequence identity) [20], but a comparison of the presently available X-ray [33–38] and NMR [20,39] structures shows a well- conserved tertiary structure exhibiting a Rossman fold [40]. It has been proposed that substrate specificity is mainly determined by the residues lining the active site. Eukaryotic and prokaryotic lmwPTPs acting on eukaryotic substrates as virulence factors show high similarity in these residues [34–37]. Endogenous prokaryotic lmwPTPs acting on pro- karyotic substrates have different specificities and correspondingly different residues lining the active site. Wzb, the only representative of type one prokaryotic lmwPTPs with an available experimental structure [20], possesses mainly hydrophobic residues GAL- VGKGA (38–45), compared with polar and aromatic residues in the case of eukaryotes. The B. taurus and human forms share the sequence SDWNVGRSP (47–55), forming the so-called W-loop lining the active site. The second type of endogenous lmwPTP, repre- sented by YwlE [39], has a loop with the sequence FASPNGKA(40–47), containing both polar and hydrophobic residues. In both types of endogenous lmwPTP, W49, suggested to be important for eukary- otic substrate recognition, is missing [20]. The YY-loop is the second flexible loop that appears over the active site in all lmwPTPs. The name comes from the two consecutive tyrosine resi- dues found in eukaryotic lmwPTPs. All known eukaryotic and prokaryotic lmwPTPs bear at least one tyrosine in this region, with the exception of YwlE, which possesses a nonphosphorylatable phen- ylalanine (F120). Thus, although phosphorylation and dimerization could be related regulatory events in most lmwPTPs, phosphorylation cannot be a regu- latory mechanism of YwlE. However, dimerization, as shown below, is conserved. The dimerization of both eukaryotic and prokaryotic lmwPTPs involves the active site, and the best docking model of the prokaryotic dimer is very similar to the crystal struc- ture of the eukaryotic dimer. Furthermore, both phosphatases evolve to similar higher order oligo- mers, as detected by NMR relaxation. The evolution- ary conservation of the oligomerization process strongly suggests a functional role in spite of the large dissociation constants measured in vitro. J. Blobel et al. Conserved weak protein interactions in lmwPTP FEBS Journal 276 (2009) 4346–4357 ª 2009 The Authors Journal compilation ª 2009 FEBS 4347 Results Concentration-dependent chemical shifts and docking calculations Figure 1A compares the 15 N– 1 H heteronuclear single- quantum correlation (HSQC) spectra of YwlE recorded at protein concentrations of 0.05 and 1.0 mm. Although most of the peaks remain unperturbed, sev- eral cross-peaks show unequivocal concentration- dependent chemical shifts suggesting oligomerization (Fig. 1A). The combined 1 H and 15 N chemical shift changes are shown in Fig. 1B. The residues showing the largest variations are located in the neighboring region of the active site (Fig. 1C). Residues V39–A51, forming what would be the W-loop in eukaryotic lmwPTPs, as well as residue T84, facing the W-loop on the opposite site of the active site crevice, are among the most affected. Residues N10 and S14, located in the crevice of the active site, also show chemical shift changes. Furthermore, the aromatic resi- dues Y127 and F120 around the YY-loop, just outside the active site, are also affected. Only one perturbed residue (V32) is placed in a remote position from the A B D C Fig. 1. Concentration-dependent chemical shifts. (A) Overlay of expanded regions of 15 N– 1 H HSQC spectra of 0.05 mM (black) and 1.0 mM (blue) YwlE. (B) Combined 15 N and 1 H chemical shift changes. (C) Chemical shift mapping. Residues showing changes above the threshold, indicated by the black broken line in (B), are highlighted on the structure of YwlE (1zgg). The W- and YY-loops are shown in blue and red, respectively. The active site residues are shown in yellow. (D) Sequence alignment of YwlE and BPTP (adapted from Lescop et al. [19]). Residues showing the largest concentration-dependent chemical shift changes are highlighted in green. The active site of BPTP is highlighted in yellow. Conserved weak protein interactions in lmwPTP J. Blobel et al. 4348 FEBS Journal 276 (2009) 4346–4357 ª 2009 The Authors Journal compilation ª 2009 FEBS active site. A similar set of residues was perturbed by concentration changes of eukaryotic BPTP [14]. Figure 1D compares the chemical shift differences associated with the same concentration changes superimposed onto the aligned sequences of YwlE and BPTP. The oligomerization of BPTP has been characterized extensively by NMR chemical shifts [14], NMR relaxa- tion [15], 129 Xe-NMR [41], analytical ultracentrifuga- tion [13], X-ray [13] and small-angle X-ray scattering (SAXS) [42]. At low concentrations (< 0.25 mm), only monomeric and dimeric forms are detectable. At higher concentrations, NMR relaxation, 129 Xe-NMR and SAXS experiments detect the formation of larger oligomers. NMR relaxation data can be explained by the presence of an additional species which is compati- ble with a tetramer, although more complex oligomeri- zation processes cannot be ruled out. Chemical shift and ultracentrifugation data of BPTP have been analyzed previously as a two-state mono- mer–dimer equilibrium, and this was shown to be a correct approximation up to a concentration of 0.5 mm. Assuming that the monomer–dimer equilib- rium is also the dominant oligomerization process in YwlE, we used the chemical shift restraints to model the structure of the dimer using the molecular docking program haddock 2.0 [43]. The NMR structure of YwlE (1zgg) was used as the model for the monomeric form. The docking protocol and the definition of active and passive restraints are described in Materials and methods. The 200 best solutions clustered into five families. The five families are related by the relative rotation of the two lmwPTP monomers around an axis going through their active sites. The RMSD values, buried surface, energy components, haddock scoring of all families (Table S1) and Ramachandran map analysis of the best family (Table S2) are given as Supporting information. The most populated family (containing more than 50% of the solutions) includes the two structures with the best haddock scoring terms (E haddock = )107.8 and )101.0 kcalÆmol )1 ). Consi- dering the average of the 10 best structures in each family, the same family also shows the largest buried surface area (1278 ± 135 A ˚ 2 ) and the largest desolva- tion energy ()38.3 ± 5.6 kcalÆmol )1 ). The best representative (BR) of the most populated family, sharing an RMSD with all other family mem- bers of 3.8 ± 1.1 A ˚ , shows very good agreement with the crystallographic dimer of BPTP [13]. The resulting superposition of both dimers is shown in Fig. 2A. In the docking model of the YwlE dimer, the flex- ible loops around the active site (W- and YY-loops) are involved in the stabilization of the dimer, in full agreement with the crystallographic dimer of BPTP. Other structural similarities between the prokaryotic model and the eukaryotic dimer are the aromatic residue F120 and the catalytically active C7 of YwlE, which occupy the positions of Y131 and C12 of BPTP. The second residue defining the active site motif of all phosphatases, namely the arginine placed six residues after the catalytic cysteine, is also located in equivalent positions in both prokaryotic and eukaryotic dimeric lmwPTPs (Fig. 2B). Residue W49 of BPTP, after which the eukaryotic W-loop is named, is replaced by F40 in the prokaryotic form (Fig. 2B). A B Fig. 2. (A) Best YwlE dimeric docking model (green) superimposed onto the crystallographic BPTP dimer (gray). (B) Expansion of the active site of YwlE (green) displaying functionally important side- chains (colored by atom type) superimposed with the equivalent side-chains of BPTP (gray). The labels show the structurally equiva- lent YwlE ⁄ BPTP residues. C7 ⁄ C12 and R13 ⁄ R18 participate in the catalytic activity, and F40 ⁄ W49 and F120 ⁄ Y131 are involved in dimerization. J. Blobel et al. Conserved weak protein interactions in lmwPTP FEBS Journal 276 (2009) 4346–4357 ª 2009 The Authors Journal compilation ª 2009 FEBS 4349 Self-association monitored by NMR relaxation The concentration-dependent chemical shift changes in YwlE are smaller in magnitude than those observed for BPTP. 15 N-NMR relaxation rates are very sensitive to self-association equilibria as they strongly depend on the rotational correlation time of all the species in solution. Therefore, we used longitudinal (R 1 ) and transverse (R 2 ) 15 N-NMR relaxation rates measured at different protein concentrations to analyze the oligo- merization of lmwPTP. Figure 3 shows the average R 2 ⁄ R 1 values (<R 2 ⁄ R 1 >) of all measured residues at different YwlE concentrations. The increase in <R 2 ⁄ R 1 > at higher concentrations confirms the self- association of YwlE. When multiple species coexist in fast exchange in solution, the measured 15 N-NMR relaxation rates are the concentration-weighted averages of the individual relaxation rates of each species. In NMR relaxation experiments, all residues in the protein are sensing the changes in the correlation time associated with the for- mation of higher molecular weight species, whereas only residues in the interface of the dimer show changes in chemical shifts. The 15 N-NMR relaxation approach makes use of a large number of independent measurements (approximately the number of residues times the number of concentrations), giving the possi- bility to distinguish between different oligomerization models. However, the structure of the monomer and dimer are required to analyze the data. The combined use of NMR spin relaxation and hydrodynamic calculations has previously been shown to be a powerful tool for the characterization of weak protein–protein interactions in solution [15, 41, 44–48]. This approach assumes that the lifetime of the differ- ent oligomeric species is significantly longer than their rotational correlation times, but the exchange is still fast on the chemical shift time-scale. Under these conditions, the measured relaxation rates are inter- preted as the weighted average of the relaxation rates of the coexisting individual species [48]. A detailed analysis of the oligomerization process was carried out by comparing the residue-specific relaxation rates obtained experimentally with those derived from hydrodynamic calculations of the experi- mental monomer structure and the haddock model for the dimer, as described below. The theoretical relaxation data of the monomer and the haddock model of the dimer were computed using HydroNMR [49,50], and the dissociation constant of the dimer (K d ), giving the relative populations of monomer and dimer at different concentrations, was fitted to reproduce the experimental R 2 ⁄ R 1 values of the individual residues (see Materials and methods). This approach gives a well-defined minimum (v 2 = 1.128) with a K d value of 5.20 ± 0.20 mm. The experi- mental and fitted R 2 ⁄ R 1 values for all four relaxation datasets are shown in Fig. 4A–D. The residue-specific residuals are given in Fig. S1. Good agreement was observed at the lower protein concentrations, but the calculated R 2 ⁄ R 1 values at higher lmwPTP concentrations were systematically lower than the experimental values (Fig. 4A). A statis- tically significant better fit was obtained by the inclu- sion of an additional species (an isotropic tetramer) in the equilibrium, as observed previously in the case of BPTP [15,41,42]. As described previously for BPTP, we modeled the higher oligomers of YwlE as a single isotropic tetra- mer, i.e. with the same relaxation rates, R 1T = 0.689 and R 2T = 42.4 s )1 , for all residues (see Materials and methods). This procedure provided a much better agreement with the experimental relaxation data (v 2 = 0.921), as shown in Fig. 4F–I,, yielding thermo- dynamic constants of K d = 7.59 ± 0.73 mm and K t = 0.27 ± 0.10 mm. The improvement derives mainly from the better reproduction of the high- concentration data (cf. Fig. 4E, J). The reported uncer- tainties were obtained from a Monte-Carlo analysis of the model as described in Materials and methods. The improvement in the figure of merit v 2 from 1.128 to 0.921 is statistically significant according to the F-test (P <10 )10 ), indicating that the YwlE oligomerization equilibrium involves at least three species. An equiva- lent situation has been demonstrated previously in the case of BPTP [15]. The dependence of the dissociation constants on the assumed values of R 1T and R 2T was checked by testing values diverging by ± 20% from the initial estimates. The variations in K d and K t were 0.23 mm and 0.11 mm, respectively, centered around the initially estimated dissociation constants. Fig. 3. Average R 2 ⁄ R 1 (<R 2 ⁄ R 1 >) values measured at different YwlE concentrations. Conserved weak protein interactions in lmwPTP J. Blobel et al. 4350 FEBS Journal 276 (2009) 4346–4357 ª 2009 The Authors Journal compilation ª 2009 FEBS Figure 5 compares the concentration dependence of the molar fractions of the different oligomers of YwlE and BPTP calculated from the dissociation constants determined by NMR. Discussion Evolutionary conservation of specific sequence or structural features in homologous proteins is consid- ered to be a relevant criterion for their physiological significance. Intermolecular interactions are also vali- dated by their conservation in phylogenetically distant species. In this work, we show that similar oligomeri- zation processes are conserved in eukaryotic and pro- karyotic lmwPTPs. Previous studies of lmwPTP from B. taurus have demonstrated the formation of oligomers in solution [13–15,41,42]. A dimer showing intermolecular contacts involving the active site and tyrosine residues in the YY-loop was observed in crystals [13] and was con- firmed to exist in solution [14]. As the active site is closed by the second molecule on dimer formation, this species is intrinsically inactive. Higher order oligomers were also detected by concentration-dependent NMR relaxation rate measurements [15], 129 Xe-NMR [41] and SAXS [42]. The oligomerization processes were found to correspond to weak interactions in vitro, with A B C D E F G H I J Fig. 4. R 2 ⁄ R 1 values of individual residues measured at the four YwlE concentrations (mM) of 1.00 [(A) and (F) in blue], 0.50 [(B) and (G) in green], 0.25 [(C) and (H) in dark yellow] and 0.10 [(D) and (I) in red]. Calculated values using the best-fitted parameters for the monomer– dimer (A–D) and monomer–dimer–tetramer (F–I) models are shown in black. The contribution of the highest concentration experiments to the fitting error of individual residues is shown in (E) for the monomer–dimer model and in (J) for the monomer–dimer–tetramer model. The individual contributions from all the concentrations are given as Supporting information. Fig. 5. Molar fractions of the different species present in the YwlE (full lines) and BPTP (broken lines) oligomerization equilibria. Mono- mers are shown in red, dimers in green and tetramers in blue. Points and circles represent the experimental points with their uncertainties. J. Blobel et al. Conserved weak protein interactions in lmwPTP FEBS Journal 276 (2009) 4346–4357 ª 2009 The Authors Journal compilation ª 2009 FEBS 4351 dissociation constants in the millimolar range, and, in spite of the putative regulatory role that can be associ- ated with an interaction that changes the phosphatase activity reversibly, the large dissociation constants shed some doubt on the physiological relevance of this mechanism. YwlE from B. subtilis is a prokaryotic lmwPTP involved in the regulation of endogenous bacterial meta- bolic processes [33], not influenced, like prokaryotic virulence factors, by the interaction of bacteria with eukaryotic hosts. As such, the comparison with BPTP is expected to genuinely display the effects of evolution in lmwPTPs of two phylogenetically distant organisms. The very similar fold of YwlE and eukaryotic lmwPTP monomers provides a strong case for a common evolu- tionary origin, in spite of only a modest sequence homol- ogy. Low sequence homology is expected between homologous proteins from very distant species, and stresses the relevance of the conservation of specific resi- dues or residue classes in particular sites. Here, we have analyzed the oligomerization of YwlE using NMR chemical shift and 15 N-NMR relaxation measurements at different protein concentrations in order to compare it with the previously reported oligomerization of BPTP. The structure of the YwlE dimer was modeled by computational docking of two monomers using experi- mental NMR chemical shifts as constraints. The BR of the family with the highest average haddock score, which is also the most populated, shows the highest similarity to the crystal structure of dimeric BPTP. A detailed comparison of the specific residues forming the dimer interface confirmed that the dimers formed by eukaryotic and prokaryotic lmwPTP are, indeed, homologous structures. Two pairs of aromatic residues seem to play equivalent roles in the formation of the dimer in YwlE and BPTP: F120 ⁄ Y131 and F40 ⁄ W49 from the prokaryotic and eukaryotic lmwPTPs, respec- tively. F120 and Y131 occupy similar positions in the interaction surface, close to the active site cysteines C7 and C12 of YwlE and BPTP, respectively. Tyrosines 131 and 132 are highly conserved in all eukaryotic lmwPTPs and are phosphorylation sites implicated in lmwPTP regulation. The other pair of aromatic residues involves the highly conserved tryptophan, which is present in nearly all eukaryotic lmwPTPs and gives the name to the W-loop. This flexible loop shows maximal variations between eukaryotic and endogenous prokaryotic lmwPTPs, in agreement with the different characteris- tics of their respective substrates. W49 is missing in prokaryotic lmwPTP, whereas the role of its indole side-chain in the stabilization of the dimer seems to be taken on by the phenyl group of F40 of YwlE. Residues N44 in YwlE and R53 in BPTP, both located in the W-loop, seem to play equivalent roles in the stabilization of the dimers by interacting with their related residues N44 and R43 of the second molecule of the dimer. The physical characteristics of the residue pairs F40 ⁄ W49, N44 ⁄ R53 and F120 ⁄ Y131 of YwlE and BPTP are conserved in PtpB, a lmwPTP from the Gram-negative bacterium Salmonella aureus (F36 and N40) [51], and Etp from E. coli (H42 and K46). Escherichia coli Wzb, a member of the first type of endogenous lmwPTPs, does not have the aromatic homolog to F40 ⁄ W49 in the substrate recognition loop. However, concentration-dependent 15 N-NMR relaxation data for this protein point to the formation of higher molecular weight species [20]. The apparent correlation time increases from 9.95 ns (0.1 mm)to 10.7 ns (0.3 mm) and, compared with the theoretical value for the monomer (9.21 ns) predicted from its NMR structure (2fek) using HydroNMR [49,50], sug- gests a weak self-association mechanism, similar to that observed for YwlE and BPTP. The similarity of the lmwPTP dimers of the prokary- otic and eukaryotic forms, involving structurally related, although not identical, residues, strongly sug- gests that the conserved feature is, indeed, the forma- tion of a dimer, and rules out the alternative explanation that dimer formation is a necessary side- effect of the conservation of the active site and sur- rounding substrate recognition and regulation loops. Phosphorylation of the adjacent tyrosine residues Y131 and Y132 in eukaryotic lmwPTP is considered to be a regulation mechanism. Interestingly, the crystal structure of the BPTP dimer, with a bound phosphate and the tyrosine side-chain pointing to the active site of the second molecule, is reminiscent of the expected reaction product of a dephosphorylation reaction, although dimerization does not require the previous phosphorylation of the tyrosine. The observation of dimeric YwlE having a phenylalanine residue in the dimer interface in place of a tyrosine confirms that lmwPTP dimerization is independent of phosphoryla- tion. However, the structure of the YwlE dimer sug- gests that the active site of each molecule is occupied by the residues of the other molecule forming the dimer, and therefore the dimer is an inactive species. Modulation of the monomer–dimer equilibrium could therefore provide a mechanism to modulate phospha- tase activity. The formation of higher oligomers also seems to be conserved between eukaryotic and prokaryotic lmwPTPs. Higher oligomers formed by the interac- tion of dimers are also expected to be enzymatically Conserved weak protein interactions in lmwPTP J. Blobel et al. 4352 FEBS Journal 276 (2009) 4346–4357 ª 2009 The Authors Journal compilation ª 2009 FEBS inactive. The W49G BPTP mutant, in which dimeri- zation is prevented, shows a much lower tendency to oligomerize (results not shown). Larger oligomers are known to be preferentially stabilized by crowding con- ditions as found inside the cell [16,52]. Crowding is expected to decrease the effective dissociation constant of a lmwPTP tetramer (72 kDa) by a factor of 10 1 –10 2 . Larger aggregates or aggregates of higher molecular weight can give reductions of 10 3 –10 5 in the dissociation constants inside the cell relative to the values that would be determined in vitro [53]. A moderate decrease in the dissociation constants of BPTP oligomers has been observed in vitro in the presence of a low-molecular-weight crowder [41]. Oligomer formation, therefore, could be a requirement to ensure that the effective dissociation constants match the protein concentrations in vivo. One can speculate on the hypothetical advantages of a regulation mechanism based on the equilibrium between active monomers and an inactive dimer, in which the active site is also the dimerization site. Pro- vided that the stability of the dimer is only moderate, increasing the concentration of possible substrates would cause an increase in the concentration of active monomers, as substrate binding would compete with dimerization. This suggests that oligomeric lmwPTP would provide a latent reservoir of phosphatase (equivalent to a proenzyme form) that could be acti- vated by phosphorylated substrates, and would return to the inactive form after the substrate has been exhausted by the activity of the phosphatase. This self-regulating mechanism would allow for the genera- tion of ‘phosphorylation pulses’ following kinase activity. Materials and methods Protein preparation and NMR experiments Sample preparation and NMR measurements, including HSQC and 15 N-NMR relaxation studies, have been described elsewhere [39]. Briefly, reducing conditions were ensured by the presence of 30 mm dithiothreitol in solution made up of 50 mm Tris ⁄ HCl buffer at pH 7.5, 50 mm NaCl and 10% D 2 O. No phosphate was present. The NMR experiments were performed at 25 °C on a 600 MHz instru- ment. Spectra were analyzed using NMRPipe [54]. 15 N-NMR relaxation rates R 1 and R 2 were measured at 0.1, 0.25, 0.5 and 1.0 mm total protein concentrations. Changes in chemical shift were monitored by comparing measure- ments at 0.05 and 1.00 mm lmwPTP. The combined changes in chemical shift (Dd) were calculated using the relationship Dd ¼½Dd 2 H þðDd N =5Þ 2  1=2 ð1Þ where Dd H and Dd N are the changes in chemical shift observed in the 1 H and 15 N dimensions, respectively. Generation of a dimeric model by computational docking Models of potential lmwPTP dimers were generated from the first model of the NMR structure 1zgg using haddock 2.0 [46]. This approach is implemented in cns [55] (cns Version 1.2 used here) and takes advantage of experimen- tally measured data reporting on perturbations in the pro- tein–protein interface. Here, concentration-dependent chemical shifts, as detected by HSQC experiments, were used. As suggested by the authors of haddock, residues showing strong chemical shift changes in combination with a solvent accessibility of greater than 50% were chosen as ‘active’ ambiguous interaction restraints (AIRs). These included the three residues F40, N44 and F120. A second group of residues serving as a less restrictive type of restraint, generally referred to as ‘passive’ AIRs, was made up of residues showing strong chemical shift changes and also their direct neighbors, both of which had to exhibit a surface accessibility of greater than 30%. The 13 residues G9, N31, N33, S42, P43, G45, T49, H50, T84, H85, G121, I126 and K128 passed the restriction criteria for passive AIRs. The solvent accessibility of all residues was deter- mined by the program naccess [56] from the monomer. haddock docking was launched using the default settings of the program, whereas python scripts derived from aria [57] were used to analyze all results and automate the pro- cess. The process consists of two docking stages: a rigid body energy minimization (EM) and a semi-rigid simulated annealing in torsion angle space. It should be noted that, during the rigid body EM, a 180° rotated model was gener- ated from each rigid-docked structure to amplify the diver- sity amongst dimeric models. In the second stage, all residues making intermolecular contacts within a 5 A ˚ cut- off were considered as flexible. No symmetry restraints were enforced. The full electrostatic binding energy was pre- served throughout the protocol. In the scoring of the final dimeric models, the contributions from van der Waals’ and electrostatic interactions, AIR distance restraints and desol- vation energies, as well as buried surface area terms, were scaled and summed in the haddock scoring term (E haddock ) as suggested by the authors. haddock 2.0 gener- ated 1000 dimers during hard docking. The best 200 dimers were retained for the semi-flexible simulated annealing step. The refined final 200 dimers were sorted into families using the program cluster_struc, a part of the haddock program package, using a maximum RMSD difference of 12 A ˚ amongst the family members in combination with a mini- mum family size of five structures. The BR of a family is J. Blobel et al. Conserved weak protein interactions in lmwPTP FEBS Journal 276 (2009) 4346–4357 ª 2009 The Authors Journal compilation ª 2009 FEBS 4353 the structure that has the lowest average RMSD to all the remaining members. The BR of the most populated family, which has the highest average haddock score, as well as the largest average buried surface area and the highest des- olvation energy amongst the 10 best members (Fig. S1), was chosen as the model of the YwlE dimer. Hydrodynamic calculations 15 N-NMR relaxation rates for monomer and dimer were calculated using the program HydroNMR [49]. Hydrogens were added to the dimer using a program from the WHAT IF server (http://swift.cmbi.ru.nl/servers/html/index.html). The atomic element radius was assigned to be 3.3 A ˚ and the atomic distance (N–H) 1.02 A ˚ [50]. The temperature was set to 25 °C, the viscosity to 8.91 · 10 )3 P and the NMR field strength to 14.09 T. For the monomer of B. subtilis, a rotational correlation time of s c Mon = 8.65 ns (<R 2 ⁄ R 1 > = 8.36) was calculated, exhibiting an anisot- ropy of D par ⁄ D per = 1.18. The dimer had a s c Dim value of 20.00 ns (<R 2 ⁄ R 1 > = 43.14) and D par ⁄ D per = 1.58, being clearly the most anisotropic species. For the tetramer, no structure is available, but it is expected to be a compact globular object. When calculating the theoretical relaxation rates for the tetramer, solvent depletion effects arising from protein–protein interfaces must be accounted for. This can be achieved using the rotational correlation time of the dimer (s c Dim ) for the calculation of the rotational correla- tion time of a solvent-depleted monomer (s c DMon ) through the relationship s c Dmon = s c Dim ⁄ 2.78, giving s c DMon = 7.19 ns [58,59]. The theoretical rotational correlation time for the tetramer can then be calculated by s c Tet = ns c DMon , with n = 4, resulting in s c Tet = 28.78 ns [15]. From s c Tet , theoretical R 1T and R 2T values for a spherical body of 0.689 and 42.4 s )1 (R 2 ⁄ R 1 = 61.52) are calculated for a magnetic field of 14.09 T. The two relaxation rates are used for all residues of the tetramer in the fitting protocol of experimental 15 N-NMR relaxation data. The rotational correlation time of E. coli lmwPTP Wzb was calculated from the best energy model of the NMR structure 2fek [20] using HydroNMR [49,50] at 25 °C and 14.09 T. 15 N-NMR relaxation data analysis Experimental 15 N-NMR relaxation data were filtered for the fitting as described elsewhere [41]. Briefly, data from individual residues were not used when any of the following three situations were encountered: (a) heteronuclear Over- hauser effect < 0.6, (b) large (> 25%) experimental errors when compared with the relaxation rates R 2 ⁄ R 1 and (c) large disagreement (> 25%) between the experimental and simulated R 2 ⁄ R 1 values using the relevant model, filtering for residues affected by chemical exchange. A total of 95 experimental R 2 ⁄ R 1 values were extracted, leaving 313 R 2 ⁄ R 1 values spanning the four protein concentrations of 0.10, 0.25, 0.50 and 1.00 mm lmwPTP in the case of the monomer–dimer model and 309 R 2 ⁄ R 1 values for the monomer–dimer–tetramer equilibrium. The average relative relaxation rates (<R 2 ⁄ R 1 >) were calculated for each protein concentration. Experimental R 1 and R 2 values are the concentration- weighted average of the relaxation rates of all participating species. Equation (2) shows the calculation of the relaxation rates for the monomer–dimer–tetramer model, with M being the molar fraction of monomer, D the molar fraction of dimer and T the molar fraction of tetramer. R n ¼ M Á R nM þ D Á R nD þ T Á R nT ð2Þ The relaxation rate R n of each species is denoted with the suf- fix corresponding to each species. n is equal to 1 and 2 in the case of longitudinal and transverse relaxation, respectively. The equilibrium parameters were determined by minimiz- ing the error function defined in Eqn (3). v 2 ¼ 1=N ÁðR i R j ½ðR 2 =R 1 Þ exp ij ÀðR 2 =R 1 Þ theo ij  2 =½EðR 2 =R 1 Þ exp ij  2 Þ ð3Þ where i and j denote different residues (i) at varying protein concentrations (j ), and E(R 2 ⁄ R 1 ) exp is the corresponding experimental error. N is the number of experimental data used. For the monomer–dimer model, the fitting of the theoret- ical relaxation rates of two species to the experimental values adjusts a single parameter, namely the dissociation constant K d connecting the relative concentrations of monomer [M] and dimer [D] over all concentration ranges through Eqn (4). The presence of a tetramer, being a dimer of dimers, is accounted for by the dissociation constant K t given by Eqn (5). [T] denotes the concentration of tetramer. K d ¼½M 2 =½Dð4Þ K t ¼½D 2 =½Tð5Þ The minimization protocol consists of a grid search for each variable, followed by the minimization of all variables together using the function fmincon as implemented in Matlabª. Errors in the calculated parameters are deter- mined using the Monte-Carlo method. Thus, a new set of synthetic relaxation rates is calculated from the determined dissociation constants, whereas a further term is added to each relative relaxation rate R 2 ⁄ R 1 calculated from the experimental error multiplied by a random chosen value obtained from a Gaussian distribution. The newly gener- ated relaxation rate data set is fitted to the applied model. The process is repeated 100 times. The final error in the dis- sociation constants is equal to the standard deviation of the 100 obtained dissociation constants for each species. For Conserved weak protein interactions in lmwPTP J. Blobel et al. 4354 FEBS Journal 276 (2009) 4346–4357 ª 2009 The Authors Journal compilation ª 2009 FEBS the fitting of the experimental relaxation data to the mono- mer–dimer–tetramer model, a further type of error estima- tion is used to prove the validity of the results obtained using the minimization protocol with Monte-Carlo error estimation with respect to the relaxation rates of the tetra- mer (R 1T and R 2T ). Thus, 100 new sets of R 1T and R 2T were generated with a variation of 20% from their theoreti- cally determined values (0.689 and 42.4 s )1 ) using a Gauss- ian distribution. Each set was used for a new fitting to the experimental relaxation data using the minimization proto- col as described above. Acknowledgements The authors thank Dr Ewen Lescop (ICSN-CNRS) for useful discussions. This work was partially supported by funds from the Spanish Ministry of Education (BIO2007-63458 to MP). 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Weak oligomerization of low-molecular-weight protein tyrosine phosphatase is conserved from mammals to bacteria Jascha Blobel 1 ,. supported by funds from the Spanish Ministry of Education (BIO2007-63458 to MP). J.B. is a recipient of a pre- doctoral fellowship from the Spanish Ministerio de Education