Solution Structure and Conformational Heterogeneity of Acylphosphatase from Bacillus Subtilis

FEBS Letters 584 (2010) 2852–2856

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Solution structure and conformational heterogeneity of acylphosphatase from Bacillus subtilis

Jicheng Hu a,b,c, Dan Li b, Xiao-Dong Su b, Changwen Jin a,b,c, Bin Xia a,b,c,* a Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing 100871, China b School of Life Sciences, Peking University, Beijing 100871, China c College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China article info abstract

Article history: Acylphosphatase is a small enzyme that catalyzes the hydrolysis of acyl phosphates. Here, we present Received 18 March 2010 the solution structure of acylphosphatase from Bacillus subtilis (BsAcP), the ﬁrst from a Gram-posi- Revised 26 April 2010 tive bacterium. We found that its active site is disordered, whereas it converted to an ordered state Accepted 26 April 2010 upon ligand binding. The structure of BsAcP is sensitive to pH and it has multiple conformations in Available online 4 May 2010 equilibrium at acidic pH (pH < 5.8). Only one main conformation could bind ligand, and the relative Edited by Miguel De la Rosa population of these states is modulated by ligand concentration. This study provides direct evidence for the role of ligand in conformational selection. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Acylphosphatase Solution structure Multiple conformation Conformational selection Nuclear magnetic resonance

1. Introduction site forms a cradle-like and ordered conformation, and a sulfate and a chloride ion near Arg23 and Asn41 side-chains make exten- Acylphosphatase (AcP, EC 3.6.1.7), a widespread enzyme that is sive interactions with last three residues (Gly-Val-Phe) of loop found from archaebacteria to human, specifically catalyses the 14–21 [7]. It was recently reported that a non-native partially hydrolysis of carboxyl-phosphate bond in acyl phosphates [1]. folded state of the AcP from Sulfolobus solfataricus is enzymatically The true physiological function of AcP has not been fully revealed. active even if its active site appears not fully structured [14].In Studies have shown that AcP is involved in the control of the ionic addition, AcP is frequently used as a model system for studying states of the cells by hydrolyzing the b-aspartyl phosphate inter- protein folding and fibril formation, as the protein may aggregate mediates formed during ATPases action [2–4]. It was also shown to form amyloids in vitro by reducing the pH to 5.5 and with the that AcP associates with male hybrid sterility in Drosophila [5]. addition of 18–35% TFE [15–17]. It was reported that the formation Structures of several AcPs from different organisms have been of amyloid fibril by AcP is retarded in the presence of inorganic solved, most of which are in the ligand-bound state [6–13]. The phosphate, a competitive inhibitor binding to the active site, first AcP (muscle type AcP) structure was determined by Nuclear although it is not clear how phosphate modulates the conforma- magnetic resonance (NMR), and the structure in the ligand-free tional state of AcP [18,19]. state revealed the presence of mobility in loop regions, although In the present study, we report a structural characterization of the overall RMS difference (RMSD) for the structure ensemble is an AcP from Bacillus subtilis (BsAcP, encoded by yflL gene) by rather high (1.5 Å) [6,12,13]. In contrast, all X-ray structures of NMR spectroscopy. We have determined the solution structure of AcPs indicated that most loops in AcPs, including the active site BsAcP in the ligand-free state at neutral pH and showed that its ac- loop, adopt well defined ordered conformation [7–9,11]. In the tive site is intrinsically disordered in absence of ligand. In addition, crystal structure of CTAcP, loop 14–21 and loop 42–45 in the active given the amyloid formation properties of AcP at acidic pH, we probed the conformational change of BsAcP induced by reducing pH, and the conformational state modulated by phosphate at acidic pH. We provide direct evidence showing that BsAcP has multiple * Corresponding author at: Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing 100871, China. Fax: +86 10 62753790. populated conformations in equilibrium at pH below 5.8, and only E-mail address: [email protected] (B. Xia). one form of BsAcP is active, which could bind phosphate.

0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.04.069 J. Hu et al. / FEBS Letters 584 (2010) 2852–2856 2853

2. Materials and methods 3.2. Solution structure of BsAcP

2.1. Sample preparation We obtained essentially complete 1H, 13C, and 15N chemical shift assignments for ligand-free BsAcP in Tris buffer at pH 7.0 15N and 15N/13C labeled BsAcP proteins were expressed from a (Supplementary Fig. 2). Spectral analysis allowed the assignments pET-21a expression plasmid (Novagen) in Escherichia coli BL21 of 81 out of 88 backbone NH resonances, while NH signals of res- 15 13 (DE3) host using NH4Cl and C-glucose-based M9 medium. idues Gly14, Val15, Gly16, Phe17, Tyr19, Asp38, and His84 are The protein was purified with cation exchange chromatography not visible in the 2D 1H–15N HSQC spectrum. In addition, the followed by Superdex 75 gel-filtration. Sample for NMR spectros- NH2 signals of Gln13 were absent and only one peak from the 1 15 copy had approximately 0.3 mM protein in 50 mM Tris–HAc buffer NH2 of Asn36 was observed in the 2D H– N HSQC spectrum. 1 15 containing 10% D2O. Overall, 91% of the backbone, and 90% of the side-chain H, N, and 13C resonances were assigned for ligand-free BsAcP. 2.2. NMR spectroscopy The structural statistics of the final structures are given in Table 1. The structure of BsAcP is a mixed a/b fold with two a helices and NMR experiments were performed at 298 K on a Bruker a five-stranded antiparallel b sheet. It is characterized by the fol- AVANCE 600 NMR spectrometer equipped with a triple resonance lowing secondary structure elements (Fig. 1): b1 (2–11), a1 (19– probe. All spectra were processed with NMRPipe [20] and analyzed 26), b2 (31–36), b3 (42–48), a2 (50–62), b4 (68–77) and b5 (86– with NMRView [21]. Backbone sequential and side-chain assign- 88). Starting from N-terminus, there are five loops connecting the ments were obtained using standard 3D NMR experiments [22]. secondary structure elements and they have been labeled loop The chemical shift assignments of ligand-free BsAcP have been 1–5 (Fig. 1). The global folding of BsAcP is well defined due to deposited at BioMagResBank under accession number 6884. For the large numbers of NOEs. The average RMS difference (RMSD), phosphate binding, 2D 1H–15N HSQC spectra were collected, and among the final 20 structures, for heavy atoms in the secondary chemical shift changes in the spectra on transition of BsAcP from structure regions is 0.3 Å. free to bound state were followed until saturation was reached.

The dissociation constant Kd for phosphate binding was calculated 3.3. Active site of BsAcP by fitting the following equation: The atomic RMSD of the Ca atoms between the mean structure Dd ¼ 0:5Dd ððK =½Eþ½PO3=½Eþ1Þ of BsAcP and CTAcP for residues in the secondary structure regions obs qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffimax d 4 is 1.5 Å, revealing a high degree of structure conservation. How- 3 2 3 ðKd=½Eþ½PO4 =½Eþ1Þ 4½PO4 =½EÞ; ever, some local differences are observed in the active site. In the crystal structure of CTAcP, loop 14–21 (loop 1) in the active site 3 where, [PO4 ] and [E] are the respective concentrations of phos- forms an ordered and cradle-like conformation [7]. However, in 2 phate and protein; Ddobs can be defined as: Ddobs =(DdHN +(DdN/ the solution structure of ligand-free BsAcP, the conformation of 2 1/2 6.5) ) ; Ddmax is the maximum chemical shift difference between residues Gly14 to Arg18 in loop 1 is very flexible due to lack of dis- the free and phosphate-bound states. tance constraints. In this region, no NMR resonance was observed for residues Gly14, Val15, Gly16 and Phe17, and the NMR signals 2.3. Structure calculation of Arg18 were much broader than the rest signals of the protein.

NOE data for the structure calculation were derived from 3D 15N-NOESY-HSQC and 3D 13C-NOESY-HSQC spectra acquired at Table 1 298 K with a mixing time of 120 ms. Structure calculations were Experimental and structural statistics for the family of 20 structures of BsAcP. performed with the torsion angle dynamics annealing simulation program CYANA [23] and refined with AMBER7 [24]. A total of Parameters 200 structures were calculated using CYANA, and 100 structures Number of distance and dihedral restraints with the lowest target function values were selected and refined Intraresidue NOEs (|i j| = 0) 688 Sequential NOEs (|i j| = 1) 323 in AMBER7 with standard generalized Born model [24]. Finally, Medium range NOEs (2 6 |i j| 6 5) 165 20 lowest-energy structures were selected to represent BsAcP. Ter- Long range NOEs (|i j| > 5) 398 tiary structure was visualized with MOLMOL [25]. The coordinates Ambiguous NOEs 527 of ligand-free BsAcP structures have been submitted to the Protein All NOEs 2101 Hydrogen bond restraints 72 Data Bank under accession code 2FHM. Dihedral angle restraints 132 Number of NOE distance violations (>0.2 Å) 0 3. Results Number of dihedral angle violations (>5°)0 Average CYANA Target function value (Å) 2.07 ± 0.31 3.1. Enzymatic characterization of BsAcP Average AMBER energy (kcal/mol) 4949.33 ± 7.33 Average RMSD from the mean structure (Å) BsAcP can catalyze the hydrolysis of benzoylphosphate very Secondary structure efficiently (Supplementary Fig. 1). The optimal pH for the acyl Backbone heavy atoms 0.29 ± 0.05 phosphate hydrolysis activity of BsAcP was determined to be pH All heavy atoms 0.76 ± 0.08 5.3. Its catalytic activity drops sharply when the pH decreases, All residues Backbone heavy atoms 0.81 ± 0.20 and the enzyme is totally inactivated when the pH is below 4.0. All heavy atoms 1.32 ± 0.17 We carried out kinetic study for the enzyme at pH 5.3, and deter- 1 Ramachandran of non-glycine/proline residues (%) mined the Km value of 2.1 mM, Vmax of 0.53 mM min , kcat of Most favoured regions 87.6 3 1 3 1 1 18.3 10 s and kcat/Km is 8.7 10 s mM . Although BsAcP Additional allowed regions 11.0 displays a lower affinity to the substrate, it has a similar kcat/Km va- Generally allowed regions 0.9 lue to those of the vertebrate AcPs (3.5–9.5 103 s1 mM1) due to Disallowed regions 0.4 a higher efficiency for hydrolyzing the substrate. Overall G-factor 0.45 2854 J. Hu et al. / FEBS Letters 584 (2010) 2852–2856

acidic pH. The pH-induced NMR signal changes are reversible over this pH range. At pH 5.3, while BsAcP achieves its highest acyl phosphatase activity, NMR resonance assignments reveals that 40 residues possess nondegenerate NH resonances that exist alternately in a major (strong peak intensity) or a minor conformation (weak peak intensity), comprising 44% of the total residue number (Supplementary Fig. 2). The relative peak volume ratios of the major to minor conformations varies with residues but remains unchanged over concentrations range from 0.1 to 0.8 mM for each residue. In addi-

tion, the major and minor signals have similar R2 values through- out the sequence (Fig. 3B), indicating the two sets of resonances should be due to intramolecular rearrangement but not intermo- lecular interactions (e.g. monomer–dimer transition). Fig. 1. Solution structure of BsAcP. (A) Backbone traces (N, Ca, CO) of 20 The residues that possess nondegenerate resonances at pH 5.3 superimposed conformers with lowest AMBER energies of BsAcP. (B) Ribbon are mapped onto the structure of BsAcP. There are approximately representation of the energy minimized mean structure of BsAcP. four patches of residues that have two NH signals, (I) one located in the vicinity of active site; (II) one located in the vicinity of b2; In addition, the lower {1H}–15N heteronuclear NOE values for res- (III) one consisting of residues in a1 and the edge of a2; and ﬁnally idues around loop 1, loop 4 and the C-terminal tail indicate that (IV) one in loop 4 (Fig. 3C). For both the major and minor confor- these regions are more mobile than residues in the secondary mations, the 13Ca and 13Cb secondary chemical shifts indicate that structures (Supplementary Fig. 3). both forms have basically the same secondary structure features as We have performed 2D 1H–15N HSQC experiment to probe the that observed at pH 7.0. The 13Ca, 13Cb and combined NH chemical conformational changes associated with the phosphate binding. shifts difference between the major and minor conformations sug- During phosphate titration, the backbone NH signals of Gly14, gest that their structures mainly differ at loop regions and the mar-

Val15, Gly16, Phe17 and Tyr19, as well as the side-chain NH2 sig- gins of secondary structures (Fig. 3A). For most residues, the nals of Gln13, started to show up and their intensities gradually in- general NOE pattern observed in the 3D 15N NOESY-HSQC spectra creased as the phosphate concentration was increased. It was also is very similar with that observed at pH 7.0, whereas signiﬁcant noticed that the single peak from the side-chain NH2 of Asn36 was NOE pattern differences between major form and minor conforma- shifted due to phosphate binding, whereas a new peak correspond- tions were only observed for residues Gly10, Gln13, Arg18, Met23, ing to the other NH2 signal of Asn36 appeared and its intensity Glu58 and Glu76, etc. (Supplementary Fig. 4). gradually increased during the phosphate titration. When the We have also investigated the effect of phosphate binding on amount of phosphate was over 30 folds of that of the protein, the conformational heterogeneity of BsAcP at pH 5.3 (Fig. 2B). For res- phosphate induced changes in the 2D 1H–15N HSQC spectra idues around the active site, their NH cross-peaks from the major stopped (Fig 2A). The appearance of these signals suggests that conformation showed continuous NH chemical shift changes as the active site structural rearrangement and stabilization associate phosphate was titrated in (Fig. 3E). NH signals of Gly14, Gly16 with phosphate binding. and Arg18 were particularly affected, each shifts by over 0.5 ppm. Both backbone and side-chain NH cross-peaks for Asn36 3.4. Conformational heterogeneity at acidic pH were also shifted substantially. In contrast, the corresponding NH signals from residues around the active site of the minor conforma- To gain insight into the pH-induced conformational transition tion showed no chemical shift change, only their intensities were of BsAcP, a series of 2D 1H–15N HSQC spectra were acquired over decreased as the phosphate concentration increases. For residues the pH range of 7.0–2.5. When the sample pH was above 6.0, only far away from the active site, no chemical shift perturbation was a single set of resonances could be observed. However, two or more observed due to the phosphate binding, while NH peak intensities sets of resonances were observed when the sample pH was below of the major conformation continued increasing and NH peak 5.8, indicating the existence of multiple conformations for BsAcP at intensities of the minor conformation continued decreasing till

Fig. 2. Phosphate titration. The overlay of 2D 1H–15N HSQC spectra for ligand-free BsAcP (blue) and phosphate-bound BsAcP (red) at pH 7.0 (left) and pH 5.3 (right). At pH 7.0, resonances which display large chemical shift changes are labeled, and resonances that are entirely absent in the unbound state but appear in the phosphate-bound state are indicated with cycle. J. Hu et al. / FEBS Letters 584 (2010) 2852–2856 2855

13 a 13 b 2 2 1/2 Fig. 3. Conformational heterogeneity at pH 5.3. (A) C , C , and combined NH chemical shift differences (Ddcomb =(DdHN +(DdN/6.5) ) ) between the major and minor conformations vs. residue numbers. (B) Comparison of R2 values for the major (red) and minor (blue) NH signals of BsAcP at pH 5.3. (C) Residues that possess at least two NH resonances are mapped onto the structure of BsAcP (red). (D) Estimation of the dissociation constant Kd for phosphate binding to the major conformation of BsAcP. The Kd was calculated from phosphate-dependent changes in the Arg18 NH resonance and Asn36 NdH resonance. (E) Selective regions displaying the changes of resonances in the 2D 1H–15N HSQC spectra of BsAcP at pH 5.3 upon the addition of phosphate. Black, blue, green, magenta and red colors indicate the ratio of phosphate/BsAcP as 0, 1.0, 3.0, 6.0 and 15.0, respectively. The residue number is indicated. disappearance with increased phosphate concentration (Fig. 3E). revealed in many phosphatases, such as low molecular weight pro- These observations suggest that only the major conformation can tein tyrosine phosphatase from Bos taurus (PDB 1BVH) [26], Cam- bind phosphate while the minor conformation could not, and the pylobacter jejuni (2GI4) [27], Tritrichomonas foetus (1P8A) [28], B. phosphate binding alters the relative population of conformational subtilis (1ZGG) [29], E. coli (2FEK) [30], and human PRL-3 (1V3Z) distribution. The equilibrium dissociation constant (Kd) for phos- [31], and human phosphohistidine phosphatase 1 (2AI6) [32], sug- phate binding to the major conformation was determined to be gesting that active site conformational flexibility should be consid- 2.8 mM, based on the chemical shift changes of Arg18 NH reso- ered to understand ligand recognition and design inhibitors. nance and Asn36 NdH resonance from the phosphate titration In addition, our study reveals that a significant portion of resi- experimental data (Fig. 3D). dues of BsAcP has more than two conformations in solution as pH below 5.8, and there is a trend towards more populated confor- 4. Discussion mations as pH further reduced. At pH 5.3, when BsAcP achieves its highest enzyme activity, we found that BsAcP coexists in (at least) In this work, we have determined the solution structure of two populated conformational states. One of the states (the major BsAcP in the ligand-free state, the first AcP from a Gram-positive conformation) represents the relatively high-affinity binding state bacterium. The overall fold of ligand-free BsAcP is similar to the for phosphate (Kd 2.8 mM); the other state (the minor conforma- crystal structures of AcPs in the ligand-bound state, but the confor- tion) represents a different state with no binding affinity to phos- mation and flexibility of the active site loop region differ signifi- phate. The equilibrium between the two different states can be cantly. Our NMR data indicate that loop 1, loop 4 and the shifted by phosphate binding. The NMR chemical shifts of the com- C-terminal residue Ser91 around the active site region are more plex with phosphate indicate that the final binding state is similar mobile than the other parts of the structure. The backbone amides but not completely identical to the major conformation of the of Gly14, Val15, Gly16, Phe17 and Tyr19 in the active site region ligand-free state, since significant chemical deviation in the bound are not observable in any NMR spectra at pH 7.0, which is mostly state from the major conformation in the free protein are observed. likely due to exchange broadening on intermediate time scale. This As clear evidence is presented for the existence of multiple observation is similar to that reported in AcP from S. solfataricus,in conformations in the ligand-free state of BsAcP and a single set which an intermediate exchange regime was also found for several of conformation in the bound state, the interaction between BsAcP peaks in the active site region [19]. Binding of phosphate is associ- and phosphate supports the recently proposed pre-existing equi- ated with marked stabilization of the active site loop 1 conforma- librium model for protein–ligand interaction [33,34]. According tion, as the invisible NMR signals gradually appear, and the peak to the theory of pre-existing equilibrium model, proteins exist as intensity of weak peaks gradually restore in this region. These an ensemble of conformations in the ligand-free state, all typically observations further supports the idea that the cradle-like and or- with similar free energies, but some of these conformations have a dered conformation of the active site loop observed in the X-ray greater binding affinity to a certain ligand than the others. The li- structure is due to ligand binding [7]. The mobility of the active site gand will bind selectively to the active conformation, thereby loops as an important structure feature in ligand binding has been altering the protein conformational distribution to favor one or 2856 J. Hu et al. / FEBS Letters 584 (2010) 2852–2856 more bound states [33,34]. As conformational flexibility in an en- [12] Saudek, V., Williams, R.J., Stefani, M. and Ramponi, G. (1989) Mobility of zyme commonly allows it to bind structurally distinct substrates, secondary structure units of horse-muscle acylphosphatase. Relation to antigenicity. Eur. J. Biochem. 185, 99–103. it would mediate broad substrate specificity for an enzyme [35]. [13] Saudek, V., Wormald, M.R., Williams, R.J., Boyd, J., Stefani, M. and Ramponi, G. The substrates for AcPs include a wide range of acyl phosphates, (1989) Identification and description of beta-structure in horse muscle such as 1,3-diphosphoglycerate, carbamyl phosphate, succinyl- acylphosphatase by nuclear magnetic resonance spectroscopy. J. Mol. Biol. 207, 405–415. phosphate, acetylphosphate, and b-aspartylphosphate etc. In addi- [14] Bemporad, F. et al. (2008) Biological function in a non-native partially folded tion, AcPs also display hydrolytic activities on aryl phosphate state of a protein. EMBO J. 27, 1525–1535. monoesters nucleoside diphosphates, nucleoside triphosphatase [15] Chiti, F., Webster, P., Taddei, N., Clark, A., Stefani, M., Ramponi, G. and Dobson, C.M. (1999) Designing conditions for in vitro formation of amyloid and polynucleotides [36–38]. Thus, in order to reach sufficient protofilaments and fibrils. Proc. Natl. Acad. Sci. USA 96, 3590–3594. affinity for distinct substrates, a certain degree of conformational [16] Plakoutsi, G., Taddei, N., Stefani, M. and Chiti, F. (2004) Aggregation of the heterogeneity that is thermally accessible in solution might be acylphosphatase from Sulfolobus solfataricus: the folded and partially unfolded states can both be precursors for amyloid formation. J. Biol. Chem. 279, required. 14111–14119. In summary, we present here structural studies of BsAcP at both [17] Soldi, G., Bemporad, F. and Chiti, F. (2008) The degree of structural protection neutral and acidic conditions. Our work demonstrates that the at the edge beta-strands determines the pathway of amyloid formation in active site region of BsAcP is highly flexible in solution, which is globular proteins. J. Am. Chem. Soc. 130, 4295–4302. [18] Soldi, G., Plakoutsi, G., Taddei, N. and Chiti, F. (2006) Stabilization of a native stabilized upon ligand binding. The discovery of a dynamic equilib- protein mediated by ligand binding inhibits amyloid formation independently rium between two or more conformations of BsAcP at acidic pH of the aggregation pathway. J. Med. Chem. 49, 6057–6064. provides a unique view of BsAcP conformational heterogeneity [19] Pagano, K., Bemporad, F., Fogolari, F., Esposito, G., Viglino, P., Chiti, F. and Corazza, A. (2010) Structural and dynamics characteristics of acylphosphatase and the role of ligand in conformational selection. The origin of from Sulfolobus solfataricus in the monomeric state and in the initial native- conformational heterogeneity of BsAcP, the function of the na- like aggregates. J. Biol. Chem. 15, 25 [Epub ahead of print]. tive-like minor conformation observed at acidic pH and whether [20] Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J. and Bax, A. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX minor conformation is relate to the amyloid fibril formation, still pipes. J. Biomol. NMR 6, 277–293. awaits future investigation. [21] Johnson, B.A. (2004) Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol. Biol. 278, 313–352. [22] Sattle, M., Schleucher, J. and Griesinger, C. (1999) Heteronuclear Acknowledgments multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. NMR Spectrosc. All NMR experiments were carried out at the Beijing Nuclear 34, 93–158. [23] Guntert, P., Mumenthaler, C. and Wuthrich, K. (1997) Torsion angle dynamics Magnetic Resonance Center (BNMRC), Peking University. This re- for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273, search was supported by grants 30125009 to BX from the National 283–298. Science Foundation of China and grant 2006CB910203 to CJ from [24] Pearlman, D.A. et al. (1995) Amber, a package of computer-programs for the 973 Program of China. applying molecular mechanics, normal-mode analysis, molecular-dynamics and free-energy calculations to simulate the structural and energetic properties of molecules. Comput. Phys. Commun. 91, 1–41. Appendix A. Supplementary data [25] Koradi, R., Billeter, M. and Wuthrich, K. (1996) MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55. [26] Logan, T.M., Zhou, M.M., Nettesheim, D.G., Meadows, R.P., Van Etten, R.L. and Supplementary data associated with this article can be found, in Fesik, S.W. (1994) Solution structure of a low molecular weight protein the online version, at doi:10.1016/j.febslet.2010.04.069. tyrosine phosphatase. Biochemistry 33, 11087–11096. [27] Tolkatchev, D., Shaykhutdinov, R., Xu, P., Plamondon, J., Watson, D.C., Young, N.M. and Ni, F. (2006) Three-dimensional structure and ligand interactions of References the low molecular weight protein tyrosine phosphatase from Campylobacter jejuni. Protein Sci. 15, 2381–2394. [1] Stefani, M., Taddei, N. and Ramponi, G. (1997) Insights into acylphosphatase [28] Gustafson, C.L., Stauffacher, C.V., Hallenga, K. and Van Etten, R.L. (2005) structure and catalytic mechanism. Cell. Mol. Life Sci. 53, 141–151. Solution structure of the low-molecular-weight protein tyrosine phosphatase [2] Nediani, C., Fiorillo, C., Marchetti, E., Pacini, A., Liguri, G. and Nassi, P. (1996) from Tritrichomonas foetus reveals a flexible phosphate binding loop. Protein Stimulation of cardiac sarcoplasmic reticulum calcium pump by Sci. 14, 2515–2525. acylphosphatase. Relationship to phospholamban phosphorylation. J. Biol. [29] Xu, H., Xia, B. and Jin, C. (2006) Solution structure of a low-molecular-weight Chem. 271, 19066–19073. protein tyrosine phosphatase from Bacillus subtilis. J. Bacteriol. 188, 1509– [3] Nassi, P., Nediani, C., Fiorillo, C., Marchetti, E., Liguri, G. and Ramponi, G. (1994) 1517. Modifications induced by acylphosphatase in the functional properties of [30] Lescop, E., Hu, Y., Xu, H., Hu, W., Chen, J., Xia, B. and Jin, C. (2006) The solution heart sarcolemma Na+,K+ pump. FEBS Lett. 337, 109–113. structure of Escherichia coli Wzb reveals a novel substrate recognition [4] Nassi, P., Nediani, C., Liguri, G., Taddei, N. and Ramponi, G. (1991) Effects of mechanism of prokaryotic low molecular weight protein–tyrosine acylphosphatase on the activity of erythrocyte membrane Ca2+ pump. J. Biol. phosphatases. J. Biol. Chem. 281, 19570–19577. Chem. 266, 10867–10871. [31] Kim, K.A. et al. (2004) Structure of human PRL-3, the phosphatase associated [5] Michalak, P. and Ma, D. (2008) The acylphosphatase (Acyp) alleles associate with cancer metastasis. FEBS Lett. 565, 181–187. with male hybrid sterility in Drosophila. Gene 416, 61–65. [32] Gong, W., Li, Y., Cui, G., Hu, J., Fang, H., Jin, C. and Xia, B. (2009) Solution [6] Pastore, A., Saudek, V., Ramponi, G. and Williams, R.J. (1992) Three- structure and catalytic mechanism of human protein histidine phosphatase 1. dimensional structure of acylphosphatase. Refinement and structure Biochem. J. 418, 337–344. analysis. J. Mol. Biol. 224, 427–440. [33] Tsai, C.J., Kumar, S., Ma, B. and Nussinov, R. (1999) Folding funnels, binding [7] Thunnissen, M.M., Taddei, N., Liguri, G., Ramponi, G. and Nordlund, P. (1997) funnels, and protein function. Protein Sci. 8, 1181–1190. Crystal structure of common type acylphosphatase from bovine testis. [34] Kumar, S., Ma, B., Tsai, C.J., Sinha, N. and Nussinov, R. (2000) Folding and Structure 5, 69–79. binding cascades: dynamic landscapes and population shifts. Protein Sci. 9, [8] Cheung, Y.Y., Lam, S.Y., Chu, W.K., Allen, M.D., Bycroft, M. and Wong, K.B. 10–19. (2005) Crystal structure of a hyperthermophilic archaeal acylphosphatase [35] Tokuriki, N. and Tawfik, D.S. (2009) Protein dynamism and evolvability. from Pyrococcus horikoshii – structural insights into enzymatic catalysis, Science 324, 203–207. thermostability, and dimerization. Biochemistry 44, 4601–4611. [36] Paoli, P., Pazzagli, L., Giannoni, E., Caselli, A., Manao, G., Camici, G. and [9] Miyazono, K., Sawano, Y. and Tanokura, M. (2005) Crystal structure and Ramponi, G. (2003) A nucleophilic catalysis step is involved in the hydrolysis structural stability of acylphosphatase from hyperthermophilic archaeon of aryl phosphate monoesters by human CT acylphosphatase. J. Biol. Chem. Pyrococcus horikoshii OT3. Proteins 61, 196–205. 278, 194–199. [10] Pagano, K., Ramazzotti, M., Viglino, P., Esposito, G., Degl’Innocenti, D., Taddei, [37] Paoli, P., Camici, G., Manao, G., Giannoni, E. and Ramponi, G. (2000) N. and Corazza, A. (2006) NMR solution structure of the acylphosphatase from Acylphosphatase possesses nucleoside triphosphatase and nucleoside Escherichia coli. J. Biomol. NMR 36, 199–204. diphosphatase activities. Biochem. J. 349, 43–49. [11] Rosano, C., Zuccotti, S., Bucciantini, M., Stefani, M., Ramponi, G. and Bolognesi, [38] Chiarugi, P., Raugei, G., Fiaschi, T., Taddei, L., Camici, G. and Ramponi, G. (1996) M. (2002) Crystal structure and anion binding in the prokaryotic hydrogenase Characterization of a novel nucleolytic activity of acylphosphatases. Biochem. maturation factor HypF acylphosphatase-like domain. J. Mol. Biol. 321, 785– Mol. Biol. Int. 40, 73–81. 796.