PDBsum entry 1h2f

Hydrolase

PDB id: 1h2f

Contents

Protein chain

207 a.a. *

Ligands

VA3

PO4

Waters ×142

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structures of phosphate and trivanadate complexes of bacillus stearothermophilus phosphatase phoe: structural and functional analysis in the cofactor-Dependent phosphoglycerate mutase superfamily.

Authors

D.J.Rigden, J.E.Littlejohn, K.Henderson, M.J.Jedrzejas.

Ref.

J Mol Biol, 2003, 325, 411-420. [DOI no: 10.1016/S0022-2836(02)01229-9]

PubMed id

12498792

Abstract

Bacillus stearothermophilus phosphatase PhoE is a member of the cofactor-dependent phosphoglycerate mutase superfamily possessing broad specificity phosphatase activity. Its previous structural determination in complex with glycerol revealed probable bases for its efficient hydrolysis of both large, hydrophobic, and smaller, hydrophilic substrates. Here we report two further structures of PhoE complexes, to higher resolution of diffraction, which yield a better and thorough understanding of its catalytic mechanism. The environment of the phosphate ion in the catalytic site of the first complex strongly suggests an acid-base catalytic function for Glu83. It also reveals how the C-terminal tail ordering is linked to enzyme activation on phosphate binding by a different mechanism to that seen in Escherichia coli phosphoglycerate mutase. The second complex structure with an unusual doubly covalently bound trivanadate shows how covalent modification of the phosphorylable His10 is accompanied by small structural changes, presumably to catalytic advantage. When compared with structures of related proteins in the cofactor-dependent phosphoglycerate mutase superfamily, an additional phosphate ligand, Gln22, is observed in PhoE. Functional constraints lead to the corresponding residue being conserved as Gly in fructose-2,6-bisphosphatases and Thr/Ser/Cys in phosphoglycerate mutases. A number of sequence annotation errors in databases are highlighted by this analysis. B. stearothermophilus PhoE is evolutionarily related to a group of enzymes primarily present in Gram-positive bacilli. Even within this group substrate specificity is clearly variable highlighting the difficulties of computational functional annotation in the cofactor-dependent phosphoglycerate mutase superfamily.

Figure 1.
Figure 1. Stereo MOLSCRIPT[23.] Figure of the PhoE-phosphate complex. Residues contacting the bound phosphate (larger spheres; shaded bonds) are drawn and labeled, along with the C-terminal residue Val208 whose carboxyl group interacts with Arg9. Residue His10 lies beneath the bound phosphate and is not labeled for reasons of clarity. Potential hydrogen bonds involving Arg9 are drawn as dotted lines. A blue C^a trace replaces part of the a-helix, which would otherwise obscure Gln22. The C-terminal tails of superimposed rat F26BPase (magenta) and phosphorylated E. coli dPGM (orange) are compared to the PhoE tail. The N terminus of the PhoE-phosphate structure is labeled in black and the C termini of the other structures labeled in their respective colors. The B. stearothermophilus PhoE protein was produced and purified as described[11.] and stored at -80 °C with preservation of activity in 10.0 mM Tris-HCl (pH 7.4), 1.0 mM DTT, 2.0 mM EDTA, 150.0 mM NaCl at a concentration of 25 mg/ml. Crystallization of the B. stearothermophilus PhoE-phosphate complex was accomplished using the vapor diffusion, hanging drop method[24.] in 24 well VDX culture plates (Hampton Research Inc.). Equal volumes of the protein sample and reservoir solution (1 µl each), and 0.5 µl of AMP substrate[11.] (25 mM) were used and the mixture was equilibrated against 1 ml of reservoir solution at 22 °C. A sparse matrix screen (Hampton Research Inc.) was used to obtain initial conditions. Diffraction quality irregularly shaped crystals were obtained after the refinement of initial conditions using 15.0% (v/v) ethylene glycol (EG), and 85.0 mM sodium cacodylate buffer (pH 4.5), as the reservoir solution, in one to two weeks (0.5 mm×0.4 mm×0.3 mm). The PhoE-vanadate complex was obtained by soaking the crystals described above in 50.0 mM of ammonium metavanadate (Sigma-Aldrich Co.), 30.0% EG, 20.0 mM sodium acetate buffer (pH 5.0) for three days in a depression dish. The PhoE-phosphate complex crystals were soaked for 20 seconds in a cryo-protecting solution (30.0% EG, 25.0 mM AMP, and 55.0 mM sodium cacodylate buffer (pH 4.5)) and flash frozen at -170 °C in liquid nitrogen immediately before data collection. The PhoE-vanadate complex crystals were also flash frozen in liquid nitrogen directly from their soaking solution. Both sets of crystals were diffracted using a synchrotron X-ray radiation source, beamline 5.0.1 at the Advanced Light Source, Lawrence Berkeley National Laboratory, with a 1.0 Å wavelength. The oscillation diffraction images were recorded using an ADSC Quantum 4u CCD detector. The diffraction data were processed with the HKL2000 package.[25.] A Free R-factor,[26.] calculated from 5% of reflections set aside at the outset, was used to monitor the progress of refinement. The free R set from the earlier crystal[17.] was transferred to the new data for the phosphate complex and completed randomly in the higher resolution range. This free R assignment was then transferred to the trivanadate complex data. The new complex structures were solved using rigid body refinement with a maximum likelihood target using CNS[27.] and atoms of the refined model subjected to a random positional shift of up to 0.3 Å in each of the x, y and z dimensions before further refinement in order to eliminate phase bias. All data were used throughout, without the application of sigma or amplitude-based cutoffs. Further rounds of positional and restrained individual B-refinement were carried out with a variety of stereochemical[28.] and other analyses [29. and 30.] periodically performed in order to locate possible model errors. Alternate conformations were introduced into the model when electron density clearly justified their inclusion. Ligands were modeled and refined with the aid of the HIC-UP database. [31.] The phosphate complex contains a phosphate ion and an ethylene glycol molecule in the catalytic site, and a second ethylene glycol molecule at the protein surface. The trivanadate complex contains trivanadate in the catalytic site and a partially occupied phosphate group at the protein surface. Water molecules were added into 3s peaks of the s-A weighted |2F[o] -F[c]| map within hydrogen bonding distance of suitable model atoms. Programs of the CCP4 package[32.] were used for manipulations and O [29.] for visualization and manual rebuilding. Structural superpositions were made with LSQMAN. [33.] The structures and structure factors have been deposited in the RCSB PDB with codes 1h2e and 1h2esf (phosphate complex) and 1h2f and 1h2fsf (trivanadate complex).

Figure 3.
Figure 3. ALSCRIPT[34.] alignment of three phylogenetically related groups of sequences, each containing Gln corresponding to PhoE Gln22. The groups, whose relationship is clear from phylogenetic analysis of a T-COFFEE alignment [35.] with PROTDIST and PROTPARS of the PHYLIP package, [36.] are separated by horizontal lines. For clarity, not all the sequences analyzed are shown. The sequences determined in the Streptococcus pyogenes MGAS315 genome[37.] represent the three determined S. pyogenes genomes. Similarly, Streptococcus pneumoniae R6[38.] and Listeria monocytogenes,[39.] were arbitrarily chosen as respective representatives of the two streptococcal and two listeria genomes sequenced. The Listeria monocytogenes sequence with Genbank code 16802560 contains 18 further residues at the N-terminal end, as indicated, which are not included in the alignment. PhoE numbering and secondary structure (determined with STRIDE[40.] for the high resolution phosphate complex) are shown above the alignment. Boxes mark residues of the catalytic core and shaded residues form the hydrophobic substrate-binding site in PhoE. Highly conserved positions are emboldened and totally conserved residues additionally italicized.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2003, 325, 411-420) copyright 2003.

Secondary reference #1

Title

Structure and mechanism of action of a cofactor-Dependent phosphoglycerate mutase homolog from bacillus stearothermophilus with broad specificity phosphatase activity.

Authors

D.J.Rigden, L.V.Mello, P.Setlow, M.J.Jedrzejas.

Ref.

J Mol Biol, 2002, 315, 1129-1143. [DOI no: 10.1006/jmbi.2001.5290]

PubMed id

11827481

Figure 5.
Figure 5. Neighbor-joining tree representation of structural relationships in the dPGM superfamily. Distances between structures were based on their structural similarities. The PDB codes were as follows: rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, 1bif; E. coli dPGM, 1e58; S. cerevisiae dPGM, 1qhf; Sch. pombe dPGM, 1fzt; human prostatic acid phosphatase, 1cvi; E. coli phytase, 1dkl; Aspergillus ficuum phytase, 1ihp; A. niger acid phosphatase, 1qfx.

Figure 6.
Figure 6. Modeled binding conformations for modeled substrates 3-phosphoglycerate and a-napthylphosphate. The same protein residues coloring scheme as that used in Figure 4 and Figure 5 is applied but only side-chain and C^a atoms of residues predicted to bind either substrate are shown. Asn16 was omitted for clarity from (a), since it lies in front of the 3-phosphoglycerate moiety. Hydrogen bonds are shown with dotted lines. (a) 3-Phosphoglycerate and (b) a-napthylphosphate.

The above figures are reproduced from the cited reference with permission from Elsevier

Secondary reference #2

Title

A cofactor-Dependent phosphoglycerate mutase homolog from bacillus stearothermophilus is actually a broad specificity phosphatase.

Authors

D.J.Rigden, I.Bagyan, E.Lamani, P.Setlow, M.J.Jedrzejas.

Ref.

Protein Sci, 2001, 10, 1835-1846.

PubMed id

11514674