Structure of the Bacteriophage λ Ser/Thr Protein Phosphatase with Sulfate Ion Bound in Two Coordination Modes^,

Abstract

The protein phosphatase encoded by bacteriophage lambda (lambda PP) belongs to a family of Ser/Thr phosphatases (Ser/Thr PPases) that includes the eukaryotic protein phosphatases 1 (PP1), 2A (PP2A), and 2B (calcineurin). These Ser/Thr PPases and the related purple acid phosphatases (PAPs) contain a conserved phosphoesterase sequence motif that binds a dinuclear metal center. The mechanisms of phosphoester hydrolysis by these enzymes are beginning to be unraveled. To utilize lambda PP more effectively as a model for probing the catalytic mechanism of the Ser/Thr PPases, we have determined its crystal structure to 2.15 A resolution. The overall fold resembles that of PP1 and calcineurin, including a conserved beta alpha beta alpha beta structure that comprises the phosphoesterase motif. Substrates and inhibitors probably bind in a narrow surface groove that houses the active site dinuclear Mn(II) center. The arrangement of metal ligands is similar to that in PP1, calcineurin, and PAP, and a bound sulfate ion is present in two novel coordination modes. In two of the three molecules in the crystallographic asymmetric unit, sulfate is coordinated to Mn2 in a monodentate, terminal fashion, and the two Mn(II) ions are bridged by a solvent molecule. Two additional solvent molecules are coordinated to Mn1. In the third molecule, the sulfate ion is triply coordinated to the metal center with one oxygen coordinated to both Mn(II) ions, one oxygen coordinated to Mn1, and one oxygen coordinated to Mn2. The sulfate in this coordination mode displaces the bridging ligand and one of the terminal solvent ligands. In both sulfate coordination modes, the sulfate ion is stabilized by hydrogen bonding interactions with conserved arginine residues, Arg 53 and Arg 162. The two different active site structures provide models for intermediates in phosphoester hydrolysis and suggest specific mechanistic roles for conserved residues.

Structural and Functional Characterization of a Novel Phosphodiesterase from Methanococcus jannaschii

“…A Dali (21) search using the monomer crystal structure found the following homologs in the Protein Data Bank (22) with Z-score higher than 6: (i) Pyrococcus furiosus mre11 nuclease (23) (Protein Data Bank code 1II7); (ii) Sus Scrofa and Phaseolus vulgaris purple acid phosphatase (24, 25) (Protein Data Bank codes 1UTE and 4KBP); (iii) Homo sapiens and Bacteriophage serine-threonine protein phosphatase (26,27) (Protein Data Bank codes 1AUI and 1G5B); and (iv) E. coli 5Ј-nucleotidase (28) (Protein Data Bank code 1USH). All of these structural homologs have low similarity with MJ0936 at the sequence level and they were not detected using a BLAST (29) search against the sequence data base.…”

The Catalytic Mechanisms of Binuclear Metallohydrolases

“…While λPP is considerably smaller than its eukaryotic counterparts (221 amino acid residues as compared to 330 residues for PP1 and 524 for PP2B), its three-dimensional fold in the catalytic core is very similar to that of other PPPs, 92 with the active site located on the surface of the protein in a shallow groove. The structure of λPP has been determined to 2.15 Å resolution in the presence of both Mn(II) and sulfate.…”

“…63 The observed µ-η 2 -η 2 -H 2 PO 4 -coordination mode in sweet potato PAP is unique among PAP structures. 64 92 The structure of rat PAP has also been determined in the presence of sulfate, 67 which coordinates monodentately to the redox-active iron and forms hydrogen bonds with Asn91 and His92. Both metal ions in the active site have six ligands, with one Fe(III)-bound and one bridging water ligand completing their first coordination spheres.…”

A Mycobacterial Cyclic AMP Phosphodiesterase That Moonlights as a Modifier of Cell Wall Permeability

“…Indeed, overexpression of Rv0805 was used to reduce intracellular levels of cAMP in M. tuberculosis to show that bacterially derived cAMP was essential to attenuate macrophage killing (4). Rv0805 is a member of the superfamily of metallophosphoesterases (MPEs) 6 that has been well characterized biochemically and structurally (7)(8)(9). MPEs contain five blocks of residues (D-(X) n -GD-(X) n -GNH(E/D)-(X) n -H-(X) n -GHXH) (10) with a conserved structural-fold, containing two metal ions in the active site.…”