Refined structures of placental alkaline phosphatase show a consistent pattern of interactions at the peripheral site

Abstract

In order to gain deeper insights into the functional sites of human placental alkaline phosphatase, the structures of the enzyme with the putative regulators L-Phe, pNPP and 5'-AMP [Llinas et al. (2005), J. Mol. Biol. 350, 441-451] were re-refined. Significant variations in ligand positioning and identity were found compared with the previous report. The multiple corrections to the model improved the phases and the electron-density maps, allowing the modeling of omitted side chains and multiple disordered residues. These improvements led to a change in the position of L-Phe at the peripheral binding site, which appeared to be reversed. The structure with pNPP contained only p-nitrophenol in three distinct sites, while the structure with 5'-AMP contained the p-nitrophenyl group in two of the sites instead of 5'-AMP. Comparison of the re-refined models shows a consistent pattern of interactions at the peripheral site.

Figure 1

The 2F _o − F _c electron density for both models of PLAP with pNPP bound (1zed): original model in mauve and the model presented in this paper (newly refined) in green. The difference electron density contoured at the −3σ level is depicted by red contours and indicated that three peptide bonds should be flipped (369, 372 and 374). The reversed peptide 369 creates a cis-Pro residue. The arrows indicate the three peptide bonds that were flipped in the process of refinement.

Figure 2

The 2F _o − F _c electron-density map contoured at the 1σ level phased with the refined model (originating from 1zef). (a) The l-Phe bound at the active site is in a position very close to the original position present in 1zef. (b) The l-Phe bound at peripheral site 2. The map shows that the l-Phe is reversed compared with its original position (in magenta) as suggested by the pattern of donor–acceptor relationships (Arg250 and Glu293).

Figure 3

The final refined model covered by a 2F _o − F _c electron-density map contoured at the 1σ level phased with the presently refined model (originating from 1zed). The difference electron density contoured at the −4σ level phased with the original model is shown in red. The map shows that the phosphate group of pNPP has been hydrolyzed and the bound species is a p-nitrophenol molecule (pNP). The blue model represents the original position of pNPP as present in 1zed. (a) The model of the active site with pNPP bound. The gray spheres represent Zn²⁺ ions, while the green sphere represents the Mg²⁺ ion. Ser104 has a covalently attached phosphate representing an active intermediate in the catalysis. (b) The peripheral site 2 near Arg250 with bound pNP molecule. (c) The third p-nitrophenol-binding site near the interface between three molecules.

Figure 4

Structure of the PLAP dimer (derived from the original 1zed structure). Red spheres indicate metal ions bound at the active site. Three molecules of p-­nitrophenol are bound at each subunit. The active site (site 1) and the peripheral site 2 have been described previously. An additional peripheral site 3 is found at the edge of the molecule, wedged between the symmetry-related molecules, as seen in Fig. 3 ▶(c).