For many years the crystallographic coordinates of the Escherichia coli AP (ECAP)  molecule provided the only source of structural information on APs, but now the three-dimensional structure of the first mammalian AP, i.e., human placental AP (PLAP; 1EW2; http://pdbbeta.rcsb.org/pdb/) has been solved . As had been predicted from sequence comparisons, the central core of PLAP, consisting of an extended β-sheet and flanking α-helices, is very similar to that of ECAP. The overall structure of PLAP is a dimer and each monomer contains 484 residues, four metal atoms, one phosphate ion, and 603 water molecules. The two monomers are related by a two-fold crystallographic axis (Figure 1). The surface of PLAP is poorly conserved with that of ECAP with only 8% residues in common. PLAP possesses additional secondary structure elements, comprising an N-terminal α-helix (residues 9 to 25), an α-helix and a β-strand in a highly divergent region (residues 208–280) and a different organization of the small β-sheet in domain 365–430. In the active site, only the residues that are essential for catalysis are preserved, i.e., the catalytic Ser, the three metal ion sites, M1 (occupied by Zn2+, also called Zn1), M2 (occupied by Zn2+; also called Zn2) and M3 (occupied by Mg2+) as well as their ligands, while most of the surrounding residues are different. Half of the enzyme surface corresponds to three clearly identifiable regions whose sequences largely vary among human APs, and are lacking in non-mammalian enzymes, i.e., the long N-terminal α-helix, an interfacial flexible loop known as the ‘crown domain’ and a fourth metal binding domain (M4). The availability of the PLAP structure facilitated modeling the human germ cell AP (GCAP), intestinal AP (IAP) and tissue-nonspecific AP (TNAP, a.k.a. liver/bone/kidney type AP) isozymes, revealing that all the novel features discovered in PLAP are conserved in those human isozymes as well . The active site Ser is conserved in all species where an AP has been sequenced to-date.
Structure/function studies comparing PLAP and the ECAP structure have found a conserved function for those residues that stabilize the active site Zn1, Zn2 and Mg metal ions, i.e., Asp42, His153, Ser155, Glu311, Asp316, His320, Asp357, His358, His360 and His432  (Figure 2). However, while mutations at Zn2 or Mg sites have similar effects in PLAP and ECAP, the environment of the Zn1 ion in PLAP is less affected by substitutions than in ECAP. This reflects the fact that, as shown on Figure 2, residue Glu429 is strategically positioned at the roof of the active site cleft where it provides additional stabilization to the active site environment, so that Zn2+ cannot easily diffuse in or out of the PLAP molecule . An additional non-catalytic metal-binding site M4 that appears to be occupied by calcium and that is not present in ECAP was revealed upon solving the PLAP 3D structure [4, 7]. This fourth metal is coordinated by carboxylates from Glu216, Glu270 and Asp285, by the carbonyl of Phe269 and a water molecule and this architecture is conserved in all human and mouse APs and presumably represents a novel feature common to all mammalian APs. However, the structural and functional significance of this new metal site remains to be established.
All mammalian APs have five cysteine residues (Cys101, Cys121, Cys183, Cys467 and Cys474 in PLAP) per subunit, not homologous to any of the four cysteines in ECAP. They form two disulfide bonds, Cys121-Cys183 and Cys467-Cys474, whereas the Cys101 residue remains in free form . The N-terminus of PLAP has an additional α-helix not present in the ECAP structure and its position is removed from the rest of the monomer, but it interacts with the second monomer with a buried surface area of 555 Å2, suggesting an involvement in enzyme dimerization. Recently, Hoylaerts et al.  have shown that the integrity of the N-terminal arm, and in particular of the α-helix comprising residues 10–25, represents a structural requirement for the active site to execute the intramolecular transition required during enzyme catalysis in both PLAP and TNAP.
The top flexible loop that constitutes the crown domain of PLAP is formed by the insertion of a 60-residue segment (366–430) from each monomer. It consists of two small interacting β-sheets, each composed of three parallel strands, and surrounded by six large and flexible loops containing a short α-helix . This region displays the least degree of sequence conservation among mammalian APs and isozyme-specific properties, such as the characteristic uncompetitive inhibition [6, 9–12], their variable heat-stability , and their allosteric behavior  have all been attributed to residue 429 located on this crown domain (Figures 2 and 3). This domain may also mediate the interaction between APs and extracellular matrix proteins, such as collagens [13, 15–17]. Another interesting structural feature of PLAP, with no counterpart in the ECAP structure, is Y367 (Figure 3). This residue is part of the subunit interface in the PLAP dimer, where it protrudes from one subunit and its hydroxyl group is located 6.1 Å from the phosphate and 3.1 Å from His432, which in turn chelates the zinc atom Zn1 in the active site of the other subunit. Kozlenkov et al. [6, 12] has shown that Y367, similar to residue 429, also plays a crucial role in determining the uncompetitive inhibition properties to the mammalian APs but very likely also the allosteric character of mammalian APs.
The monomer-monomer interface in PLAP has a strong hydrophobic character in contrast to that of ECAP. Less than 30% of the residues are involved in hydrogen-bonding interactions, which confer flexibility on the interface. Both the amino terminal arm and the crown domain take part in stabilizing the AP dimeric structure [4, 5]. Hoylaerts et al.  investigated whether the monomers in a given PLAP or GCAP dimer are subject to cooperativity during catalysis following an allosteric model or act via a half-of-sites model, in which at any time only a single monomer is operative. The authors used wt, single and double mutant PLAP homodimers and heterodimers for the analysis and found that mammalian APs are non-cooperative allosteric enzymes, but the stability and catalytic properties of each monomer are controlled by the conformation of the second AP subunit. The 3D structure of PLAP determined by Le Du et al.  contributed the visualization of the residues postulated to be involved in conferring the allosteric character to PLAP. The allosteric behavior is probably further favored by the quality of the dimer interface, by the long N-terminal α-helix from one monomer that embraces the other subunit, and by the protrusion of Tyr367 from one monomer into the active site of the other.
Recently, Llinas et al.  identified a novel, non-catalytic peripheral site on the surface of PLAP. This newly identified peripheral site is 28 Å from the active site but only 13 Å from the calcium binding site. The residues that constitute this site are located on two α-helices, 250–257 and 287–297. The loop between Ser257 and Ser287 contains the three residues that coordinate the Ca2+ ion, namely Phe269, Glu270, and Asp285 located only two residues from Ser287. Arg250 is located two residues away from Trp248 that interacts with the Ca2+ through a water molecule. Therefore, the large loop, comprising residues 250–297, that belongs to the peripheral domain of PLAP includes both the M4 site, and the peripheral binding site. The location of the Ca2+ ion in this loop suggests that the function of this novel M4 site could be related to the conformational stabilization of the two α-helices that form the peripheral site.
Finally, while ECAP is located in the periplasmic space of the bacterium, mammalian APs are ectoenzymes bound to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor . The GPI-glycan moiety, with general structure Protein-(ethanolamine-PO4-6Manα1-2Manα1-6Manα1-4 GlcN1-6myo-inositol-1-P-lipid, is added post-translationally to the nascent PLAP peptide chain in a transamidation reaction that involves the removal of a stretch of 29 hydrophobic amino acids from the COOH-terminus and the concomitant transfer of the preassembled GPI anchor to the new carboxyterminal amino acid, Asp484 in PLAP . To-date, all mammalian APs are thought to be anchored to the plasma membrane with each monomer carrying one unit of the GPI anchor.