Structural studies of human alkaline phosphatase in complex with strontium: implication for its secondary effect in bones

Abstract

Strontium is used in the treatment of osteoporosis as a ranelate compound, and in the treatment of painful scattered bone metastases as isotope. At very high doses and in certain conditions, it can lead to osteomalacia characterized by impairment of bone mineralization. The osteomalacia symptoms resemble those of hypophosphatasia, a rare inherited disorder associated with mutations in the gene encoding for tissue-nonspecific alkaline phosphatase (TNAP). Human alkaline phosphatases have four metal binding sites--two for zinc, one for magnesium, and one for calcium ion--that can be substituted by strontium. Here we present the crystal structure of strontium-substituted human placental alkaline phosphatase (PLAP), a related isozyme of TNAP, in which such replacement can have important physiological implications. The structure shows that strontium substitutes the calcium ion with concomitant modification of the metal coordination. The use of the flexible and polarizable force-field TCPEp (topological and classical polarization effects for proteins) predicts that calcium or strontium has similar interaction energies at the calcium-binding site of PLAP. Since calcium helps stabilize a large area that includes loops 210-228 and 250-297, its substitution by strontium could affect the stability of this region. Energy calculations suggest that only at high doses of strontium, comparable to those found for calcium, can strontium substitute for calcium. Since osteomalacia is observed after ingestion of high doses of strontium, alkaline phosphatase is likely to be one of the targets of strontium, and thus this enzyme might be involved in this disease.

Figure 1.

Stereo view of the 2F_o − F_c electron density map (blue) at the 1σ level and the F_o − F_c electron density map (red) at the 5σ level, at the fourth metal binding site of alkaline phosphatase in the presence of calcium (A) or strontium (B).

Figure 2.

Coordination network of the calcium (A) or strontium (B) at the fourth metal binding site of alkaline phosphatase showing the residues in interaction with the metal. (C) Superimposition of the two binding sites showing the deviation between the calcium-containing AP (white structure) and the strontium-containing AP (yellow structure).

Figure 3.

(A) Location of Lys275 (in blue) in the active-site valley of PLAP. (B) Network of interaction connecting the active site to the fourth metal binding site in the presence of strontium.

Figure 4.

(A,B) Interaction of loop 210–228 (in blue) with the fourth metal binding site. (C,D) Succession of stacking interaction between loop 210–228 (in blue) and the core of PLAP structure.