Determination of the structure form of the fourth ligand of zinc in Acutolysin A using combined quantum mechanical and molecular mechanical simulation

Abstract

Acutolysin A, which is isolated from the snake venom of Agkistrodon acutus, is a member of the SVMPs subfamily of the metzincin family, and it is a snake venom zinc metalloproteinase possessing only one catalytic domain. The catalytic zinc ion, in the active site, is coordinated in a tetrahedral manner with three imidazole nitrogen atoms of histidine and one oxygen atom. It is uncertain whether this oxygen atom is a water molecule or a hydroxide ion just from the three-dimensional X-ray crystal structure. The identity of the fourth ligand of zinc is theoretically determined for the first time by performing both combined quantum mechanical and molecular mechanical (QM/MM) simulation and high-level quantum mechanical calculations. All of the results obtained indicate that the fourth ligand in the active site of the reported X-ray crystal structure is a water molecule rather than a hydroxide anion. On the basis of these theoretical results, we note that the experimental observed pH dependence of the proteolytic and hemorrhagic activity of Acutolysin A can be attributed to the deprotonation of the zinc-bound water to yield a better nucleophile, the hydroxide ion. Structural analyses revealed structural details useful for the understanding of acutolysin catalytic mechanism.

Figure 1

Left picture is the crystal structure of Acutolysin A from 1bsw.pdb. The molecule is shown in standard orientation. The calcium ion, the zinc ion (filled circles), and the three histidine zinc ligation residues are also shown. The right one is the enlargement for the structure of the active site, and it is also the schematic representation of the partition of the protein system into a quantum mechanical region and a classical region. The boundary atoms are treated using the generalized hybrid orbital method, which are indicated in yellow.

Figure 2

Root-mean-square deviation (Å) from the experimental structure for backbone atoms, atoms within 10 Å of zinc and atoms in QM region versus the simulation time: (a) for 1bsw_water, (b) for 1bsw_hydroxide.

Figure 3

Definitions of atom names in the active site: (a) for 1bsw_water, (b) for 1bsw_hydroxide.

Figure 4

Selected distances as obtained from MD simulation: (a) for 1bsw_water, (b) for 1bsw_hydroxide. (D1) Zn-OH2 (wat300 or hydroxide ion300), (D2) Zn-His142:NE2, (D3) Zn-His146:NE2, (D4) Zn-His152:NE2, (D5) OH2 (wat300 or hydroxide ion300)-Glu143:OE1, (D6) OH2 (wat300 or hydroxide ion300)-Glu143:OE2. Residue number corresponds to that in the X-ray structure.

Figure 5

Selected dihedral angles of atoms in QM region as obtained MD simulation: (a) for 1bsw_water, (b) for 1bsw_hydroxide. (DIHE1) CE1-NE2-Zn-OH2, (DIHE2) CE3-NE4-Zn-OH2, (DIHE3) CE5-NE5-Zn-OH2 (see Figure 3 for atom names).

Figure 6

Selected hydrogen bond distances between donor and acceptor in the active site as obtained from MD simulation: (a) for 1bsw_water, (b) for 1bsw_hydroxide. (HB1) ND1-Ile165:O, (HB2) ND3-Val150:O, (HB3) ND3-Glu143:OE1.

Figure 7

Hydrogen bonds in the active site: (a) X-ray structure in 1bsw_water, (b) snapshot of 250 ps extracted from MD trajectory in 1bsw_water, (c) X-ray structure in 1bsw_hydroxide, and (d) snapshot of 900 ps extracted from MD trajectory in 1bsw_hydroxide.

Figure 8

Root-mean-square deviation (Å) from the experimental structure for the residues of Glu143 in the active site: (a) for 1bsw_water, (b) for 1bsw_hydroxide.

Figure 9

Optimized structure of active site residues using the high-level QM calculations, which clearly shows the polarization of Glu143 to the catalytic water.

Figure 10

(a) Plot of distances between zinc and waters in the active site (within 4.5 Å of zinc) versus the simulation time for 1bsw_water. (b) Selected hydrogen bond distances between oxygen atoms of waters in the active site and protein oxygen atoms as obtained from 1bsw_water simulation.