A proposed structural model of human protein Z

Abstract

Analysis of structures and sequence similarities of the vitamin K-dependent (VKD) proteins in the blood coagulation pathway has shown that these proteins have evolved through a series of gene duplications and diversification, acquiring a degree of functional diversity in the process [1][2][3]. Protein Z (PZ) is a VKD plasma glycoprotein that is highly conserved across different species. It is homologous to the blood coagulation factors VII (FVII), FIX, FX, and protein C (PC). However, PZ differs from other coagulation proteins in that it lacks the critical histidine and serine residues of the catalytic triad, and is therefore not a zymogen of a serine protease (SP) [4,5].Human PZ was first isolated in 1984 [6], and its gene was characterized in 1998 [7]. PZ is relatively abundant in humans, with a wide plasma concentration range, and circulates as a complex with PZ-dependent protease inhibitor (ZPI) [8][9][10]. It has been shown to function as a cofactor in the inhibition of activated FX (FXa) by ZPI, causing a thousandfold increase in inhibition in a Ca 2+ -dependent manner [11]. The relative abundance in plasma and sequence similarity with other VKD proteins serve to pique our interest in the PZ system.In order to gain a better understanding of the function and evolutionary significance of PZ, it is important to start from structural information at an atomic level. Although PZ was isolated over 20 years ago, its physiologic role in relation to its molecular structure is still not clear. In this study, we propose a solvent-equilibrated structural model of human PZ using homology modeling and molecular dynamics (MD) simulation. The possibility of using this approach is supported by recent findings [12].Human PZ is a single-chain molecule of 360 residues with four domains: a Gla domain (residues 1-46) with 13 ccarboxyglutamic acid (Gla) residues (residues 7, 8, 11, 15, 17, 20, 21, 26, 27, 30, 33, 35 and 40), two epidermal growth factor (EGF)-like domains -an EGF1 domain (residues 47-83) with a b-hydroxyaspartate (Bha) residue at 64, and an EGF2 domain (residues 85-126) -and an SP-like domain (residues 135-360) with high homology to the catalytic domain of other VKD SPs [4,5].We have employed an iterative process of homology modeling and structure evaluation followed by MD simulation to construct a solvent-equilibrated structural model of PZ. Two different schemes were adopted to construct the homology model for PZ: one using multiple sequence alignment of human FVIIa [13] 18-23, 51-62, 56-71, 73-82, 89-101, 97-110, 112-125, 163-179 and 287-301). To obtain a refined solvent-equilibrated model and to remove bad contacts in the homology model, we performed an MD simulation using PMEMD9 in the AMBER9 [20] suite with the ff99SB forcefield. The total system was composed of 99 459 atoms, including 12 calcium ions, four sodium counterions for neutralizing the system, and 31 307 TIP3P water molecules. The seven conserved calcium ions in the Gla domain were placed on the basis of FVIIa, and the additional calcium ions w...

Computational study of the putative active form of protein Z (PZa): Sequence design and structural modeling

“…2A), which brings the orientation of the SP domain close to the EGF1 domain. This bend was also observed in our previous extended The multiple sequence alignment for Gla, EGF1, and EGF2 domains is the same as our previous PZ paper ( Lee et al 2007) (part of EGF2 is shown in the alignment). The modified regions are color coded as shown in the legend.…”

“…For substrate recognition and catalytic activity, the serine protease domain must have the following key characteristics (chymotrypsinogen numbering is used): the His57-Asp102-Ser195 catalytic triad, a selectivity S1 site composed of Asp189, Gly216, and Gly226, an oxyanion hole formed by the backbone NHs of Gly193 and Ser195, a proximal hydrophobic S2 pocket, a distal hydrophobic S3 pocket, and the salt bridge between Asp194 and N-terminal residue (Ile16) (Perona and Craik 1995;Czapinska and Otlewski 1999;Hedstrom 2002). In order to identify a reasonable primary sequence for the activated form of PZ, the multiple sequence alignment of PZ with FVII, FIX, FX, and PC was used as the starting point (Lee et al 2007). The amino acid residues in PZ corresponding to the key residues (above) that confer these serine protease characteristics were first modified based on the multiple sequence alignment.…”

“…The overall procedure for the homology modeling was similar to that employed in a previous study (Lee et al 2007). A multiple sequence alignment of human FVIIa, FIXa, FXa, and PC sequences was created with CLUSTALW, using the BLOSUM matrices for scoring the alignments (Thompson et al 1994).…”

“…However, PZ lacks the critical histidine and serine residues of the catalytic triad, and is therefore not a zymogen of a serine protease. Based on its homology with the other coagulation factors, we proposed a solvent-equilibrated model of human PZ using comparative modeling and molecular dynamics (MD) simulation (Lee et al 2007). One of the striking features in this model was that even though PZ lacked two of the critical catalytic triad residues, the relative spatial arrangement of the putative active site region and the distances between these residues were similar to the other serine proteases after a long time simulation.…”

Basis for the Specificity and Activation of the Serpin Protein Z-dependent Proteinase Inhibitor (ZPI) as an Inhibitor of Membrane-associated Factor Xa

“…Disulfides are present at the predicted positions of 131-132, 133-241, 163-179, and 287-301. Also as expected, the residues that substitute for the catalytic His and Ser of functional trypsin family proteinases (here Lys-178 and Asp-313, respectively) in the pseudo-active site are close together, and near the top of the molecule (27) (Fig. 1A), with C␣-C␣ separation of 8.3 Å, the same as in fXa, although the third residue of the triad is the normal Asp (Asp-221).…”