Modulation of catalytic function by differential plasticity of the active site: case study of Trypanosoma cruzi trans-sialidase and Trypanosoma rangeli sialidase

Abstract

trans-Sialidase is an essential enzyme for Trypanosoma cruzi, the causative agent of Chagas' disease, to escape from the host immune system and to invade the host cells. Therefore, T. cruzi trans-sialidase (TcTS) presents a potential and appealing therapeutic target for this lethal disease. The availability of a structurally very similar enzyme with strict hydrolase activity (Trypanosoma rangeli sialidase, TrSA) provides us a unique opportunity to understand the determinants of their structure and catalytic mechanism. In this study, we compare the catalytic cleft plasticity of free (apo) and ligand-bound (holo) forms of the two enzymes using molecular dynamics simulations. We focus on the mouth of the catalytic cleft that is defined by two residues: W312 and Y119 in TcTS and W312 and S119 in TrSA. Our results indicate that TcTS has a very flexible, widely open catalytic cleft, mostly due to W312 loop motion, in apo form. However, when the catalytic cleft is occupied by a ligand, the flexibility and solvent exposure of TcTS is significantly reduced. On the other hand, TrSA maintains a more open catalytic cleft compared to its crystal structures in both apo and holo forms (and compared to TcTS in holo forms). The reduced solvent exposure of TcTS catalytic cleft might be partially or fully responsible for TcTS to be a less efficient hydrolase than TrSA.

Figure 1

Chemical structures of sialic acid (1) and 2, 3-dehydro-3-deoxy-Nacetylneuraminic acid (2, abbreviated as DANA). The star shows the location of F atom insertion mentioned in the text.

Figure 2

The proposed mechanism for trans-sialidase catalysis reaction of TcTS. The donor and acceptor sugar moieties are colored in red and orange, respectively. In sialidase catalysis reaction of TcTS, a water molecule takes the role of the acceptor lactose.

Figure 3

The interactions that clamp W312 loop (in yellow) to R7 shown for TcTS bound to sialyl-lactose (sialic acid is colored orange and lactose is colored ice blue). K309 and E338 connect the two flexible loops (in yellow and purple) to R7 that lies on a β-sheet structure. R314 serves to fix W312 loop by interacting with the carboxylate group of sialic acid in the presence of a ligand.

Figure 4

Closed and open forms of W312 loop. The initial closed form of the loop (red) rearranges and creates a more open catalytic cleft (blue) in apo form of TcTS. W312 visits all possible different conformations once W312 loop leaves the closed form.

Figure 5

W312 C_α-Y119 C_α distance (in Å) in the molecular dynamics simulations of apo and sialyl-lactose bound forms of TcTS.

Figure 6

The correlation of Y119 χ₁ dihedral angle change (in the top panel) with the opening of W312 loop (in the bottom panel) monitored by the distance between C_α atoms of W312 and Y119 in two separate MD simulations of DANA-bound TcTS. W312 loop opening does not occur unless Y119 side chain swings into the catalytic cleft (for which χ₁=180°). The dihedral angles are in degrees and distances are in Angstroms.

Figure 7

The correlation of Y119 χ₁ dihedral angle change (in the top panel) with the opening of W312 loop (in the bottom panel) monitored by the distance between C_α atoms of W312 and Y119 in two separate MD simulations of TcTS covalent intermediate. The dihedral angles are in degrees and distances are in Angstroms.

Figure 8

Water network around R245 that connects Y119 conformational motion to W312 loop motion. The water network at the beginning of simulation (shown in green) is distorted (colored according to atom type) when Y119 moves from χ1=−60° to χ1=180°. The third possible Y119 conformation is also shown (in orange).

Figure 9

Catalytic cleft opening in TrSA MD simulations monitored by the distance between W312 CA and S119 CA (in Å). A) TrSA B) TrSA covalent intermediate C) DANA-bound TrSA D) Sialyl-lactose-bound TrSA

Figure 10

Closed and open forms of TrSA catalytic cleft. The initial closed form and the more open average structure of the apo form of TrSA (in the 50-ns simulation) are superimposed and the important regions of the catalytic cleft are depicted in red and blue, respectively.

Figure 11

Close crystal contacts in the unit cell of apo form of TrSA. Orange and blue ribbons show the two neighboring enzymes. K495 side chain lies inside the active site of TrSA hindering W312 to move freely. S119 is also shown to clarify the relative orientation.