Functions of tryptophan residues in EWGWS insert of Plasmodium falciparum enolase

Abstract

Plasmodium falciparum enolase (Pfeno) is a dimeric enzyme with multiple moonlighting functions. This enzyme is thus a potential target for anti‐malarial treatments. A unique feature of Pfeno is the presence of a pentapeptide insert 104 EWGWS 108 . The functional role of tryptophan residues in this insert was investigated using site‐directed mutagenesis. Replacement of these two Trp residues with alanines (or lysines) resulted in a near complete loss of enolase activity and dissociation of the normal dimeric form into monomers. Molecular modeling indicated that 340 R forms π‐cation bonds with the aromatic rings of 105 W and 46 Y. Mutation induced changes in the interactions among these three residues were presumably relayed to the inter‐subunit interface via a coil formed by 46 Y : 59 Y, resulting in the disruption of a salt bridge between 11 R : 425 E and a π‐cation interaction between 11 R : 59 Y. This led to a drop of ~ 4 kcal·mole −1 in the inter‐subunit docking energy in the mutant, causing a ~ 10 3 fold decrease in affinity. Partial restoration of the inter‐subunit interactions led to reformation of dimers and also restored a significant fraction of the lost enzyme activity. These results suggested that the perturbations in the conformation of the surface loop containing the insert sequence were relayed to the interface region, causing dimer dissociation that, in turn, disrupted the enzyme's active site. Since Plasmodium enolase is a moonlighting protein with multiple parasite‐specific functions, it is likely that these functions may map on to the highly conserved unique insert region of this protein. Enzymes Enolase( EC4.2.1.11 ).

Strain-transcending neutralization of malaria parasite by antibodies against Plasmodium falciparum enolase

“…The effect of varying the insert sequence in Pfeno on binding of H12E1 was examined by monitoring the extent of antibody reactivity with different variants. Figure 2 a shows the sequence changes made in these variants using site directed mutagenesis [ 21 , 24 , 39 , 40 ]. Replacement of S108 with glycine (S108G-rPfeno) had no effect on antibody binding.…”

“…These variants differ from each other in their oligomeric structure as well as in enzymatic activity. WT and S108G-rPfeno are enzymatically active and dimeric while the other three variants are monomeric and largely inactive [ 24 , 39 , 40 ]. To rule out the possibility that non-reactivity of the ∆ 5 -rPfeno and Trp variants (W105, 107A-rPfeno and W105, 107K-rPfeno) was due to the altered oligomeric structure of the protein, an ELISA was performed on the GST-tagged forms of the different variants.…”

“…To rule out the possibility that non-reactivity of the ∆ 5 -rPfeno and Trp variants (W105, 107A-rPfeno and W105, 107K-rPfeno) was due to the altered oligomeric structure of the protein, an ELISA was performed on the GST-tagged forms of the different variants. In the GST tagged form, all these variants form dimers (or oligomers) and acquire active conformation [ 24 , 39 ]. ELISA data presented in Fig.…”