Table 1.
Data collection, processing and refinement statistics.
Fig 1.
Sequence alignment of MTAPs from S. mansoni (SmMTAP), S. japonicum (SjMTAP) and human (HsMTAP).
SmMTAP shares 77% and 47% identity with SjMTAP and HsMTAP, respectively. The SmMTAP secondary structure elements are labeled and shown as arrows and helices. One sequence insertion also present in both Schistosoma species could be observed between beta strands 10 and 11.
Fig 2.
A. SmMTAP trimeric structure in cartoon scheme. Beta strands are colored blue and helices in red. B. Superposition of 66 monomers of 22 independent trimers from 13 different SmMTAP structures. The central beta sheet core is the invariant part of the structure. Gate loop suffers high conformational plasticity.
Fig 3.
Stereo image of gate loop movement between adenine complex structure (red) and APO (blue).
The D230 main residue in the active site is shown in yellow. The movement involves residues 229–253. In the APO structure, D230 points away from the active site. The presence of the base or base moiety in the base bind site appears to be necessary to "close" the gate loop in the base interacting conformation.
Fig 4.
A. Composite omit map contoured at 1σ for the region containing the SS bond in SmMTAP. B. Stick model for the same region showed in A. This disulphide bond is formed by the residues Cys 233 and Cys 242 only in some Apo mutant structures. This SS bond helps maintain the gate loop in open conformation and could be involved in the nucleoside accessibility of the active site.
Fig 5.
Ligplus+ diagrams for ligands in the SmMTAP active site.
A. adenine; B. tubercidin; C., different binding modes of tubercidin in the active site interacting with both D230 and Q289 residues. D. MTA.
Fig 6.
Different binding modes of bases in the main part of BBS residues, showing stick model for residues S118 and 229–232.
A. H-bond scheme for adenine in the BBS of wild-type SmMTAP. B. adenine in S12T/N87T chain B. D230 does not form direct interaction with the base. C. In S12T chain C the orientation of D230 is also different. D. Tubercidin in wild-type SmMTAP, the side chain of D230 points away to active site. E. In Q289L Tubercidin chain A D230 form canonical contacts within the base even being 7-deaza-adenosine. F. MTA molecule in S12Tmutant active site. The active site shows high conformational plasticity especially for the side chain of D230 residue.
Fig 7.
Stereo image for residues 229–232 showing the large movement involved in ligand binding.
The SmMTAP adenine complex is shown in cyan, and the Apo form is shown in yellow. In Apo form, D230 points away to the active site, and F231 points towards to the base binding site. This conformational change was not observed in human MTAP and could explain the low KM for adenosine for Schistosoma enzyme.
Fig 8.
Ligplus+ plots for the sulphate molecule.
A. wt SmMTAP. B. Double mutant S12T/N87T and their consequence in sulphate/phosphate binding, where 3 new H-bonds are formed.
Fig 9.
Part of the phosphate and ribose binding sites showing the interactions between the side chain of Q289 and both S12 and O5' of tubercidin.
Human enzymes possess leucine in this position and thus do not form these interactions.
Fig 10.
Superposition of wild-type SmMTAP (white) and double mutant S12T/N887T showing the consequence of these mutations in phosphate binding.
The side chain of T87 allows an extra H-bond with the phosphate (sulphate), and the presence of T12 only permits one side chain conformation for phosphate interaction; intriguingly, the presence of these mutations increased the phosphate KM.
Table 2.
Kinetic parameters for wt SmMTAP and their mutants for adenosine, MTA, 2'-deoxyadenosine, and for adenosine hydrolysis.