Abstract
BackgroundHuman triosephosphate isomerase (HsTIM) deficiency is a genetic disease caused often by the pathogenic mutation E104D. This mutation, located at the side of an abnormally large cluster of water in the inter-subunit interface, reduces the thermostability of the enzyme. Why and how these water molecules are directly related to the excessive thermolability of the mutant have not been investigated in structural biology.ResultsThis work compares the structure of the E104D mutant with its wild type counterparts. It is found that the water topology in the dimer interface of HsTIM is atypical, having a "wet-core-dry-rim" distribution with 16 water molecules tightly packed in a small deep region surrounded by 22 residues including GLU104. These water molecules are co-conserved with their surrounding residues in non-archaeal TIMs (dimers) but not conserved across archaeal TIMs (tetramers), indicating their importance in preserving the overall quaternary structure. As the structural permutation induced by the mutation is not significant, we hypothesize that the excessive thermolability of the E104D mutant is attributed to the easy propagation of atoms' flexibility from the surface into the core via the large cluster of water. It is indeed found that the B factor increment in the wet region is higher than other regions, and, more importantly, the B factor increment in the wet region is maintained in the deeply buried core. Molecular dynamics simulations revealed that for the mutant structure at normal temperature, a clear increase of the root-mean-square deviation is observed for the wet region contacting with the large cluster of interfacial water. Such increase is not observed for other interfacial regions or the whole protein. This clearly suggests that, in the E104D mutant, the large water cluster is responsible for the subunit interface flexibility and overall thermolability, and it ultimately leads to the deficiency of this enzyme.ConclusionsOur study reveals that a large cluster of water buried in protein interfaces is fragile and high-maintenance, closely related to the structure, function and evolution of the whole protein.
Highlights
Human triosephosphate isomerase (TIM) deficiency, first reported by Schneider et al in 1965 [1], is a genetic disease caused by the dysfunction of TIM
Despite of these conflicting statements in the past research, the common understanding on the pathogenesis of the mutation E104D shares the following points: (i) There is an alteration of the binding of the two subunits in the E104D mutant [16]; (ii) The altered binding of the two subunits harms thermostability of the protein [1]; and (iii) the excessive thermolability causes the dysfunction of the enzyme [1]
Our pathogenesis study for the mutation E104D is to affirm: (i) this mutation can lead to an excessive thermolability of the enzyme, and (ii) the binding between the two subunits is harmed by the increased flexibility of the interfacial atoms, which are amplified by the large cluster of buried water near the mutation site
Summary
We first present results to illustrate how exactly atypical the HsTIM dimer interface is, followed by results on the evolution of the water and residues in the interface of TIMs. As can be seen from Figure 5(A), none of these residue has a large RMSD, indicating that the mutation does not alter the active site of the enzyme This is supported by the observation that the mutant enzyme has almost the same activity as wild type enzyme at normal temperature [17]. In order to better understand the role of the large water cluster in region A in enhancing the interface mobility after the mutation, the average change in normalized B factor of interfacial non water atoms is plotted at different burial levels (the burial levels in the wild type enzyme); see Figure 7(B). The mobility change of the interfacial water molecules and atoms indicates that after the mutation, water and Figure 6 Structural alignment between wild type E104D mutant HsTIM dimer interface. In HsTIM, we believe that this large cluster of water may play important role in the pathogenesis of other point mutations in this enzyme and this water cluster could be one of the reasons why there are so many pathogenic point mutations in this enzyme [6]
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