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Structural analysis on mutation residues and interfacial water molecules for human TIM disease understanding.

ABSTRACT: BACKGROUND: Human 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. RESULTS: This 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. CONCLUSIONS: Our 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.

SUBMITTER: Li Z

PROVIDER: S-EPMC3853089 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Structural analysis on mutation residues and interfacial water molecules for human TIM disease understanding.

Li Zhenhua Z He Ying Y Liu Qian Q Zhao Liang L Wong Limsoon L Kwoh Chee Keong CK Nguyen Hung H Li Jinyan J

BMC bioinformatics 20131022

<h4>Background</h4>Human 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.<h4>Results</h4>This work compares the structure of the E104D ...[more]

PMID: 24564410

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Project description:Exploring the potential functions of nonconserved residues on the outer side of α-helices and systematically optimizing them are pivotal for their application in protein engineering. Based on the evolutionary structural conservation analysis of GH5_5 cellulases, a practical molecular improvement strategy was developed. Highly variable sites on the outer side of the α-helices of the GH5_5 cellulase from Aspergillus niger (AnCel5A) were screened, and 14 out of the 34 highly variable sites were confirmed to exert a positive effect on the activity. After the modular combination of the positive mutations, the catalytic efficiency of the mutants was further improved. By using CMC-Na as the substrate, the catalytic efficiency and specific activity of variant AnCel5A_N193A/T300P/D307P were approximately 2.0-fold that of AnCel5A (227 ± 21 versus 451 ± 43 ml/s/mg and 1,726 ± 19 versus 3,472 ± 42 U/mg, respectively). The half-life (t1/2) of variant AnCel5A_N193A/T300P/D307P at 75°C was 2.36 times that of AnCel5A. The role of these sites was successfully validated in other GH5_5 cellulases. Computational analyses revealed that the flexibility of the loop 6-loop 7-loop 8 region was responsible for the increased catalytic performance. This work not only illustrated the important role of rapidly evolving positions on the outer side of the α-helices of GH5_5 cellulases but also revealed new insights into engineering the proteins that nature left as clues for us to find. IMPORTANCE A comprehensive understanding of the residues on the α-helices of the GH5_5 cellulases is important for catalytic efficiency and stability improvement. The main objective of this study was to use the evolutionary conservation and plasticity of the TIM-barrel fold to probe the relationship between nonconserved residues on the outer side of the α-helices and the catalytic efficiency of GH5_5 cellulases by conducting structure-guided protein engineering. By using a four-step nonconserved residue screening strategy, the functional role of nonconserved residues on the outer side of the α-helices was effectively identified, and a variant with superior performance and capability was constructed. Hence, this study proved the effectiveness of this strategy in engineering GH5_5 cellulases and provided a potential competitor for industrial applications. Furthermore, this study sheds new light on engineering TIM-barrel proteins.

Project description:BackgroundT-cell immunoglobulin and mucin domain (Tim) proteins are immunomodulatory molecules that play key roles in the regulation of T-cell activation. Published studies have reported that Tim molecules are involved in the pathogenesis of certain autoimmune diseases. Type 1 diabetes (T1D) is an autoimmune disease in which T cells mediate the destruction of islet β cells. However, the expression of Tim molecules in T1D remains unclear. In this study, we measured the expression of Tim family molecules as well as T-cell subset-specific transcription factors in T1D patients, and we explored the possible involvement of Tim molecules in the pathogenesis of T1D.MethodsNinety T1D patients, Thirty-six type 2 diabetes (T2D) patients and forty healthy controls (HCs) were recruited for this study. Peripheral blood mononuclear cells (PBMCs) were isolated, RNA was extracted from the PBMCs and reverse transcribed into cDNA, and gene expression patterns were analysed by RT-qPCR. The expression of Tim molecules in different T-cell subsets was analysed by flow cytometry.ResultsCompared with that in HCs, the mRNA expression of Tim-1 and RORC was increased in T1D patients (P=0.0355 and P=0.0423, respectively), while the expression of Tim-3 was decreased (P=0.0013). In addition, compared with HCs, the ratio of Tim-3 to Tim-1 expression in diabetic patients was decreased (P<0.0001 for T1D and P=0.0387 for T2D). The ratios of T-Bet to GATA3 expression and RORC to FOXP3 expression were higher in T1D patients than in HCs (P=0.0042 and P=0.0066, respectively). Furthermore, the T1D patients with defective islet function had more significant imbalances in the Tim-3/Tim-1 and RORC/FOXP3 ratios (P<0.0001, and P=0.001, respectively). Moreover, Both Tim-3 expression in CD4+ T cells and the Tim-3 to Tim-1 ratio were elevated in T1D in the remission phase compared to T1D.ConclusionOur study revealed altered expression of Tim molecules in T1D patients. The imbalanced ratios of Tim-3/Tim-1 expression were more pronounced in T1D patients with defective islet function. However, alterations in Tim molecule expression are mitigated in T1D in the remission phase. All these findings suggest that Tim family molecules may be involved in the pathogenesis of T1D.

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Structural analysis on mutation residues and interfacial water molecules for human TIM disease understanding.

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Structural analysis on mutation residues and interfacial water molecules for human TIM disease understanding.

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