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Structural insights into glucan phosphatase dynamics using amide hydrogen-deuterium exchange mass spectrometry.

ABSTRACT: Laforin and starch excess 4 (SEX4) are founding members of a class of phosphatases that dephosphorylate phosphoglucans. Each protein contains a carbohydrate binding module (CBM) and a dual-specificity phosphatase (DSP) domain. The gene encoding laforin is mutated in a fatal neurodegenerative disease called Lafora disease (LD). In the absence of laforin function, insoluble glucans that are hyperphosphorylated and exhibit sparse branching accumulate. It is hypothesized that these accumulations trigger the neurodegeneration and premature death of LD patients. We recently demonstrated that laforin removes phosphate from phosphoglucans and hypothesized that this function inhibits insoluble glucan accumulation. Loss of SEX4 function in plants yields a similar cellular phenotype; an excess amount of insoluble, hyperphosphorylated glucans accumulates in cells. While multiple groups have shown that these phosphatases dephosphorylate phosphoglucans, there is no structure of a glucan phosphatase and little is known about the mechanism whereby they perform this action. We utilized hydrogen-deuterium exchange mass spectrometry (DXMS) and structural modeling to probe the conformational and structural dynamics of the glucan phosphatase SEX4. We found that the enzyme does not undergo a global conformational change upon glucan binding but instead undergoes minimal rearrangement upon binding. The CBM has improved protection from deuteration when bound to glucans, confirming its role in glucan binding. More interestingly, we identified structural components of the DSP that also have improved protection from deuteration upon glucan addition. To determine the position of these regions, we generated a homology model of the SEX4 DSP. The homology model shows that all of these regions are adjacent to the DSP active site. Therefore, our results suggest that these regions of the DSP participate in the presentation of the phosphoglucan to the active site and provide the first structural analysis and mode of action of this unique class of phosphatases.

SUBMITTER: Hsu S

PROVIDER: S-EPMC2767375 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Structural insights into glucan phosphatase dynamics using amide hydrogen-deuterium exchange mass spectrometry.

Hsu Simon S Kim Youngjun Y Li Sheng S Durrant Eric S ES Pace Rachel M RM Woods Virgil L VL Gentry Matthew S MS

Biochemistry 20091001 41

Laforin and starch excess 4 (SEX4) are founding members of a class of phosphatases that dephosphorylate phosphoglucans. Each protein contains a carbohydrate binding module (CBM) and a dual-specificity phosphatase (DSP) domain. The gene encoding laforin is mutated in a fatal neurodegenerative disease called Lafora disease (LD). In the absence of laforin function, insoluble glucans that are hyperphosphorylated and exhibit sparse branching accumulate. It is hypothesized that these accumulations tri ...[more]

PMID: 19754155

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Project description:The use of Fourier transform mass spectrometry (FTMS) to monitor noncovalent complex formation in the gas phase under native conditions between the Link module from human tumor necrosis factor stimulated gene-6 (Link_TSG6) and hyaluronan (HA) oligosaccharides is reported. In particular, a titration experiment with increasing concentrations of octasaccharide (HA(8)) to protein produced a noncovalent complex with 1:1 stoichiometry when the oligosaccharide was in molar excess. However, in the presence of a molar excess of tetrasaccharide (HA(4)) nearly all proteins and oligosaccharides were observed in their unbound charge states. These results are consistent with solution-phase properties for this interaction in which HA(8), but not HA(4), supports high affinity Link_TSG6 binding. Hydrogen/deuterium amide exchange mass spectrometry (H/D-EX MS) was also utilized to investigate the level of global deuterium incorporation, over time, for Link_TSG6 in both the absence and presence of HA(8). After dilution into quenching conditions, deuterium incorporation reached limiting asymptotic values of 37 and 26 deuterons for the free and bound protein at 240 and 480 min, respectively, indicating that the oligosaccharide interferes with amide exchange on binding. To detect sequence-specific deuterium incorporation, pepsin digestion of Link_TSG6 in both the absence and presence of HA(8) was performed. A level of deuterium incorporation of 10-30% was observed for peptides analyzed in free Link_TSG6. Interestingly, HA(8) blocked some sites of proteolysis in Link_TSG6 compared to the free protein. Molecular modeling indicated that amino acids proximal to the ligand correlated with regions of the protein that were resistant to enzymatic digestion. Of the peptides that could be analyzed by H/D-EX MS in the presence of the ligand, a 30-60% reduction in deuterium incorporation, relative to the free protein, was observed, even for those sequences not directly involved in HA binding. These results support the utility of FTMS as a method for the characterization of protein-carbohydrate interactions.

Dataset Information

Structural insights into glucan phosphatase dynamics using amide hydrogen-deuterium exchange mass spectrometry.

Publications

Structural insights into glucan phosphatase dynamics using amide hydrogen-deuterium exchange mass spectrometry.

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