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

Biochemistry. 2009 Oct 20;48(41):9891-902. doi: 10.1021/bi9008853.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / metabolism
  • Arabidopsis Proteins / chemistry
  • Arabidopsis Proteins / metabolism
  • Chromatography, Gel
  • Deuterium
  • Glucans / chemistry*
  • Glucans / metabolism*
  • Hydrogen
  • Mass Spectrometry
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Phosphoric Monoester Hydrolases / chemistry*
  • Phosphoric Monoester Hydrolases / metabolism
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism

Substances

  • Arabidopsis Proteins
  • Glucans
  • Peptide Fragments
  • Recombinant Proteins
  • glucan phosphate
  • Hydrogen
  • Deuterium
  • Phosphoric Monoester Hydrolases