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Title: A Hydrophobic Pocket in the Active Site of Glycolytic Aldolase Mediates Interactions with Wiskott-Aldrich Syndrome Protein

Abstract

Aldolase plays essential catalytic roles in glycolysis and gluconeogenesis. However, aldolase is a highly abundant protein that is remarkably promiscuous in its interactions with other cellular proteins. In particular, aldolase binds to highly acidic amino acid sequences, including the C-terminus of the Wiskott-Aldrich syndrome protein, an actin nucleation promoting factor. Here we report the crystal structure of tetrameric rabbit muscle aldolase in complex with a C-terminal peptide of Wiskott-Aldrich syndrome protein. Aldolase recognizes a short, 4-residue DEWD motif (residues 498-501), which adopts a loose hairpin turn that folds about the central aromatic residue, enabling its tryptophan side chain to fit into a hydrophobic pocket in the active site of aldolase. The flanking acidic residues in this binding motif provide further interactions with conserved aldolase active site residues, Arg-42 and Arg-303, aligning their side chains and forming the sides of the hydrophobic pocket. The binding of Wiskott-Aldrich syndrome protein to aldolase precludes intramolecular interactions of its C-terminus with its active site, and is competitive with substrate as well as with binding by actin and cortactin. Finally, based on this structure a novel naphthol phosphate-based inhibitor of aldolase was identified and its structure in complex with aldolase demonstrated mimicry of the Wiskott-Aldrichmore » syndrome protein-aldolase interaction. The data support a model whereby aldolase exists in distinct forms that regulate glycolysis or actin dynamics.« less

Authors:
; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930430
Report Number(s):
BNL-81173-2008-JA
Journal ID: ISSN 0021-9258; JBCHA3; TRN: US200904%%706
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; Journal Volume: 282; Journal Issue: 19
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; ACTIN; ALDOLASES; AMINO ACID SEQUENCE; AROMATICS; CHAINS; CRYSTAL STRUCTURE; GLYCOLYSIS; MUSCLES; NAPHTHOLS; NUCLEATION; PEPTIDES; PROTEINS; RABBITS; RESIDUES; SUBSTRATES; TRYPTOPHAN; national synchrotron light source

Citation Formats

St-Jean,M., Izard, T., and Sygusch, J. A Hydrophobic Pocket in the Active Site of Glycolytic Aldolase Mediates Interactions with Wiskott-Aldrich Syndrome Protein. United States: N. p., 2007. Web. doi:10.1074/jbc.M611505200.
St-Jean,M., Izard, T., & Sygusch, J. A Hydrophobic Pocket in the Active Site of Glycolytic Aldolase Mediates Interactions with Wiskott-Aldrich Syndrome Protein. United States. doi:10.1074/jbc.M611505200.
St-Jean,M., Izard, T., and Sygusch, J. Mon . "A Hydrophobic Pocket in the Active Site of Glycolytic Aldolase Mediates Interactions with Wiskott-Aldrich Syndrome Protein". United States. doi:10.1074/jbc.M611505200.
@article{osti_930430,
title = {A Hydrophobic Pocket in the Active Site of Glycolytic Aldolase Mediates Interactions with Wiskott-Aldrich Syndrome Protein},
author = {St-Jean,M. and Izard, T. and Sygusch, J.},
abstractNote = {Aldolase plays essential catalytic roles in glycolysis and gluconeogenesis. However, aldolase is a highly abundant protein that is remarkably promiscuous in its interactions with other cellular proteins. In particular, aldolase binds to highly acidic amino acid sequences, including the C-terminus of the Wiskott-Aldrich syndrome protein, an actin nucleation promoting factor. Here we report the crystal structure of tetrameric rabbit muscle aldolase in complex with a C-terminal peptide of Wiskott-Aldrich syndrome protein. Aldolase recognizes a short, 4-residue DEWD motif (residues 498-501), which adopts a loose hairpin turn that folds about the central aromatic residue, enabling its tryptophan side chain to fit into a hydrophobic pocket in the active site of aldolase. The flanking acidic residues in this binding motif provide further interactions with conserved aldolase active site residues, Arg-42 and Arg-303, aligning their side chains and forming the sides of the hydrophobic pocket. The binding of Wiskott-Aldrich syndrome protein to aldolase precludes intramolecular interactions of its C-terminus with its active site, and is competitive with substrate as well as with binding by actin and cortactin. Finally, based on this structure a novel naphthol phosphate-based inhibitor of aldolase was identified and its structure in complex with aldolase demonstrated mimicry of the Wiskott-Aldrich syndrome protein-aldolase interaction. The data support a model whereby aldolase exists in distinct forms that regulate glycolysis or actin dynamics.},
doi = {10.1074/jbc.M611505200},
journal = {Journal of Biological Chemistry},
number = 19,
volume = 282,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • The mouse WASP gene, the homolog of the gene mutation in Wiskott-Aldrich syndrome, has been isolated and sequenced. The predicted amino acid sequence is 86% identical to human WASP sequence. A distinct feature of the mouse gene is an expanded polymorphic GGA trinucleotide repeat that codes for polyglycine and varies from 15 to 17 triplets in Mus musculus strains. The genomic structure of the mouse gene closely resembles the human with respect to exon-intron positions and intron lengths. The mouse WASP gene is expressed as an {approx}2.4-kb mRNA in thymus and spleen. Chromosomal mapping in an interspecific M. musculus/M. spretusmore » backcross placed in the WASP locus near the centromere of the mouse X chromosome, inseparable form Gata1, Tcfe3, and scurfy (sf). This localization makes WASP a candidate for involvement in scurfy, a T cell-mediated fatal lymphoreticular disease of mice that has previously been proposed as a mouse homolog of Wiskott-Aldrich syndrome. Northern analysis of sf tissue samples indicated the presence of a consequence of lymphocytic infiltration, but no abnormalities in the amount or size of mRNA present. 34 refs., 5 figs.« less
  • The Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency combined with thrombocytopenia in which the molecular defect is still unknown. Initial linkage data placed the WAS gene between TIMP and the marker DXS255 in Xp11.23 to Xp11.22. As no recombination was detected between the disease locus closely linked to DXS255 and the marker loci OATL1, SYP and TFE3, the position of WAS relative to these polymorphic loci could not yet be determined. In this study, further segregation analysis has been performed using additional (CA)n repeats DXS1367, DXS6616 and DXS1126. While DXS1367 and DXS6616 could be mapped adjacent to OATL1, location ofmore » DXS1126 between OATL1 and TFE3 is demonstrated. In a WAS pedigree of three generations (4 affected males, 10 obligate female carriers, 7 non-carriers) we observed a recombination event between the disease and the locus TIMP, DXSS1367, and DXS6616 in a patient manifesting WAS and the daughter of his female cousin. The carrier status of the female relative was confirmed or excluded by X inactivation analysis. No recombination was detected by the marker DXS6616 containing the zinc finger genes ZNF21 and ZNF81 as a candidate region of the Wiskott-Aldrich syndrome and narrows the boundaries to an interval bracketed by DXS6616 and DXS255. In addition, the current results identify the DXS1367 probe as a useful diagnostic marker for indirect genotype analysis of the Wiskott-Aldrich syndrome.« less
  • We report two sisters in a family representing manifestations of Wiskott-Aldrich syndrome (WAS), an X-linked immunodeficiency disorder. An elder sister had suffered from recurrent infections, small thrombocytopenic petechiae, purpura, and eczema for 7 years. The younger sister had the same manifestations as the elder sister`s for a 2-year period, and died of intracranial bleeding at age 2 years. All the laboratory data of the two patients were compatible with WAS, although they were females. Sialophorin analysis with the selective radioactive labeling method of this protein revealed that in the elder sister a 115-KD band that should be specific for sialophorinmore » was reduced in quantity, and instead an additional 135-KD fragment was present as a main band. Polymerase chain reaction (PCR) analysis of the sialophorin gene and single-strand conformation polymorphism (SSCP) analysis of the PCR product demonstrated that there were no detectable size-change nor electrophoretic mobility change in the DNA from both patients. The results indicated that their sialophorin gene structure might be normal. Studies on the mother-daughter transmission of X chromosome using a pERT84-MaeIII polymorphic marker mapped at Xp21 and HPRT gene polymorphism at Xq26 suggested that each sister had inherited a different X chromosome from the mother. Two explanations are plausible for the occurrence of the WAS in our patients: the WAS in the patients is attributable to an autosomal gene mutation which may regulate the sialophorin gene expression through the WAS gene, or, alternatively, the condition in this family is an autosomal recessive disorder separated etiologically from the X-linked WAS. 17 refs., 6 figs., 1 tab.« less
  • Sialophorin (CD43) of leukocytes and platelets is a surface sialoglycoprotein that is phenotypically defective on lymphocytes of patients with the X chromosome-linked immunodeficiency Wiskott-Aldrich syndrome. Previous studies with monoclonal antibodies indicate that sialophorin is a component of a T-lymphocyte activation pathway. Here the authors describe the cDNA cloning and derived amino acid sequence of human sialophorin. The sequence predicts an integral membrane polypeptide with an N-terminal hydrophobic signal region followed by a mucin-like 235-residue extracellular region with a uniform distribution of 46 serine, 47 threonine, and 24 proline residues. This is followed by a 23-residue transmembrane region and a 123-residuemore » C-terminal intracellular region. These latter regions have been highly conserved during evolution; the intracellular region contains a number of potential phosphorylation sites that might mediate transduction of activation signals. The chromosomal location of the sialophorin gene was determined and the implications of this assignment for the pathogenesis of the Wiskott-Aldrich syndrome are discussed.« less
  • The Wiskott-Aldrich syndrome (WAS) is one of several human immunodeficiency diseases inherited as an X-linked trait. The location of WAS on the X chromosome is unknown. The authors have studied 10 kindreds segregating for WAS for linkage with cloned, polymorphic DNA markers and have demonstrated significant linkage between WAS and two loci, DXS14 and DXS7, that map to the proximal short arm of the X chromosome. Maximal logarithm of odds (lod scores) for WAS-DXS14 and WAS-DWS7 were 4.29 (at 0 = 0.03) and 4.12 (at 0 = 0.00), respectively. Linkage data between WAS and six markers loci indicate the ordermore » of the loci to be (DXYS1-DXS1)-WAS-DXS14-DXS7-(DXS84-OTC). These results suggest that the WAS locus lies within the pericentric region of the X chromosome and provide an initial step toward identifying the WAS gene and improving the genetic counselling WAS families.« less