The ATPase domain of hsp70 possesses a unique binding specificity for 3'-sulfogalactolipids.
The region(s) of hsp70 critical for sulfogalactolipid (SGL) recognition has been defined through deletion analysis and site-directed mutagenesis. Truncated polymerase chain reaction products of hsp70 generated N-terminal fragments of 43, 35, 29, and 22 kDa. The C terminus substrate-binding domain (28 kDa) was also expressed. The N-terminal ATPase domain (rP43) shared the binding specificity of hsp70, because only sulfogalactosyl ceramide and sulfogalactosyl glycerolipid were recognized by both TLC overlay and RELISA. The C-terminal domain showed no binding. SGL binding of rP29 and rP22 was severely reduced. The loss of SGL binding for rP35 by RELISA but not TLC overlay was considered as a function of receptor presentation. The truncation of rP43 to rP35 demonstrates that residues 318-387 (the base of the ATP binding cleft) are critical for high affinity SGL binding. Mutagenesis showed that Arg(342) and Phe(198) are crucial for this process. SGL binding, mediated by these conserved residues within the ATPase domain of hsp70, implies that this binding specificity is evolutionarily conserved.
Mamelak
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D
Division of Infection, Immunity, Injury, and Repair, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Lingwood
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C
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002518:Ceramides / Q000378:metabolism; D002855:Chromatography, Thin Layer; D004797:Enzyme-Linked Immunosorbent Assay; D038983:Galactolipids; D006017:Glycolipids / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013379:Substrate Specificity; D013433:Sulfoglycosphingolipids / Q000378:metabolism; D013737:Testis / Q000737:chemistry
D002518:Ceramides; D038983:Galactolipids; D006017:Glycolipids; D018840:HSP70 Heat-Shock Proteins; C490434:Hspa2 protein, mouse; D010446:Peptide Fragments; D013433:Sulfoglycosphingolipids; C414346:sulfogalactosylglycerolipid; D000251:Adenosine Triphosphatases