Mapping of a substrate binding site in the protein disulfide isomerase-related chaperone wind based on protein function and crystal structure.
The protein disulfide isomerase (PDI)-related protein Wind is essential in Drosophila melanogaster, and is required for correct targeting of Pipe, an essential Golgi transmembrane 2-O-sulfotransferase. Apart from a thioredoxin fold domain present in all PDI proteins, Wind also has a unique C-terminal D-domain found only in PDI-D proteins. Here, we show that Pipe processing requires dimeric Wind, which interacts directly with the soluble domain of Pipe in vitro, and we map an essential substrate binding site in Wind to the vicinity of an exposed cluster of tyrosines within the thioredoxin fold domain. In vitro, binding occurs to multiple sites within the Pipe polypeptide and shows specificity for two consecutive aromatic residues. A second site in Wind, formed by a cluster of residues within the D-domain, is likewise required for substrate processing. This domain, expressed separately, impairs Pipe processing by the full-length Wind protein, indicating competitive binding to substrate. Our data represent the most accurate map of a peptide binding site in a PDI-related protein available to date and directly show peptide specificity for a naturally occurring substrate.
Barnewitz
Kathrin
K
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Germany.
Guo
Chaoshe
C
Sevvana
Madhumati
M
Ma
Qingjun
Q
Sheldrick
George M
GM
Söling
Hans-Dieter
HD
Ferrari
David M
DM
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D003461:Crystallography; D019281:Dimerization; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D057927:Hydrophobic and Hydrophilic Interactions; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000235:genetics; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D014709:Vero Cells
D029721:Drosophila Proteins; D018832:Molecular Chaperones; C110185:wbl protein, Drosophila; D001224:Aspartic Acid; D014443:Tyrosine; D013879:Thioredoxins; D015238:Sulfotransferases; C115847:pip protein, Drosophila; D019704:Protein Disulfide-Isomerases