Identification of elements of the peptide binding site of DnaK by peptide cross-linking.
We used photocross-linking of peptides to DnaK to identify elements of the peptide binding site of DnaK. We attached a photoactivatable group (N-hydroxysuccinimidyl-4-azido-salicylic acid (NHS-ASA) or N-iodoacetamidobutyl-4-azido-salicylic acid (I-ABASA)) to different positions on peptide C of the vesicular stomatitis virus glycoprotein, 125I-radiolabeled the cross-linker, cross-linked the peptide to DnaK by UV irradiation, and then determined the amino acid residues of DnaK that were cross-linked to the peptide. Limited trypsin digestion of the DnaK-peptide complex revealed that the derivatives modified with photoactivatable cross-linker peptide C cross-linked to a C-terminal fragment of DnaK and that the N-terminal 45-kDa fragment of DnaK was not cross-linked by these modified peptides. The attachment points of the three peptide C derivatives carrying photoactivatable cross-linkers at different locations on the peptide, PepC-ASA, PepC-S7C-ABASA, and PepC-S8C-ABASA, have been identified as Arg-536, Arg-527, and His-541 of DnaK, respectively. Thus all three peptides cross-linked to amino acids located close together in a sequence that includes one end of the long alpha-helix in the NMR-based secondary structure model of the peptide binding domain of Hsp70 family (Morshauser, R., Wang, H., Flynn, G., and Zuiderweg, E. (1995) Biochemistry 34, 6261-6266).
Zhang
J
J
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Walker
G C
GC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry; D003432:Cross-Linking Reagents; D000091342:Endoplasmic Reticulum Chaperone BiP; D029968:Escherichia coli Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry*; D006360:Heat-Shock Proteins; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D016415:Sequence Alignment; D014357:Trypsin / Q000737:chemistry; D014466:Ultraviolet Rays; D014721:Vesicular stomatitis Indiana virus / Q000737:chemistry; D014759:Viral Envelope Proteins / Q000737:chemistry
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D000091342:Endoplasmic Reticulum Chaperone BiP; D029968:Escherichia coli Proteins; C028391:G protein, vesicular stomatitis virus; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C494373:Hspa8 protein, rat; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D014759:Viral Envelope Proteins; D014357:Trypsin; C063842:dnaK protein, E coli