The tryptophan bridge is a critical feature of the papillomavirus E2 DNA binding domain.
The papillomavirus E2 protein is a DNA binding protein that regulates viral transcription and replication. E2 binds DNA as a dimer. Recent crystallographic data for E2 complexed to DNA revealed that novel peptide structures in E2 mediated dimerization and DNA binding. To identify important features of these motifs we have used limited proteolysis and urea denaturation as biochemical probes for structure, applying these techniques to E2 alone, E2 bound to DNA, cross-linked products, and mutants that were targeted at Trp360, a contact point along the dimer interface. DNA binding stabilized E2 structure, shifting the point at which it denatures from 5 to 7.6 M urea. In contrast, Trp360 mutant proteins, while dimeric, were more sensitive to denaturation by urea when bound to DNA. The most striking results came from uv cross-linking studies in which Trp360 was targeted as the site of cross-linking. Ultraviolet cross-linking dramatically increased the resistance of E2 to proteolysis regardless of the protease tested and with no deleterious effect on the affinity of E2 for DNA. Cross-linking through Cys356 with bismaleimidohexane did not promote stabilization. The ability to stabilize or destabilize E2 by Trp360-targeted modifications demonstrates the importance of the Trp360-Trp360 interaction, which may represent a general feature of the beta-barrel motif.
Corina
K
K
Biogen, Inc., Cambridge, Massachusetts 02142.
Grossman
S R
SR
Barsoum
J
J
Prakash
S S
SS
Androphy
E J
EJ
Pepinsky
R B
RB
D001483:Base Sequence; D001665:Binding Sites; D052688:Bovine papillomavirus 1 / Q000378:metabolism*; D002918:Chymotrypsin; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism / Q000528:radiation effects; D014357:Trypsin / Q000378:metabolism; D014364:Tryptophan; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects
D017931:DNA Primers; D004268:DNA-Binding Proteins; C051550:E2 protein, Bovine papillomavirus; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014764:Viral Proteins; D014364:Tryptophan; D002918:Chymotrypsin; D014357:Trypsin