Determinants of stability for the E6 protein of papillomavirus type 16.
E6 is an oncoprotein produced by human papillomavirus (HPV). The E6 protein from high-risk HPV type 16 contains two zinc-binding domains with two C-x-x-C motifs each. E6 exerts its transforming functions through formation of a complex with E6AP, which binds p53 and stimulates its degradation. There have been few biophysical and structural studies due to difficulty in preparation of soluble protein; here we describe the preparation of soluble E6 constructs including the two separated zinc-binding domains of E6. These proteins are used to examine the extent to which the two domains cooperate to mediate E6 function, how zinc influences the behavior of E6 protein, and which domains mediate aggregation. We demonstrate, using p53 degradation, E6AP binding, and hDlg (human homolog of the Drosophila discs large tumor suppressor protein) PDZ (postsynaptic density/disc large/zonula occludens) protein binding assays, that these soluble proteins are active, and, using NMR, circular dichroism, and fluorescence spectroscopies, we show that they are folded and stable. We show that the separated N-terminal and C-terminal domains interact, but nonproductively, for E6 function. The two domains bind zinc differently with higher affinity associated with the C-terminal domain. Analyses using surface plasmon resonance and circular dichroism and fluorescence spectroscopies show that aggregation is mediated more through the N-terminal domain than through the C-terminal domain. These studies allow a model in which the C-terminal zinc-binding domain of E6 recruits a target protein such as hDlg and the N-terminal domain is mostly responsible for recruiting a ubiquitin ligase to mediate target protein degradation.
Liu
Yuqi
Y
Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.
Cherry
Jonathan J
JJ
Dineen
Joseph V
JV
Androphy
Elliot J
EJ
Baleja
James D
JD
D009682:Magnetic Resonance Spectroscopy; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility / Q000187:drug effects; D013696:Temperature; D014508:Urea / Q000494:pharmacology; D015032:Zinc / Q000378:metabolism
C068651:E6 protein, Human papillomavirus type 16; D050505:Mutant Proteins; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014508:Urea; D015032:Zinc