Conformational Dissection of a Viral Intrinsically Disordered Domain Involved in Cellular Transformation

Abstract

Intrinsic disorder is abundant in viral genomes and provides conformational plasticity to its protein products. In order to gain insight into its structure-function relationships, we carried out a comprehensive analysis of structural propensities within the intrinsically disordered N-terminal domain from the human papillomavirus type-16 E7 oncoprotein (E7N). Two E7N segments located within the conserved CR1 and CR2 regions present transient α-helix structure. The helix in the CR1 region spans residues L8 to L13 and overlaps with the E2F mimic linear motif. The second helix, located within the highly acidic CR2 region, presents a pH-dependent structural transition. At neutral pH the helix spans residues P17 to N29, which include the retinoblastoma tumor suppressor LxCxE binding motif (residues 21–29), while the acidic CKII-PEST region spanning residues E33 to I38 populates polyproline type II (PII) structure. At pH 5.0, the CR2 helix propagates up to residue I38 at the expense of loss of PII due to charge neutralization of acidic residues. Using truncated forms of HPV-16 E7, we confirmed that pH-induced changes in α-helix content are governed by the intrinsically disordered E7N domain. Interestingly, while at both pH the region encompassing the LxCxE motif adopts α-helical structure, the isolated 21–29 fragment including this stretch is unable to populate an α-helix even at high TFE concentrations. Thus, the E7N domain can populate dynamic but discrete structural ensembles by sampling α-helix-coil-PII-ß-sheet structures. This high plasticity may modulate the exposure of linear binding motifs responsible for its multi-target binding properties, leading to interference with key cell signaling pathways and eventually to cellular transformation by the virus.

Structural investigation on the intrinsically disordered N-terminal region of HPV16 E7 protein

“…The spectrum has narrow chemical-shift dispersion in both 15 N and 1 H dimensions, indicating that N-E7 does not form a globular structure under the non-denaturing experimental condition used (aqueous buffer at pH 6.5). Such an overall disordered nature of N-E7 is consistent with the results of the previous metal-free CD spectrum (11) and NMR experiments on N-E7 (29) , and also with bioinformatics predictions (30) . We achieved a full NMR resonance assignment for backbone 15 N and amide protons of N-E7 by following the standard triple-resonance assignment procedure, except for 3 prolines which do not have backbone amide NH nitrogens and protons.…”

“…Attempts to understand the role of the disordered N-terminal region of E7 were made by bioinformatics analyses using various independent disorder prediction programs; it suggested that the N-terminal region of HPV16 E7 was not entirely disordered (30) . In consistent with such prediction results, our investigation, together with the previous CD and NMR studies, show that the N-terminal half of E7, either N-E7 (this study) or the 40-residue N-terminal fragment (29) , is disordered and contains two helical PreSMos that mediate binding of N-E7 with partners; the first PreSMo is an E2F-mimic motif and the second encompasses the LXCXE motif allowing the binding of E7 with the retinoblastoma tumor suppressor protein, pRB (38) . We conclude that the N-terminal half of E7 displays promiscuity using its multiple PreSMos.…”

“…Note that the residues 33-34, potentially forming non-helical transient structures suggested by the SSP scores, also have small temperature coefficients. Presence of non-helical transient structures was noted in a previous 13 C-based NMR study which used a similar-sized N-terminal fragment (residues 1-40) of E7 (29) . This 13 C-HMQC based NMR study concluded that two helical PreSMos were formed by residues 8-13 and 17-29, which was slightly different from our results.…”

Interactions by Disorder – A Matter of Context

“…By increasing the number of positive charges in the flanking regions, the affinity for PCNA was modulated by up to four orders of magnitude (Figure 3 and Table 1) (Prestel et al, 2019). A similar effect was seen for pocket proteins of the retinoblastoma protein (Rb) family, that bind the SLiM LxCxE, present in host and viral interactors of the Rb family (Jones et al, 1990;Noval et al, 2013). Here, negative charges in the flanking regions act as affinity and specificity modulators ( Figure 1B) (Palopoli et al, 2018), conversely regulated by introduction of negative charge from phosphorylation of binding regions on the Rbs (Knudsen and Wang, 1996).…”

The High-Risk HPV16 E7 Oncoprotein Mediates Interaction between the Transcriptional Coactivator CBP and the Retinoblastoma Protein pRb

“…Cross peaks associated with the CR3 zinc finger dimerization domain are weak and broad (Figure S7). Concentration- and pH-dependent broadening of the CR3 resonances in full-length HPV16 E7 has recently been reported and has been attributed to flexibility and aggregation of the CR3 domain [13, 14]. Since the cross peaks for most residues in the CR1 and CR2 regions appear to coincide in the spectra of the peptide and the full length protein, and since the pulldown results were the same for E7(1-98) and E7(1-51) (Figure 1c, d), we deduced that the N-terminal region of the protein remained similarly disordered in the shorter construct and the full-length protein.…”