Structure of High-Risk Papillomavirus 31 E6 Oncogenic Protein and Characterization of E6/E6AP/p53 Complex Formation.

The degradation of p53 is a hallmark of high-risk human papillomaviruses (HPVs) of the alpha genus and HPV-related carcinogenicity. The oncoprotein E6 forms a ternary complex with the E3 ubiquitin ligase E6-associated protein (E6AP) and tumor suppressor protein p53 targeting p53 for ubiquitination. The extent of p53 degradation by different E6 proteins varies greatly, even for the closely related HPV16 and HPV31. HPV16 E6 and HPV31 E6 display high sequence identity (∼67%). We report here, for the first time, the structure of HPV31 E6 bound to the LxxLL motif of E6AP. HPV16 E6 and HPV31 E6 are structurally very similar, in agreement with the high sequence conservation. Both E6 proteins bind E6AP and degrade p53. However, the binding affinities of 31 E6 to the LxxLL motif of E6AP and p53, respectively, are reduced 2-fold and 5.4-fold compared to 16 E6. The affinity of E6-E6AP-p53 ternary complex formation parallels the efficacy of the subsequent reaction, namely, degradation of p53. Therefore, closely related E6 proteins addressing the same cellular targets may still diverge in their binding efficiencies, possibly explaining their different phenotypic or pathological impacts.IMPORTANCE Variations of carcinogenicity of human papillomaviruses are related to variations of the E6 and E7 interactome. While different HPV species and genera are known to target distinct host proteins, the fine differences between E6 and E7 of closely related HPVs, supposed to target the same cellular protein pools, remain to be addressed. We compare the oncogenic E6 proteins of the closely related high-risk HPV31 and HPV16 with regard to their structure and their efficiency of ternary complex formation with their cellular targets p53 and E6AP, which results in p53 degradation. We solved the crystal structure of 31 E6 bound to the E6AP LxxLL motif. HPV16 E6 and 31 E6 structures are highly similar, but a few sequence variations lead to different protein contacts within the ternary complex and, as quantified here, an overall lower binding affinity of 31 E6 than 16 E6. These results align with the observed lower p53 degradation potential of 31 E6.

Benchtop holdup assay for quantitative affinity-based analysis of sequence determinants of protein-motif interactions.

Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the "holdup" principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities.

One-step affinity purification of fusion proteins with optimal monodispersity and biological activity: application to aggregation-prone HPV E6 proteins.

BACKGROUND: Bacterial expression and purification of recombinant proteins under homogeneous active form is often challenging. Fusion to highly soluble carrier proteins such as Maltose Binding Protein (MBP) often improves their folding and solubility, but self-association may still occur. For instance, HPV E6 oncoproteins, when produced as MBP-E6 fusions, are expressed as mixtures of biologically inactive oligomers and active monomers. While a protocol was previously developed to isolate MBP-E6 monomers for structural studies, it allows the purification of only one MBP-E6 construct at the time. Here, we explored a parallelizable strategy more adapted for biophysical assays aiming at comparing different E6 proteins. RESULTS: In this study, we took advantage of the distinct size and diffusion properties of MBP-E6 monomers and oligomers to separate these two species using a rapid batch preparation protocol on affinity resins. We optimized resin reticulation, contact time and elution method in order to maximize the proportion of monomeric MBP-E6 in the final sample. Analytical size-exclusion chromatography was used to quantify the different protein species after purification. Thus, we developed a rapid, single-step protocol for the parallel purification of highly monomeric MBP-E6 samples. MBP-fused HPV16 E6 samples obtained by this approach were validated by testing the binding to their prototypical peptide targets (the LXXLL motif from ubiquitine ligase E6AP) by BIAcore-SPR assay. CONCLUSIONS: We have designed a rapid single-step batch affinity purification approach to isolate biologically active monomers of MBP-fused E6 proteins. This protocol should be generalizable to isolate the monomer (or the minimal biologically active oligomer) of other proteins prone to self-association.

Small p53 derived peptide suitable for robust nanobodies dimerization.

Bivalent VHHs have been shown to display better functional affinity compared with their monovalent counterparts. Bivalency can be achieved either by inserting a hinge region between both VHHs units or by using modules that lead to dimerization. In this report, a small self-associating peptide originating from the tetramerization domain of p53 was developed as a tool for devicing nanobody dimerization. This E3 peptide was evaluated for the dimerization of an anti-eGFP nanobody (nano-eGFP-E3) whose activity was compared to a bivalent anti-eGFP constructed in tandem using GS rich linker. The benefit of bivalency in terms of avidity and specificity was assessed in different in vitro and in cellulo assays. In ELISA and SPR, the dimeric and tandem formats were nearly equivalent in terms of gain of avidity compared to the monovalent counterpart. However, in cellulo, the nano-eGFP-E3 construct showed its superiority over the tandem format in terms of specificity with a highest and better ratio signal-to-noise. All together, the E3 peptide provides a universal suitable tool for the construction of dimeric biomolecules, in particular antibody fragments with improved functional affinity.

SynAggreg: A Multifunctional High-Throughput Technology for Precision Study of Amyloid Aggregation and Systematic Discovery of Synergistic Inhibitor Compounds.

Numerous proteins can coalesce into amyloid self-assemblies, which are responsible for a class of diseases called amyloidoses, but which can also fulfill important biological functions and are of great interest for biotechnology. Amyloid aggregation is a complex multi-step process, poorly prone to detailed structural studies. Therefore, small molecules interacting with amyloids are often used as tools to probe the amyloid aggregation pathway and in some cases to treat amyloidoses as they prevent pathogenic protein aggregation. Here, we report on SynAggreg, an in vitro high-throughput (HT) platform dedicated to the precision study of amyloid aggregation and the effect of modulator compounds. SynAggreg relies on an accurate bi-fluorescent amyloid-tracer readout that overcomes some limitations of existing HT methods. It allows addressing diverse aspects of aggregation modulation that are critical for pathomechanistic studies, such as the specificity of compounds toward various amyloids and their effects on aggregation kinetics, as well as the co-assembly propensity of distinct amyloids and the influence of prion-like seeding on self-assembly. Furthermore, SynAggreg is the first HT technology that integrates tailored methodology to systematically identify synergistic compound combinations-an emerging strategy to improve fatal amyloidoses by targeting multiple steps of the aggregation pathway. To this end, we apply analytical combinatorial scores to rank the inhibition efficiency of couples of compounds and to readily detect synergism. Finally, the SynAggreg platform should be suited for the characterization of a broad class of amyloids, whether of interest for drug development purposes, for fundamental research on amyloid functions, or for biotechnological applications.