Quantifying domain-ligand affinities and specificities by high-throughput holdup assay.

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this end, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to 1,000 domain-motif equilibrium binding affinities per day.

Disorder-to-order transition of MAGI-1 PDZ1 C-terminal extension upon peptide binding: thermodynamic and dynamic insights.

PDZ domains are highly abundant protein-protein interaction modules commonly found in multidomain scaffold proteins. The PDZ1 domain of MAGI-1, a protein present at cellular tight junctions that contains six PDZ domains, is targeted by the E6 oncoprotein of the high-risk human papilloma virus. Thermodynamic and dynamic studies using complementary isothermal titration calorimetry and nuclear magnetic resonance (NMR) (15)N heteronuclear relaxation measurements were conducted at different temperatures to decipher the molecular mechanism of this interaction.

Chemical library screening using a SPR-based inhibition in solution assay: simulations and experimental validation.

We have developed a surface plasmon resonance (SPR)-based inhibition in solution assay (ISA) to search for inhibitors of the medium affinity (KD = 0.8 μM) interaction between an E6-derived peptide (E6peptide) immobilized on the sensor and a PDZ domain (MAGI-1 PDZ1) in the mobile phase. DZ domains are widespread protein-protein interaction modules that recognize the C-terminus of various partners.

Structural basis for hijacking of cellular LxxLL motifs by papillomavirus E6 oncoproteins.

E6 viral oncoproteins are key players in epithelial tumors induced by papillomaviruses in vertebrates, including cervical cancer in humans. E6 proteins target many host proteins by specifically interacting with acidic LxxLL motifs. We solved the crystal structures of bovine (BPV1) and human (HPV16) papillomavirus E6 proteins bound to LxxLL peptides from the focal adhesion protein paxillin and the ubiquitin ligase E6AP, respectively.

The emerging contribution of sequence context to the specificity of protein interactions mediated by PDZ domains.

The canonical binding mode of PDZ domains to target motifs involves a small interface, unlikely to fully account for PDZ-target interaction specificities. Here, we review recent work on sequence context, defined as the regions surrounding not only the PDZ domains but also their target motifs. We also address the theoretical problem of defining the core of PDZ domains and the practical issue of designing PDZ constructs.

Proteasomal degradation of p53 by human papillomavirus E6 oncoprotein relies on the structural integrity of p53 core domain.

The E6 oncoprotein produced by high-risk mucosal HPV stimulates ubiquitinylation and proteasome-dependent degradation of the tumour suppressor p53 via formation of a trimeric complex comprising E6, p53, and E6-AP. p53 is also degraded by its main cellular regulator MDM2. The main binding site of p53 to MDM2 is situated in the natively unfolded N-terminal region of p53.

The structural and dynamic response of MAGI-1 PDZ1 with noncanonical domain boundaries to the binding of human papillomavirus E6.

PDZ domains are protein interaction domains that are found in cytoplasmic proteins involved in signaling pathways and subcellular transport. Their roles in the control of cell growth, cell polarity, and cell adhesion in response to cell contact render this family of proteins targets during the development of cancer. Targeting of these network hubs by the oncoprotein E6 of "high-risk" human papillomaviruses (HPVs) serves to effect the efficient disruption of cellular processes.

Surface plasmon resonance analysis of the binding of high-risk mucosal HPV E6 oncoproteins to the PDZ1 domain of the tight junction protein MAGI-1.

The E6 oncoproteins from high-risk mucosal human papillomavirus (HPV) induce cervical cancer via two major activities, the binding and the degradation of the p53 protein and PDZ domain-containing proteins. Human MAGI-1 is a multi-PDZ domain protein implicated into protein complex assembly at cell-cell contacts. High-risk mucosal HPV E6 proteins interact with the PDZ1 domain of MAGI-1 via a C-terminal consensus binding motif.

Prevalence of intrinsic disorder in the hepatitis C virus ARFP/Core+1/S protein.

The hepatitis C virus (HCV) Core+1/S polypeptide, also known as alternative reading frame protein (ARFP)/S, is an ARFP expressed from the Core coding region of the viral genome. Core+1/S is expressed as a result of internal initiation at AUG codons (85-87) located downstream of the polyprotein initiator codon, and corresponds to the C-terminal part of most ARFPs. Core+1/S is a highly basic polypeptide, and its function still remains unclear.

The social network of a cell: recent advances in interactome mapping.

Proteins very rarely act in isolation. Biomolecular interactions are central to all biological functions. In human, for example, interference with biomolecular networks often lead to disease.

Defining the minimal interacting regions of the tight junction protein MAGI-1 and HPV16 E6 oncoprotein for solution structure studies.

The oncoprotein E6 produced by tumorigenic high-risk genital human papillomaviruses targets a number of cellular proteins containing PDZ domains for proteasome-mediated degradation. In particular, E6 targets the tight junction protein MAGI-1 by binding to its PDZ1 domain. Using light scattering and NMR, we explored different fragments of both the HPV16 E6 and the MAGI-1 PDZ1 domain to define the best-behaving complex for solution structure studies.

Formation of well-defined soluble aggregates upon fusion to MBP is a generic property of E6 proteins from various human papillomavirus species.

Protein aggregation is a main barrier hindering structural and functional studies of a number of interesting biological targets. The E6 oncoprotein of Human Papillomavirus strain 16 (E6(16)) is difficult to express under a native soluble form in bacteria. Produced as an unfused sequence, it forms inclusion bodies.

Capturing protein-protein complexes at equilibrium: the holdup comparative chromatographic retention assay.

The popular pulldown chromatographic assay detects complexes mediated by fusion proteins retained on affinity resin. The main limitation of this method is that it does not analyze complexes at equilibrium but after several washing steps. Consequently, fast-dissociating complexes may remain undetected.

Structural and functional analysis of E6 oncoprotein: insights in the molecular pathways of human papillomavirus-mediated pathogenesis.

Oncoprotein E6 is essential for oncogenesis induced by human papillomaviruses (HPVs). The solution structure of HPV16-E6 C-terminal domain reveals a zinc binding fold. A model of full-length E6 is proposed and analyzed in the context of HPV evolution.

Kinetic analysis of the interactions of human papillomavirus E6 oncoproteins with the ubiquitin ligase E6AP using surface plasmon resonance.

Cervical cancers evolve from lesions generated by genital human papillomaviruses (HPV). "Low-risk" genital HPVs cause benign proliferations whereas "high-risk" types have the potential to progress into cancer. High-risk HPV E6 oncoproteins interact with the ubiquitin ligase E6AP and target several cellular proteins, including p53 and proteins of the MAGI family, towards ubiquitin-mediated degradation.

Binding of human papillomavirus 16 E6 to p53 and E6AP is impaired by monoclonal antibodies directed against the second zinc-binding domain of E6.

The E6 protein of cancer-associated human papillomavirus type 16 (16E6) binds to p53 and, in association with E6AP, promotes its degradation through the ubiquitin-proteasome pathway. The aim of this work was to develop monoclonal antibodies against 16E6 and to test their effect on the binding of 16E6 to p53 and E6AP, and on the degradation of p53. It was shown that an antibody directed against the N terminus of 16E6 inhibited E6AP-dependent binding to p53 and degradation of p53, whereas two different antibodies directed to the second zinc-binding domain of 16E6 reduced 16E6 E6AP-independent binding to p53 and binding to E6AP but not degradation of p53.

1H and 15N resonance assignment, secondary structure and dynamic behaviour of the C-terminal domain of human papillomavirus oncoprotein E6.

E6 is a viral oncoprotein implicated in cervical cancers, produced by human papillomaviruses (HPVs). E6 contains two putative zinc-binding domains of about 75 residues each. The difficulty in producing recombinant E6 has long hindered the obtention of structural data.

Domain substructure of HPV E6 oncoprotein: biophysical characterization of the E6 C-terminal DNA-binding domain.

E6 is a viral oncoprotein implicated in cervical cancers, produced by high-risk human papillomaviruses (HPVs). Structural data concerning this protein are scarce due to the difficulty of producing recombinant E6. Recently, we described the expression and purification of a stable, folded, and biologically active HPV16 E6 mutant called E6 6C/6S.