Alain Ibáñez de Opakua

Structure of p15 PAF –PCNA complex and implications for clamp sliding during DNA replication and repair

The intrinsically disordered protein p15(PAF) regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15(PAF)-PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP-box) of p15(PAF) tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15(PAF) also contacts the inside of, and passes through, the PCNA ring. The disordered p15(PAF) termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound p15(PAF) binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that p15(PAF) acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding.

The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets

Human antigen R (HuR) is a 32 kDa protein with 3 RNA Recognition Motifs (RRMs), which bind to Adenylate and uridylate Rich Elements (AREs) of mRNAs. Whereas the N-terminal and central domains (RRM1 and RRM2) are essential for AREs recognition, little is known on the C-terminal RRM3 beyond its implication in HuR oligomerization and apoptotic signaling. We have developed a detergent-based strategy to produce soluble RRM3 for structural studies. We have found that it adopts the typical RRM fold, does not interact with the RRM1 and RRM2 modules, and forms dimers in solution. Our NMR measurements, combined with Molecular Dynamics simulations and Analytical Ultracentrifugation experiments, show that the protein dimerizes through a helical region that contains the conserved W261 residue. We found that HuR RRM3 binds to 5′-mer U-rich RNA stretches through the solvent exposed side of its β-sheet, located opposite to the dimerization site. Upon mimicking phosphorylation by the S318D replacement, RRM3 mutant shows less ability to recognize RNA due to an electrostatic repulsion effect with the phosphate groups. Our study brings new insights of HuR RRM3 as a domain involved in protein oligomerization and RNA interaction, both functions regulated by 2 surfaces on opposite sides of the RRM domain.

Molecular mechanism of Gαi activation by non-GPCR proteins with a Gα-Binding and Activating motif

Heterotrimeric G proteins are quintessential signalling switches activated by nucleotide exchange on Gα. Although activation is predominantly carried out by G-protein-coupled receptors (GPCRs), non-receptor guanine-nucleotide exchange factors (GEFs) have emerged as critical signalling molecules and therapeutic targets. Here we characterize the molecular mechanism of G-protein activation by a family of non-receptor GEFs containing a Gα-binding and -activating (GBA) motif. We combine NMR spectroscopy, computational modelling and biochemistry to map changes in Gα caused by binding of GBA proteins with residue-level resolution. We find that the GBA motif binds to the SwitchII/α3 cleft of Gα and induces changes in the G-1/P-loop and G-2 boxes (involved in phosphate binding), but not in the G-4/G-5 boxes (guanine binding). Our findings reveal that G-protein-binding and activation mechanisms are fundamentally different between GBA proteins and GPCRs, and that GEF-mediated perturbation of nucleotide phosphate binding is sufficient for Gα activation.

Membrane Partitioning of the Pore-Forming Domain of Colicin A. Role of the Hydrophobic Helical Hairpin

The colicins are bacteriocins that target Escherichia coli and kill bacterial cells through different mechanisms. Colicin A forms ion channels in the inner membranes of nonimmune bacteria. This activity resides exclusively in its C-terminal fragment (residues 387-592). The soluble free form of this domain is a 10 α-helix bundle. The hydrophobic helical hairpin, H8-H9, is buried inside the structure and shielded by eight amphipathic surface helices. The interaction of the C-terminal colicin A domain and several chimeric variants with lipidic vesicles was examined here by isothermal titration calorimetry. In the mutant constructions, natural sequences of the hydrophobic helices H8 and H9 were either removed or substituted by polyalanine or polyleucine. All the constructions fully associated with DOPG liposomes including the mutant that lacked helices H8 and H9, indicating that amphipathic rather than hydrophobic helices were the major determinants of the exothermic binding reactions. Alanine is not specially favored in the lipid-bound form; the chimeric construct with polyalanine produced lower enthalpy gain. On the other hand, the large negative heat capacities associated with partitioning, a characteristic feature of the hydrophobic effect, were found to be dependent on the sequence hydrophobicity of helices H8 and H9.

The Gαi-GIV binding interface is a druggable protein-protein interaction

Heterotrimeric G proteins are usually activated by the guanine-nucleotide exchange factor (GEF) activity of GPCRs. However, some non-receptor proteins are also GEFs. GIV (a.k.a Girdin) was the first non-receptor protein for which the GEF activity was ascribed to a well-defined protein sequence that directly binds Gαi. GIV expression promotes metastasis and disruption of its binding to Gαi blunts the pro-metastatic behavior of cancer cells. Although this suggests that inhibition of the Gαi-GIV interaction is a promising therapeutic strategy, protein-protein interactions (PPIs) are considered poorly “druggable” targets requiring case-by-case validation. Here, we set out to investigate whether Gαi-GIV is a druggable PPI. We tested a collection of >1,000 compounds on the Gαi-GIV PPI by in silico ligand screening and separately by a chemical high-throughput screening (HTS) assay. Two hits, ATA and NF023, obtained in both screens were confirmed in secondary HTS and low-throughput assays. The binding site of NF023, identified by NMR spectroscopy and biochemical assays, overlaps with the Gαi-GIV interface. Importantly, NF023 did not disrupt Gαi-Gβγ binding, indicating its specificity toward Gαi-GIV. This work establishes the Gαi-GIV PPI as a druggable target and sets the conceptual and technical framework for the discovery of novel inhibitors of this PPI.

The tumor suppressor inhibitor of growth 4 binds double‐stranded DNA through its disordered central region

The tumor suppressor inhibitor of growth 4 (ING4) regulates chromatin structure by recruiting the histone acetyl transferase complex HBO1 to sites with histone H3 trimethylated at K4. ING4 dimerizes through its N‐terminal domain and recognizes H3K4me3 by the C‐terminal plant homeodomain (PHD). The central region of ING4 is disordered and contains the nuclear localization signal. Here, utilizing electrophoresis and nuclear magnetic resonance, we show that ING4 binds double‐stranded DNA through its central region with micromolar affinity. Our findings suggest that the cooperativity arising from the presence of two DNA‐binding regions in the ING4 dimer, as well as two H3K4me3‐binding PHD fingers, may strengthen nucleosome binding and HBO1 complex recruitment.

The metastasis suppressor KISS1 is an intrinsically disordered protein slightly more extended than a random coil.

The metastasis suppressor KISS1 is reported to be involved in the progression of several solid neoplasias, making it a promising molecular target for controlling their metastasis. The KISS1 sequence contains an N-terminal secretion signal and several dibasic sequences that are proposed to be the proteolytic cleavage sites. We present the first structural characterization of KISS1 by circular dichroism, multi-angle light scattering, small angle X-Ray scattering and NMR spectroscopy. An analysis of the KISS1 backbone NMR chemical shifts does not reveal any preferential conformation and deviation from a random coil ensemble. The backbone 15N transverse relaxation times indicate a mildly reduced mobility for two regions that are rich in bulky residues. The small angle X-ray scattering curve of KISS1 is likewise consistent with a predominantly random coil ensemble, although an ensemble optimization analysis indicates some preference for more extended conformations possibly due to positive charge repulsion between the abundant basic residues. Our results support the hypothesis that KISS1 mostly samples a random coil conformational space, which is consistent with its high susceptibility to proteolysis and the generation of Kisspeptin fragments.

NMR assignment and backbone dynamics of the pore-forming domain of colicin A

Colicin A protein kills cells by opening voltage-dependent ion channels in the cytoplasmic membrane. The C-terminal domain of colicin A retains the full protein’s ability to form membrane pores, making it an excellent model for in vitro studies of protein-membrane interaction. We report here the NMR assignment and backbone dynamics of this domain in solution. The chemical shifts identify ten α-helices that match those observed in the crystal structure, while the 15N{1H} NOEs show differential fast mobility for some of the inter-helical loops and the chain ends. This analysis provides the basis for further NMR studies of this channel forming protein and its interactions.