Nucleotide binding triggers a conformational change of the CBS module of the magnesium transporter CNNM2 from a twisted towards a flat structure.

Recent studies suggest CNNM2 (cyclin M2) to be part of the long-sought basolateral Mg2+ extruder at the renal distal convoluted tubule, or its regulator. In the present study, we explore structural features and ligand-binding capacities of the Bateman module of CNNM2 (residues 429-584), an intracellular domain structurally equivalent to the region involved in Mg2+ handling by the bacterial Mg2+ transporter MgtE, and AMP binding by the Mg2+ efflux protein CorC. Additionally, we studied the structural impact of the pathogenic mutation T568I located in this region. Our crystal structures reveal that nucleotides such as AMP, ADP or ATP bind at only one of the two cavities present in CNNM2429-584. Mg2+ favours ATP binding by alleviating the otherwise negative charge repulsion existing between acidic residues and the polyphosphate group of ATP. In crystals CNNM2429-584 forms parallel dimers, commonly referred to as CBS (cystathionine ß-synthase) modules. Interestingly, nucleotide binding triggers a conformational change in the CBS module from a twisted towards a flat disc-like structure that mostly affects the structural elements connecting the Bateman module with the transmembrane region. We furthermore show that the T568I mutation, which causes dominant hypomagnesaemia, mimics the structural effect induced by nucleotide binding. The results of the present study suggest that the T568I mutation exerts its pathogenic effect in humans by constraining the conformational equilibrium of the CBS module of CNNM2, which becomes 'locked' in its flat form.

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 ß-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.

Conformational diversity of human HP1α.

Heterochromatin protein 1 alpha (HP1α) is an evolutionarily conserved protein that binds chromatin and is important for gene silencing. The protein comprises 191 residues arranged into three disordered regions and two structured domains, the chromo and chromoshadow domain, which associates into a homodimer. While high-resolution structures of the isolated domains of HP1 proteins are known, the structural properties of full-length HP1α remain largely unknown. Using a combination of NMR spectroscopy and structure predictions by AlphaFold2 we provide evidence that the chromo and chromoshadow domain of HP1α engage in direct contacts resulting in a compact chromo/chromoshadow domain arrangement. We further show that HP1ß and HP1γ have increased interdomain dynamics when compared to HP1α which may contribute to the distinct roles of different Hp1 isoforms in gene silencing and activation.

PCNA molecular recognition of different PIP motifs: Role of Tyr211 phosphorylation.

The coordination of enzymes and regulatory proteins for eukaryotic DNA replication and repair is largely achieved by Proliferating Cell Nuclear Antigen (PCNA), a toroidal homotrimeric protein that embraces the DNA duplex. Many proteins bind PCNA through a conserved sequence known as the PCNA interacting protein motif (PIP). PCNA is further regulated by different post-translational modifications. Phosphorylation at residue Y211 facilitates unlocking stalled replication forks to bypass DNA damage repair processes but increasing nucleotide misincorporation. We explore here how phosphorylation at Y211 affects PCNA recognition of the canonical PIP sequences of the regulatory proteins p21 and p15, which bind with nM and µM affinity, respectively. For that purpose, we have prepared PCNA with p-carboxymethyl-L-phenylalanine (pCMF, a mimetic of phosphorylated tyrosine) at position 211. We have also characterized PCNA binding to the non-canonical PIP sequence of the catalytic subunit of DNA polymerase δ (p125), and to the canonical PIP sequence of the enzyme ubiquitin specific peptidase 29 (USP29) which deubiquitinates PCNA. Our results show that Tyr211 phosphorylation has little effect on the molecular recognition of p21 and p15, and that the PIP sequences of p125 and USP29 bind to the same site on PCNA as other PIP sequences, but with very low affinity.

Acetylation discriminates disease-specific tau deposition.

Pathogenic aggregation of the protein tau is a hallmark of Alzheimer's disease and several other tauopathies. Tauopathies are characterized by the deposition of specific tau isoforms as disease-related tau filament structures. The molecular processes that determine isoform-specific deposition of tau are however enigmatic. Here we show that acetylation of tau discriminates its isoform-specific aggregation. We reveal that acetylation strongly attenuates aggregation of four-repeat tau protein, but promotes amyloid formation of three-repeat tau. We further identify acetylation of lysine 298 as a hot spot for isoform-specific tau aggregation. Solid-state NMR spectroscopy demonstrates that amyloid fibrils formed by unmodified and acetylated three-repeat tau differ in structure indicating that site-specific acetylation modulates tau structure. The results implicate acetylation as a critical regulator that guides the selective aggregation of three-repeat tau and the development of tau isoform-specific neurodegenerative diseases.

Small-molecule targeting of GPCR-independent non-canonical G protein signaling inhibits cancer progression.

Activation of heterotrimeric G-proteins (Gαßγ) by G-protein-coupled receptors (GPCRs) is a quintessential mechanism of cell signaling widely targeted by clinically-approved drugs. However, it has become evident that heterotrimeric G-proteins can also be activated via GPCR-independent mechanisms that remain untapped as pharmacological targets. GIV/Girdin has emerged as a prototypical non-GPCR activator of G proteins that promotes cancer metastasis. Here, we introduce IGGi-11, a first-in-class smallmolecule inhibitor of non-canonical activation of heterotrimeric G-protein signaling. IGGi-11 binding to G-protein α-subunits (Gαi) specifically disrupted their engagement with GIV/Girdin, thereby blocking non-canonical G-protein signaling in tumor cells, and inhibiting pro-invasive traits of metastatic cancer cells in vitro and in mice. In contrast, IGGi-11 did not interfere with canonical G-protein signaling mechanisms triggered by GPCRs. By revealing that small molecules can selectively disable non-canonical mechanisms of G-protein activation dysregulated in disease, these findings warrant the exploration of therapeutic modalities in G-protein signaling that go beyond targeting GPCRs.

Proline/arginine dipeptide repeat polymers derail protein folding in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis and frontotemporal dementia are two neurodegenerative diseases with overlapping clinical features and the pathological hallmark of cytoplasmic deposits of misfolded proteins. The most frequent cause of familial forms of these diseases is a hexanucleotide repeat expansion in the non-coding region of the C9ORF72 gene that is translated into dipeptide repeat polymers. Here we show that proline/arginine repeat polymers derail protein folding by sequestering molecular chaperones. We demonstrate that proline/arginine repeat polymers inhibit the folding catalyst activity of PPIA, an abundant molecular chaperone and prolyl isomerase in the brain that is altered in amyotrophic lateral sclerosis. NMR spectroscopy reveals that proline/arginine repeat polymers bind to the active site of PPIA. X-ray crystallography determines the atomic structure of a proline/arginine repeat polymer in complex with the prolyl isomerase and defines the molecular basis for the specificity of disease-associated proline/arginine polymer interactions. The combined data establish a toxic mechanism that is specific for proline/arginine dipeptide repeat polymers and leads to derailed protein homeostasis in C9orf72-associated neurodegenerative diseases.

Co-factor-free aggregation of tau into seeding-competent RNA-sequestering amyloid fibrils.

Pathological aggregation of the protein tau into insoluble aggregates is a hallmark of neurodegenerative diseases. The emergence of disease-specific tau aggregate structures termed tau strains, however, remains elusive. Here we show that full-length tau protein can be aggregated in the absence of co-factors into seeding-competent amyloid fibrils that sequester RNA. Using a combination of solid-state NMR spectroscopy and biochemical experiments we demonstrate that the co-factor-free amyloid fibrils of tau have a rigid core that is similar in size and location to the rigid core of tau fibrils purified from the brain of patients with corticobasal degeneration. In addition, we demonstrate that the N-terminal 30 residues of tau are immobilized during fibril formation, in agreement with the presence of an N-terminal epitope that is specifically detected by antibodies in pathological tau. Experiments in vitro and in biosensor cells further established that co-factor-free tau fibrils efficiently seed tau aggregation, while binding studies with different RNAs show that the co-factor-free tau fibrils strongly sequester RNA. Taken together the study provides a critical advance to reveal the molecular factors that guide aggregation towards disease-specific tau strains.

Double Monoubiquitination Modifies the Molecular Recognition Properties of p15PAF Promoting Binding to the Reader Module of Dnmt1.

The proliferating cell nuclear antigen (PCNA)-associated factor p15PAF is a nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15PAF gene is overexpressed in several types of human cancer, and its function is regulated by monoubiquitination of two lysines (K15 and K24) at the protein N-terminal region. We have previously shown that p15PAF is an intrinsically disordered protein which partially folds upon binding to PCNA and independently contacts DNA through its N-terminal tail. Here we present an NMR conformational characterization of p15PAF monoubiquitinated at both K15 and K24 via a disulfide bridge mimicking the isopeptide bond. We show that doubly monoubiquitinated p15PAF is monomeric, intrinsically disordered, and binds to PCNA as nonubiquitinated p15PAF does but interacts with DNA with reduced affinity. Our SAXS-derived conformational ensemble of doubly monoubiquitinated p15PAF shows that the ubiquitin moieties, separated by eight disordered residues, form transient dimers because of the high local effective ubiquitin concentration. This observation and the sequence similarity with histone H3 N-terminal tail suggest that doubly monoubiquitinated p15PAF is a binding target of DNA methyl transferase Dnmt1, as confirmed by calorimetry. Therefore, doubly monoubiquitinated p15PAF directly interacts with PCNA and recruits Dnmt1 for maintenance of DNA methylation during replication.

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-Gßγ 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.

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.

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.