The structural properties of full-length annexin A11.

Annexin A11 (ANXA11) is a calcium-dependent phospholipid-binding protein belonging to the annexin protein family and implicated in the neurodegenerative amyotrophic lateral sclerosis. Structurally, ANXA11 contains a conserved calcium-binding C-terminal domain common to all annexins and a putative intrinsically unfolded N-terminus specific for ANXA11. Little is known about the structure and functions of this region of the protein. By analogy with annexin A1, it was suggested that residues 38 to 59 within the ANXA11 N-terminus could form a helical region that would be involved in interactions. Interestingly, this region contains residues that, when mutated, may lead to clinical manifestations. In the present study, we have studied the structural features of the full-length protein with special attention to the N-terminal region using a combination of biophysical techniques which include nuclear magnetic resonance and small angle X-ray scattering. We show that the N-terminus is intrinsically disordered and that the overall features of the protein are not markedly affected by the presence of calcium. We also analyzed the 38-59 helix hypothesis using synthetic peptides spanning both the wild-type sequence and clinically relevant mutations. We show that the peptides have a remarkable character typical of a native helix and that mutations do not alter the behaviour suggesting that they are required for interactions rather than being structurally important. Our work paves the way to a more thorough understanding of the ANXA11 functions.

The Legionella collagen-like protein employs a unique binding mechanism for the recognition of host glycosaminoglycans.

Bacterial adhesion is a fundamental process which enables colonisation of niche environments and is key for infection. However, in Legionella pneumophila, the causative agent of Legionnaires' disease, these processes are not well understood. The Legionella collagen-like protein (Lcl) is an extracellular peripheral membrane protein that recognises sulphated glycosaminoglycans (GAGs) on the surface of eukaryotic cells, but also stimulates bacterial aggregation in response to divalent cations. Here we report the crystal structure of the Lcl C-terminal domain (Lcl-CTD) and present a model for intact Lcl. Our data reveal that Lcl-CTD forms an unusual dynamic trimer arrangement with a positively charged external surface and a negatively charged solvent exposed internal cavity. Through Molecular Dynamics (MD) simulations, we show how the GAG chondroitin-4-sulphate associates with the Lcl-CTD surface via unique binding modes. Our findings show that Lcl homologs are present across both the Pseudomonadota and Fibrobacterota-Chlorobiota-Bacteroidota phyla and suggest that Lcl may represent a versatile carbohydrate binding mechanism.

Affinity-enhanced RNA-binding domains as tools to understand RNA recognition.

Understanding how the RNA-binding domains of a protein regulator are used to recognize its RNA targets is a key problem in RNA biology, but RNA-binding domains with very low affinity do not perform well in the methods currently available to characterize protein-RNA interactions. Here, we propose to use conservative mutations that enhance the affinity of RNA-binding domains to overcome this limitation. As a proof of principle, we have designed and validated an affinity-enhanced K-homology (KH) domain mutant of the fragile X syndrome protein FMRP, a key regulator of neuronal development, and used this mutant to determine the domain's sequence preference and to explain FMRP recognition of specific RNA motifs in the cell. Our results validate our concept and our nuclear magnetic resonance (NMR)-based workflow. While effective mutant design requires an understanding of the underlying principles of RNA recognition by the relevant domain type, we expect the method will be used effectively in many RNA-binding domains.

Attenuation of reverse transcriptase facilitates SAMHD1 restriction of HIV-1 in cycling cells.

BACKGROUND: SAMHD1 is a deoxynucleotide triphosphohydrolase that restricts replication of HIV-1 in differentiated leucocytes. HIV-1 is not restricted in cycling cells and it has been proposed that this is due to phosphorylation of SAMHD1 at T592 in these cells inactivating the enzymatic activity. To distinguish between theories for how SAMHD1 restricts HIV-1 in differentiated but not cycling cells, we analysed the effects of substitutions at T592 on restriction and dNTP levels in both cycling and differentiated cells as well as tetramer stability and enzymatic activity in vitro. RESULTS: We first showed that HIV-1 restriction was not due to SAMHD1 nuclease activity. We then characterised a panel of SAMHD1 T592 mutants and divided them into three classes. We found that a subset of mutants lost their ability to restrict HIV-1 in differentiated cells which generally corresponded with a decrease in triphosphohydrolase activity and/or tetramer stability in vitro. Interestingly, no T592 mutants were able to restrict WT HIV-1 in cycling cells, despite not being regulated by phosphorylation and retaining their ability to hydrolyse dNTPs. Lowering dNTP levels by addition of hydroxyurea did not give rise to restriction. Compellingly however, HIV-1 RT mutants with reduced affinity for dNTPs were significantly restricted by wild-type and T592 mutant SAMHD1 in both cycling U937 cells and Jurkat T-cells. Restriction correlated with reverse transcription levels. CONCLUSIONS: Altogether, we found that the amino acid at residue 592 has a strong effect on tetramer formation and, although this is not a simple "on/off" switch, this does correlate with the ability of SAMHD1 to restrict HIV-1 replication in differentiated cells. However, preventing phosphorylation of SAMHD1 and/or lowering dNTP levels by adding hydroxyurea was not enough to restore restriction in cycling cells. Nonetheless, lowering the affinity of HIV-1 RT for dNTPs, showed that restriction is mediated by dNTP levels and we were able to observe for the first time that SAMHD1 is active and capable of inhibiting HIV-1 replication in cycling cells, if the affinity of RT for dNTPs is reduced. This suggests that the very high affinity of HIV-1 RT for dNTPs prevents HIV-1 restriction by SAMHD1 in cycling cells.

Characterisation of a cyclic peptide that binds to the RAS binding domain of phosphoinositide 3-kinase p110α.

P110α is a member of the phosphoinositide 3-kinase (PI3K) enzyme family that functions downstream of RAS. RAS proteins contribute to the activation of p110α by interacting directly with its RAS binding domain (RBD), resulting in the promotion of many cellular functions such as cell growth, proliferation and survival. Previous work from our lab has highlighted the importance of the p110α/RAS interaction in tumour initiation and growth. Here we report the discovery and characterisation of a cyclic peptide inhibitor (cyclo-CRVLIR) that interacts with the p110α-RBD and blocks its interaction with KRAS. cyclo-CRVLIR was discovered by screening a "split-intein cyclisation of peptides and proteins" (SICLOPPS) cyclic peptide library. The primary cyclic peptide hit from the screen initially showed a weak affinity for the p110α-RBD (Kd about 360 µM). However, two rounds of amino acid substitution led to cyclo-CRVLIR, with an improved affinity for p110α-RBD in the low µM (Kd 3 µM). We show that cyclo-CRVLIR binds selectively to the p110α-RBD but not to KRAS or the structurally-related RAF-RBD. Further, using biophysical, biochemical and cellular assays, we show that cyclo-CRVLIR effectively blocks the p110α/KRAS interaction in a dose dependent manner and reduces phospho-AKT levels in several oncogenic KRAS cell lines.

CP-MAS and Solution NMR Studies of Allosteric Communication in CA-assemblies of HIV-1.

Solution and solid-state NMR spectroscopy are highly complementary techniques for studying structure and dynamics in very high molecular weight systems. Here we have analysed the dynamics of HIV-1 capsid (CA) assemblies in presence of the cofactors IP6 and ATPγS and the host-factor CPSF6 using a combination of solution state and cross polarisation magic angle spinning (CP-MAS) solid-state NMR. In particular, dynamical effects on ns to µs and µs to ms timescales are observed revealing diverse motions in assembled CA. Using CP-MAS NMR, we exploited the sensitivity of the amide/Cα-Cß backbone chemical shifts in DARR and NCA spectra to observe the plasticity of the HIV-1 CA tubular assemblies and also map the binding of cofactors and the dynamics of cofactor-CA complexes. In solution, we measured how the addition of host- and co-factors to CA -hexamers perturbed the chemical shifts and relaxation properties of CA-Ile and -Met methyl groups using transverse-relaxation-optimized NMR spectroscopy to exploit the sensitivity of methyl groups as probes in high-molecular weight proteins. These data show how dynamics of the CA protein assembly over a range of spatial and temporal scales play a critical role in CA function. Moreover, we show that binding of IP6, ATPγS and CPSF6 results in local chemical shift as well as dynamic changes for a significant, contiguous portion of CA, highlighting how allosteric pathways communicate ligand interactions between adjacent CA protomers.

Sequence-specific 1H, 13C and 15N backbone NMR assignments for the N-terminal IgV-like domain (D1) and full extracellular region (D1D2) of PD-L1.

The co-inhibitory immune checkpoint interaction between programmed cell death-protein 1 (PD-1) and programmed cell death-ligand 1 (PD-L1) serves to regulate T-cell activation, promoting self-tolerance. Over-expression of PD-L1 is a mechanism through which tumour cells can evade detection by the immune system. Several therapeutic antibodies targeting PD-L1 or PD-1 have been approved for the treatment of a variety of cancers, however, the discovery and development of small-molecule inhibitors of PD-L1 remains a challenge. Here we report comprehensive sequence-specific backbone resonance assignments (1H, 13C, and 15N) obtained for the N-terminal IgV-like domain of PD-L1 (D1) and the full two domain extracellular region (D1D2). These NMR assignments will serve as a useful tool in the discovery of small-molecule therapeutics targeting PD-L1 and in the characterisation of functional interactions with other protein partners, such as CD80.

Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of Rp- and Sp-dNTPαS Diastereomers.

SAMHD1 is a fundamental regulator of cellular dNTPs that catalyzes their hydrolysis into 2'-deoxynucleoside and triphosphate, restricting the replication of viruses, including HIV-1, in CD4+ myeloid lineage and resting T-cells. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutières syndrome (AGS) and certain cancers. More recently, SAMHD1 has been linked to anticancer drug resistance and the suppression of the interferon response to cytosolic nucleic acids after DNA damage. Here, we probe dNTP hydrolysis and inhibition of SAMHD1 using the Rp and Sp diastereomers of dNTPαS nucleotides. Our biochemical and enzymological data show that the α-phosphorothioate substitution in Sp-dNTPαS but not Rp-dNTPαS diastereomers prevents Mg2+ ion coordination at both the allosteric and catalytic sites, rendering SAMHD1 unable to form stable, catalytically active homotetramers or hydrolyze substrate dNTPs at the catalytic site. Furthermore, we find that Sp-dNTPαS diastereomers competitively inhibit dNTP hydrolysis, while Rp-dNTPαS nucleotides stabilize tetramerization and are hydrolyzed with similar kinetic parameters to cognate dNTPs. For the first time, we present a cocrystal structure of SAMHD1 with a substrate, Rp-dGTPαS, in which an Fe-Mg-bridging water species is poised for nucleophilic attack on the Pα. We conclude that it is the incompatibility of Mg2+, a hard Lewis acid, and the α-phosphorothioate thiol, a soft Lewis base, that prevents the Sp-dNTPαS nucleotides coordinating in a catalytically productive conformation. On the basis of these data, we present a model for SAMHD1 stereospecific hydrolysis of Rp-dNTPαS nucleotides and for a mode of competitive inhibition by Sp-dNTPαS nucleotides that competes with formation of the enzyme-substrate complex.

Targeting the nucleotide salvage factor DNPH1 sensitizes BRCA-deficient cells to PARP inhibitors.

Mutations in the BRCA1 or BRCA2 tumor suppressor genes predispose individuals to breast and ovarian cancer. In the clinic, these cancers are treated with inhibitors that target poly(ADP-ribose) polymerase (PARP). We show that inhibition of DNPH1, a protein that eliminates cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine (hmdU) monophosphate, potentiates the sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi). Synthetic lethality was mediated by the action of SMUG1 glycosylase on genomic hmdU, leading to PARP trapping, replication fork collapse, DNA break formation, and apoptosis. BRCA1-deficient cells that acquired resistance to PARPi were resensitized by treatment with hmdU and DNPH1 inhibition. Because genomic hmdU is a key determinant of PARPi sensitivity, targeting DNPH1 provides a promising strategy for the hypersensitization of BRCA-deficient cancers to PARPi therapy.

Backbone chemical shift spectral assignments of SARS coronavirus-2 non-structural protein nsp9.

As part of an International consortium aiming at the characterization by NMR of the proteins of the SARS-CoV-2 virus, we have obtained the virtually complete assignment of the backbone atoms of the non-structural protein nsp9. This small (12 kDa) protein is encoded by ORF1a, binds to RNA and seems to be essential for viral RNA synthesis. The crystal structures of the SARS-CoV-2 protein and other homologues suggest that the protein is dimeric as also confirmed by analytical ultracentrifugation and dynamic light scattering. Our data constitute the prerequisite for further NMR-based characterization, and provide the starting point for the identification of small molecule lead compounds that could interfere with RNA binding and prevent viral replication.

Mining the PDB for Tractable Cases Where X-ray Crystallography Combined with Fragment Screens Can Be Used to Systematically Design Protein-Protein Inhibitors: Two Test Cases Illustrated by IL1ß-IL1R and p38α-TAB1 Complexes.

Nowadays, it is possible to combine X-ray crystallography and fragment screening in a medium throughput fashion to chemically probe the surfaces used by proteins to interact and use the outcome of the screens to systematically design protein-protein inhibitors. To prove it, we first performed a bioinformatics analysis of the Protein Data Bank protein complexes, which revealed over 400 cases where the crystal lattice of the target in the free form is such that large portions of the interacting surfaces are free from lattice contacts and therefore accessible to fragments during soaks. Among the tractable complexes identified, we then performed single fragment crystal screens on two particular interesting cases: the Il1ß-ILR and p38α-TAB1 complexes. The result of the screens showed that fragments tend to bind in clusters, highlighting the small-molecule hotspots on the surface of the target protein. In most of the cases, the hotspots overlapped with the binding sites of the interacting proteins.

Crystal structures of SAMHD1 inhibitor complexes reveal the mechanism of water-mediated dNTP hydrolysis.

SAMHD1 regulates cellular 2'-deoxynucleoside-5'-triphosphate (dNTP) homeostasis by catalysing the hydrolysis of dNTPs into 2'-deoxynucleosides and triphosphate. In CD4+ myeloid lineage and resting T-cells, SAMHD1 blocks HIV-1 and other viral infections by depletion of the dNTP pool to a level that cannot support replication. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutières syndrome and hypermutated cancers. Furthermore, SAMHD1 sensitises cancer cells to nucleoside-analogue anti-cancer therapies and is linked with DNA repair and suppression of the interferon response to cytosolic nucleic acids. Nevertheless, despite its requirement in these processes, the fundamental mechanism of SAMHD1-catalysed dNTP hydrolysis remained unknown. Here, we present structural and enzymological data showing that SAMHD1 utilises an active site, bi-metallic iron-magnesium centre that positions a hydroxide nucleophile in-line with the Pα-O5' bond to catalyse phosphoester bond hydrolysis. This precise molecular mechanism for SAMHD1 catalysis, reveals how SAMHD1 down-regulates cellular dNTP and modulates the efficacy of nucleoside-based anti-cancer and anti-viral therapies.

Genome Analysis of Melon Necrotic Spot Virus Incursions and Seed Interceptions in Australia.

Melon necrotic spot virus (MNSV) was detected in field-grown Cucumis melo (rockmelon) and Citrullus lanatus (watermelon) plants in the Sunraysia district of New South Wales and Victoria, Australia, in 2012, 2013, and 2016, and in two watermelon seed lots tested at the Australian border in 2016. High-throughput sequencing was used to generate near full-length genomes of six isolates detected during the incursions and seed testing. Phylogenetic analysis of the genomes suggests that there have been at least two incursions of MNSV into Australia and none of the field isolates were the same as the isolates detected in seeds. The analysis indicated that one watermelon field sample (L10), the Victorian rockmelon field sample, and two seed interception samples may have European origins. The results showed that two isolates (L8 and L9) from watermelon were divergent from the type MNSV strain (MNSV-GA, D12536.2) and had 99% nucleotide identity to two MNSV isolates from human stool collected in the United States (KY124135.1, KY124136.1). These isolates also had high nucleotide pairwise identity (96%) to a partial sequence from a Spanish MNSV isolate (KT962848.1). The analysis supported the identification of three previously described MNSV genotype groups: EU-LA, Japan melon, and Japan watermelon. To account for the greater diversity of hosts and geographic regions of the MNSV isolates used in this study, it is suggested that the genotype groups EU-LA, Japan melon, and Japan watermelon be renamed to groups I, II, and III, respectively. The divergent isolates L8 and L9 from this study and the stool isolates from the United States formed a fourth genotype group, group IV. Soil collected from the site of the Victorian rockmelon MNSV outbreak was found to contain viable MNSV and the virus vector, a chytrid fungus, Olpidium bornovanus (Sahtiyanci) Karling, 18 months after the initial MNSV detection. This is a first report of O. bornovanus from soil sampled from an MNSV-contaminated site in Australia.

D-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition.

The broad-spectrum antibiotic D-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analog of D-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and D-Ala:D-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5'-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.

IMP1 KH1 and KH2 domains create a structural platform with unique RNA recognition and re-modelling properties.

IGF2 mRNA-binding protein 1 (IMP1) is a key regulator of messenger RNA (mRNA) metabolism and transport in organismal development and, in cancer, its mis-regulation is an important component of tumour metastasis. IMP1 function relies on the recognition of a diverse set of mRNA targets that is mediated by the combinatorial action of multiple RNA-binding domains. Here, we dissect the structure and RNA-binding properties of two key RNA-binding domains of IMP1, KH1 and KH2, and we build a kinetic model for the recognition of RNA targets. Our data and model explain how the two domains are organized as an intermolecular pseudo-dimer and that the important role they play in mRNA target recognition is underpinned by the high RNA-binding affinity and fast kinetics of this KH1KH2-RNA recognition unit. Importantly, the high-affinity RNA-binding by KH1KH2 is achieved by an inter-domain coupling 50-fold stronger than that existing in a second pseudo-dimer in the protein, KH3KH4. The presence of this strong coupling supports a role of RNA re-modelling in IMP1 recognition of known cancer targets.

LARP4A recognizes polyA RNA via a novel binding mechanism mediated by disordered regions and involving the PAM2w motif, revealing interplay between PABP, LARP4A and mRNA.

LARP4A belongs to the ancient RNA-binding protein superfamily of La-related proteins (LARPs). In humans, it acts mainly by stabilizing mRNAs, enhancing translation and controlling polyA lengths of heterologous mRNAs. These activities are known to implicate its association with mRNA, protein partners and translating ribosomes, albeit molecular details are missing. Here, we characterize the direct interaction between LARP4A, oligoA RNA and the MLLE domain of the PolyA-binding protein (PABP). Our study shows that LARP4A-oligoA association entails novel RNA recognition features involving the N-terminal region of the protein that exists in a semi-disordered state and lacks any recognizable RNA-binding motif. Against expectations, we show that the La module, the conserved RNA-binding unit across LARPs, is not the principal determinant for oligoA interaction, only contributing to binding to a limited degree. Furthermore, the variant PABP-interacting motif 2 (PAM2w) featured in the N-terminal region of LARP4A was found to be important for both RNA and PABP recognition, revealing a new role for this protein-protein binding motif. Our analysis demonstrates the mutual exclusive nature of the PAM2w-mediated interactions, thereby unveiling a tantalizing interplay between LARP4A, polyA and PABP.

Resonance assignment of human LARP4A La module.

Human LARP4A belongs to a superfamily of RNA binding proteins called La-related proteins (LARPs). Whilst being a positive regulator of protein synthesis and a promoter of mRNA stability, LARP4A also controls cell morphology and motility in human breast and prostate cancer cells. All LARPs share a characteristic RNA binding unit named the La-module, which despite a high level of primary structure conservation exhibits a great versatility in RNA target selection. Human LARP4A La-module is the most divergent compared with other LARPs and its RNA recognition properties have only recently started to be revealed. Given the key role of LARP4A protein in cancer cell biology, we have initiated a complete NMR characterisation of its La-module and here we report the assignment of 1H, 15N and 13C resonances resulting from our studies.

The Structural Properties in Solution of the Intrinsically Mixed Folded Protein Ataxin-3.

It has increasingly become clear over the last two decades that proteins can contain both globular domains and intrinsically unfolded regions that can both contribute to function. Although equally interesting, the disordered regions are difficult to study, because they usually do not crystallize unless bound to partners and are not easily amenable to cryo-electron microscopy studies. NMR spectroscopy remains the best technique to capture the structural features of intrinsically mixed folded proteins and describe their dynamics. These studies rely on the successful assignment of the spectrum, a task not easy per se given the limited spread of the resonances of the disordered residues. Here, we describe the structural properties of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. Ataxin-3 is a 42-kDa protein containing a globular N-terminal Josephin domain and a C-terminal tail that comprises 13 polyglutamine repeats within a low complexity region. We developed a strategy that allowed us to achieve 87% assignment of the NMR spectrum using a mixed protocol based on high-dimensionality, high-resolution experiments and different labeling schemes. Thanks to the almost complete spectral assignment, we proved that the C-terminal tail is flexible, with extended helical regions, and interacts only marginally with the rest of the protein. We could also, for the first time to our knowledge, observe the structural propensity of the polyglutamine repeats within the context of the full-length protein and show that its structure is stabilized by the preceding region.

Chemical shift assignment of a thermophile frataxin.

Frataxin is the protein responsible for the genetically-inherited neurodegenerative disease Friedreich's ataxia caused by partial silencing of the protein and loss of function. Although the frataxin function is not yet entirely clear, it has been associated to the machine that builds iron-sulfur clusters, essential prosthetic groups involved in several processes and is strongly conserved in organisms from bacteria to humans. Two of its important molecular partners are the protein NFS1 (or IscS in bacteria), that is the desulfurase which converts cysteine to alanine and produces sulfur, and ISU (or IscU), the scaffold protein which transiently accepts the cluster. While bacterial frataxin has been extensively characterized, only few eukaryotic frataxins have been described. Here we report the 1H, 13C and 15N backbone and side-chain chemical shift assignments of frataxin from Chaetomium thermophilum, a thermophile increasingly used by virtue of its stability.

Fragment-based de novo design of a cystathionine γ-lyase selective inhibitor blocking hydrogen sulfide production.

Hydrogen sulfide is an essential catabolite that intervenes in the pathophysiology of several diseases from hypertension to stroke, diabetes and pancreatitis. It is endogenously synthesized mainly by two pyridoxal-5'-phosphate-dependent enzymes involved in L-cysteine metabolism: cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE). Research in this field is currently impaired by the lack of pharmacological tools such as selective enzymatic inhibitors that could target specifically only one of these pathways. We used a novel approach based on a hybrid method that includes drug design, synthetic biology, metabolomics and pharmacological assays to rationally design a new inhibitor selective for the CSE enzyme. The identification of this compound opens new frontiers towards a better understanding of the role of CSE over CBS in the pathophysiology of diseases where a role for the H2S pathway has been proposed and the development of new lead compounds that could target the CSE enzyme.