A new class of capsid-targeting inhibitors that specifically block HIV-1 nuclear import.

HIV-1 capsids cross nuclear pore complexes (NPCs) by engaging with the nuclear import machinery. To identify compounds that inhibit HIV-1 nuclear import, we screened drugs in silico on a three-dimensional model of a CA hexamer bound by Transportin-1 (TRN-1). Among hits, compound H27 inhibited HIV-1 with a low micromolar IC.

Direct m6A recognition by IMP1 underlays an alternative model of target selection for non-canonical methyl-readers.

m6A methylation provides an essential layer of regulation in organismal development, and is aberrant in a range of cancers and neuro-pathologies. The information encoded by m6A methylation is integrated into existing RNA regulatory networks by RNA binding proteins that recognise methylated sites, the m6A readers. m6A readers include a well-characterised class of dedicated proteins, the YTH proteins, as well as a broader group of multi-functional regulators where recognition of m6A is only partially understood.

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.

The distinct RNA-interaction modes of a small ZnF domain underlay TUT4(7) diverse action in miRNA regulation.

TUT4 and the closely related TUT7 are non-templated poly(U) polymerases required at different stages of development, and their mis-regulation or mutation has been linked to important cancer pathologies. While TUT4(7) interaction with its pre-miRNA targets has been characterized in detail, the molecular bases of the broader target recognition process are unclear. Here, we examine RNA binding by the ZnF domains of the protein.

Structural basis for Fullerene geometry in a human endogenous retrovirus capsid.

The HML2 (HERV-K) group constitutes the most recently acquired family of human endogenous retroviruses, with many proviruses less than one million years old. Many maintain intact open reading frames and provirus expression together with HML2 particle formation are observed in early stage human embryo development and are associated with pluripotency as well as inflammatory disease, cancers and HIV-1 infection. Here, we reconstruct the core structural protein (CA) of an HML2 retrovirus, assemble particles in vitro and employ single particle cryogenic electron microscopy (cryo-EM) to determine structures of four classes of CA Fullerene shell assemblies.

Cyclic AMP signalling controls key components of malaria parasite host cell invasion machinery.

Cyclic AMP (cAMP) is an important signalling molecule across evolution, but its role in malaria parasites is poorly understood. We have investigated the role of cAMP in asexual blood stage development of Plasmodium falciparum through conditional disruption of adenylyl cyclase beta (ACβ) and its downstream effector, cAMP-dependent protein kinase (PKA). We show that both production of cAMP and activity of PKA are critical for erythrocyte invasion, whilst key developmental steps that precede invasion still take place in the absence of cAMP-dependent signalling.

Mechanism of β-actin mRNA Recognition by ZBP1.

Zipcode binding protein 1 (ZBP1) is an oncofetal RNA-binding protein that mediates the transport and local translation of β-actin mRNA by the KH3-KH4 di-domain, which is essential for neuronal development. The high-resolution structures of KH3-KH4 with their respective target sequences show that KH4 recognizes a non-canonical GGA sequence via an enlarged and dynamic hydrophobic groove, whereas KH3 binding to a core CA sequence occurs with low specificity. A data-informed kinetic simulation of the two-step binding reaction reveals that the overall reaction is driven by the second binding event and that the moderate affinities of the individual interactions favor RNA looping.

Structure of a Spumaretrovirus Gag Central Domain Reveals an Ancient Retroviral Capsid.

The Spumaretrovirinae, or foamy viruses (FVs) are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function.

HIV-1 capsid is involved in post-nuclear entry steps.

Background: HIV-1 capsid influences viral uncoating and nuclear import. Some capsid is detected in the nucleus but it is unclear if it has any function. We reported that the antibiotic Coumermycin-A1 (C-A1) inhibits HIV-1 integration and that a capsid mutation confers resistance to C-A1, suggesting that capsid might affect post-nuclear entry steps.

KH-RNA interactions: back in the groove.

The hnRNP K-homology (KH) domain is a single stranded nucleic acid binding domain that mediates RNA target recognition by a large group of gene regulators. The structure of the KH fold is well characterised and some initial rules for KH-RNA recognition have been drafted. However, recent findings have shown that these rules need to be revisited and have now provided a better understanding of how the domain can recognise a sequence landscape larger than previously thought as well as revealing the diversity of structural expansions to the KH domain.

Terminal loop-mediated regulation of miRNA biogenesis: selectivity and mechanisms.

Regulating the expression of individual miRNAs (microRNAs) is important for cell development and function. The up- or down-regulation of the processing of specific miRNA precursors to the mature active form represents one tool to control miRNA concentration and is mediated by proteins that recognize the terminal loop of the RNA precursors. Terminal loop recognition is achieved by the combined action of several RNA-binding domains.

Noncanonical G recognition mediates KSRP regulation of let-7 biogenesis.

Let-7 is an important tumor-suppressive microRNA (miRNA) that acts as an on-off switch for cellular differentiation and regulates the expression of a set of human oncogenes. Binding of the human KSRP protein to let-7 miRNA precursors positively regulates their processing to mature let-7, thereby contributing to control of cell proliferation, apoptosis and differentiation. Here we analyze the molecular basis for KSRP-let-7 precursor selectivity and show how the third KH domain of the protein recognizes a G-rich sequence in the pre-let-7 terminal loop and dominates the interaction.

Understanding the role of the Josephin domain in the PolyUb binding and cleavage properties of ataxin-3.

Ataxin-3, the disease protein in the neurodegenerative disorder Spinocerebellar Ataxia Type 3 or Machado Joseph disease, is a cysteine protease implicated in the ubiquitin proteasome pathway. It contains multiple ubiquitin binding sites through which it anchors polyubiquitin chains of different linkages that are then cleaved by the N-terminal catalytic (Josephin) domain. The properties of the ubiquitin interacting motifs (UIMs) in the C-terminus of ataxin-3 are well established.

Functional interactions as a survival strategy against abnormal aggregation.

Protein aggregation is under intense scrutiny because of its role in human disease. Although increasing evidence indicates that protein native states are highly protected against aggregation, the specific protection mechanisms are poorly understood. Insight into such mechanisms can be gained through study of the relatively few proteins that aggregate under native conditions.

Solution structure of the phytotoxic protein PcF: the first characterized member of the Phytophthora PcF toxin family.

The PcF protein from Phytophthora cactorum is the first member of the "PcF toxin family" from the plant pathogens Phytophthora spp. It is able to induce withering in tomato and strawberry leaves. The lack of sequence similarity with other proteins hampers the identification of the molecular mechanisms responsible for its toxicity.

Josephin domain of ataxin-3 contains two distinct ubiquitin-binding sites.

Joseph-Machado is an incurable neurodegenerative disease caused by toxic aggregation of ataxin-3, a ubiquitin-specific cysteine protease, involved in the ubiquitin-proteasome pathway and known to bind poly-ubiquitin chains of four or more subunits. The enzymatic site resides in the N-terminal josephin domain of ataxin-3. We have characterized the ubiquitin-binding properties of josephin and showed that, unexpectedly, josephin contains two contiguous but distinct ubiquitin-binding sites.

Discovery of subnanomolar arginine-glycine-aspartate-based alphaVbeta3/alphaVbeta5 integrin binders embedding 4-aminoproline residues.

The embodiment of 4-aminoproline residues (Amp) into the arginine-glycine-aspartate (RGD) sequence led to the discovery of a novel class of high-affinity alpha Vbeta 3/alpha Vbeta 5 integrin binders [IC 50 h (alpha Vbeta 3) 0.03-5.12 nM; IC 50 h (alpha Vbeta 5) 0.

Diastereoselective synthesis of 4,5'-bis-proline compounds via reductive dimerization of N-acyloxyiminium ions.

A short, practical synthesis of novel, unsymmetrical 4,5'-bis-proline compounds has been achieved, highlighted by the application of an unprecedented samarium diiodide-driven regio- and diastereocontrolled reductive dimerization of N-acyloxyiminium ions generated from readily available 2-methoxy-5-silyloxymethyl-N-Boc pyrrolidines. The title proline dimers proved to be pertinent metal-free catalysts in aldol and Mannich reactions.

Structure validation of the Josephin domain of ataxin-3: conclusive evidence for an open conformation.

The availability of new and fast tools in structure determination has led to a more than exponential growth of the number of structures solved per year. It is therefore increasingly essential to assess the accuracy of the new structures by reliable approaches able to assist validation. Here, we discuss a specific example in which the use of different complementary techniques, which include Bayesian methods and small angle scattering, resulted essential for validating the two currently available structures of the Josephin domain of ataxin-3, a protein involved in the ubiquitin/proteasome pathway and responsible for neurodegenerative spinocerebellar ataxia of type 3.

Grafting aminocyclopentane carboxylic acids onto the RGD tripeptide sequence generates low nanomolar alphaVbeta3/alphaVbeta5 integrin dual binders.

Eleven gamma-aminocyclopentane carboxylic acid (Acpca) platforms, including four dihydroxy representatives (19-22), three hydroxy analogues (34-36), and four deoxy derivatives (30-33), were prepared in a chiral nonracemic format. These simple units were then grafted onto an Arg-Gly-Asp (RGD) tripeptide framework by a mixed solid phase/solution protocol delivering an ensemble of 11 macrocyclic analogues of type cyclo-[-Arg-Gly-Asp-Acpca-], 1-11. The individual compounds were evaluated for their binding affinity toward the alphaVbeta3 and alphaVbeta5 integrin receptors.

The solution structure of the Josephin domain of ataxin-3: structural determinants for molecular recognition.

The Josephin domain plays an important role in the cellular functions of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. We have determined the solution structure of Josephin and shown that it belongs to the family of papain-like cysteine proteases, sharing the highest degree of structural similarity with bacterial staphopain. A currently unique structural feature of Josephin is a flexible helical hairpin formed by a 32-residue insertion, which could determine substrate specificity.

Characterization of the structure and the amyloidogenic properties of the Josephin domain of the polyglutamine-containing protein ataxin-3.

Expansion of the polyglutamine (polyQ) region in the protein ataxin-3 is associated with spinocerebellar ataxia type 3, an inherited neurodegenerative disorder that belongs to the family of polyQ diseases. Increasing evidence indicates that protein aggregation and fibre formation play an important role in these pathologies. In a previous study, we determined the domain architecture of ataxin-3, suggesting that it comprises a globular domain, named Josephin, and a more flexible C-terminal region, that includes the polyQ tract.