ABSTRACT
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.
Subject(s)
Proto-Oncogene Proteins c-myc/genetics , RNA, Messenger/chemistry , RNA-Binding Proteins/chemistry , Amino Acid Sequence , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins c-myc/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Exosomal miRNA transfer is a mechanism for cell-cell communication that is important in the immune response, in the functioning of the nervous system and in cancer. Syncrip/hnRNPQ is a highly conserved RNA-binding protein that mediates the exosomal partition of a set of miRNAs. Here, we report that Syncrip's amino-terminal domain, which was previously thought to mediate protein-protein interactions, is a cryptic, conserved and sequence-specific RNA-binding domain, designated NURR (N-terminal unit for RNA recognition). The NURR domain mediates the specific recognition of a short hEXO sequence defining Syncrip exosomal miRNA targets, and is coupled by a non-canonical structural element to Syncrip's RRM domains to achieve high-affinity miRNA binding. As a consequence, Syncrip-mediated selection of the target miRNAs implies both recognition of the hEXO sequence by the NURR domain and binding of the RRM domains 5' to this sequence. This structural arrangement enables Syncrip-mediated selection of miRNAs with different seed sequences.
Subject(s)
Aptamers, Nucleotide/chemistry , Drosophila Proteins/chemistry , Heterogeneous-Nuclear Ribonucleoproteins/chemistry , MicroRNAs/chemistry , RNA-Binding Proteins/chemistry , RNA/chemistry , Amino Acid Sequence , Animals , Aptamers, Nucleotide/genetics , Aptamers, Nucleotide/metabolism , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Exosomes/chemistry , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , RNA/genetics , RNA/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
ATP-phosphoribosyltransferase (ATP-PRT) is a hexameric enzyme in conformational equilibrium between an open and seemingly active state and a closed and presumably inhibited form. The structure-function relationship of allosteric regulation in this system is still not fully understood. Here, we develop a screening strategy for modulators of ATP-PRT and identify 3-(2-thienyl)-L-alanine (TIH) as an allosteric activator of this enzyme. Kinetic analysis reveals co-occupancy of the allosteric sites by TIH and L-histidine. Crystallographic and native ion-mobility mass spectrometry data show that the TIH-bound activated form of the enzyme closely resembles the inhibited L-histidine-bound closed conformation, revealing the uncoupling between ATP-PRT open and closed conformations and its functional state. These findings suggest that dynamic processes are responsible for ATP-PRT allosteric regulation and that similar mechanisms might also be found in other enzymes bearing a ferredoxin-like allosteric domain.Active and inactive state ATP-phosphoribosyltransferases (ATP-PRTs) are believed to have different conformations. Here the authors show that in both states, ATP-PRT has a similar structural arrangement, suggesting that dynamic alterations are involved in ATP-PRT regulation by allosteric modulators.
Subject(s)
ATP Phosphoribosyltransferase/chemistry , ATP Phosphoribosyltransferase/genetics , ATP Phosphoribosyltransferase/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Allosteric Regulation , Allosteric Site , Histidine/chemistry , Histidine/metabolism , Kinetics , Models, MolecularABSTRACT
Influenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans.
Subject(s)
Alphainfluenzavirus/immunology , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Antibody Specificity , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal, Humanized , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/isolation & purification , Antibodies, Viral/chemistry , Antibodies, Viral/isolation & purification , Binding Sites, Antibody , Crystallography, X-Ray , Epitopes/immunology , Ferrets , Humans , Influenza Vaccines , Mice , Orthomyxoviridae Infections/prevention & control , Protein ConformationABSTRACT
In 2004 an hemagglutinin 3 neuraminidase 8 (H3N8) equine influenza virus was transmitted from horses to dogs in Florida and subsequently spread throughout the United States and to Europe. To understand the molecular basis of changes in the antigenicity of H3 hemagglutinins (HAs) that have occurred during virus evolution in horses, and to investigate the role of HA in the equine to canine cross-species transfer, we used X-ray crystallography to determine the structures of the HAs from two antigenically distinct equine viruses and from a canine virus. Structurally all three are very similar with the majority of amino acid sequence differences between the two equine HAs located on the virus membrane-distal molecular surface. HAs of canine viruses are distinct in containing a Trp-222 â Leu substitution in the receptor binding site that influences specificity for receptor analogs. In the fusion subdomain of canine and recent equine virus HAs a unique difference is observed by comparison with all other HAs examined to date. Analyses of site-specific mutant HAs indicate that a single amino acid substitution, Thr-30 â Ser, influences interactions between N-terminal and C-terminal regions of the subdomain that are important in the structural changes required for membrane fusion activity. Both structural modifications may have facilitated the transmission of H3N8 influenza from horses to dogs.
Subject(s)
Amino Acid Substitution , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Influenza A Virus, H3N8 Subtype/chemistry , Animals , Crystallography, X-Ray , Dog Diseases/genetics , Dog Diseases/metabolism , Dog Diseases/virology , Dogs , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Horse Diseases/genetics , Horse Diseases/metabolism , Horse Diseases/virology , Horses , Influenza A Virus, H3N8 Subtype/metabolism , Orthomyxoviridae Infections/genetics , Orthomyxoviridae Infections/metabolism , Protein Structure, TertiaryABSTRACT
The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Crystallography, X-Ray/methods , Molecular Chaperones/metabolism , Amino Acid Sequence , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/genetics , Cell Line, Tumor , HEK293 Cells , HSP90 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/metabolism , Humans , Models, Molecular , Molecular Chaperones/genetics , Molecular Sequence Data , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Phosphorylation , Protein Structure, Tertiary , Proto-Oncogene Proteins c-ets/chemistry , Proto-Oncogene Proteins c-ets/metabolism , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
The Spumaretrovirinae, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. In addition, PFV Gag interacts with the FV Envelope (Env) protein to facilitate budding of infectious particles. Presently, there is a paucity of structural information with regards FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. Therefore, in order to probe the functional overlap of FV and orthoretroviral Gag and learn more about FV egress and replication we have undertaken a structural, biophysical and virological study of PFV-Gag. We present the crystal structure of a dimeric amino terminal domain from PFV, Gag-NtD, both free and in complex with the leader peptide of PFV Env. The structure comprises a head domain together with a coiled coil that forms the dimer interface and despite the shared function it is entirely unrelated to either the capsid or matrix of Gag from other retroviruses. Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5α restriction of PFV. These data provide the first information with regards to FV structural proteins and suggest a model for convergent evolution of gag genes where structurally unrelated molecules have become functionally equivalent.
Subject(s)
Biological Evolution , Capsid/metabolism , Gene Products, gag/chemistry , Gene Products, gag/metabolism , Simian foamy virus/metabolism , Amino Acid Sequence , Capsid/chemistry , Cell Line , Gene Products, gag/genetics , Humans , Molecular Sequence Data , Protein Structure, Quaternary , Protein Structure, Tertiary , Simian foamy virus/chemistry , Simian foamy virus/genetics , TransfectionABSTRACT
Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such signal transduction pathway called the mitotic exit network (MEN) governs the transition from mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be activated by Cdc15. This mechanism of pathway activation has implications for signal transmission through other kinase cascades and might represent a general principle in scaffold-assisted signaling.
