Mutations in the N-domain of aryl hydrocarbon receptor interacting protein affect interactions with heat shock protein 90ß and phosphodiesterase 4A5.

The aryl hydrocarbon receptor interacting protein (AIP) is a cytoplasmic molecular co-chaperone and tumour suppressor that assists in protein stability and complex formation involving the aryl hydrocarbon receptor. Germline mutations in the AIP gene predispose to pituitary tumourigenesis with patients exhibiting an aggressive clinical phenotype. Full length AIP proteins harbouring N-domain mutations (R9Q, R16H, V49 M and K103R) were purified from E.coli utilizing a methodology that maintained structural integrity and monomeric stability. Mutations did not significantly affect the thermal stability of the protein and caused no overall disruptive effect in the protein structure. The mutations studied lowered the binding affinity of AIP towards two of its binding partners; heat shock protein 90ß and phosphodiesterase 4A5 (PDE4A5). The inhibition of phosphodiesterase activity by AIP was also greatly reduced by all mutants. While previously published data has mainly concentrated on the tetratricopeptide repeats of the C-domain of AIP, we present clear evidence that AIP N-domain mutations play a significant role in two protein:protein interactions with partner proteins. The complex interactome of AIP suggests that any observable change in one or more of its binding partners cannot be disregarded as it may have repercussions on other biochemical pathways.

Crystal structure and solution state of the C-terminal head region of the narmovirus receptor binding protein.

IMPORTANCE: Genetically diverse paramyxoviruses are united in their presentation of a receptor-binding protein (RBP), which works in concert with the fusion protein to facilitate host-cell entry. The C-terminal head region of the paramyxoviral RBP, a primary determinant of host-cell tropism and inter-species transmission potential, forms structurally distinct classes dependent upon protein and glycan receptor specificity. Here, we reveal the architecture of the C-terminal head region of the RBPs from Nariva virus (NarV) and Mossman virus (MosV), two archetypal rodent-borne paramyxoviruses within the recently established genus Narmovirus, family Paramyxoviridae. Our analysis reveals that while narmoviruses retain the general architectural features associated with paramyxoviral RBPs, namely, a six-bladed ß-propeller fold, they lack the structural motifs associated with known receptor-mediated host-cell entry pathways. This investigation indicates that the RBPs of narmoviruses exhibit pathobiological features that are distinct from those of other paramyxoviruses.

Primary and secondary functions of HLA-E are determined by stability and conformation of the peptide-bound complexes.

MHC-E regulates NK cells by displaying MHC class Ia signal peptides (VL9) to NKG2A:CD94 receptors. MHC-E can also present sequence-diverse, lower-affinity, pathogen-derived peptides to T cell receptors (TCRs) on CD8+ T cells. To understand these affinity differences, human MHC-E (HLA-E)-VL9 versus pathogen-derived peptide structures are compared. Small-angle X-ray scatter (SAXS) measures biophysical parameters in solution, allowing comparison with crystal structures. For HLA-E-VL9, there is concordance between SAXS and crystal parameters. In contrast, HLA-E-bound pathogen-derived peptides produce larger SAXS dimensions that reduce to their crystallographic dimensions only when excess peptide is supplied. Further crystallographic analysis demonstrates three amino acids, exclusive to MHC-E, that not only position VL9 close to the α2 helix, but also allow non-VL9 peptide binding with re-configuration of a key TCR-interacting α2 region. Thus, non-VL9-bound peptides introduce an alternative peptide-binding motif and surface recognition landscape, providing a likely basis for VL9- and non-VL9-HLA-E immune discrimination.

Solution structures of human myeloma IgG3 antibody reveal extended Fab and Fc regions relative to the other IgG subclasses.

Human immunoglobulin G subclass 3 (IgG3) possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role of the two conserved Fc glycosylation sites are unknown. To address these issues, we subjected glycosylated and deglycosylated human myeloma IgG3 to multidisciplinary solution structure studies. Using analytical ultracentrifugation, the elongated structure of IgG3 was determined from the reduced sedimentation coefficients s020,w of 5.82 to 6.29 S for both glycosylated and deglycosylated IgG3. X-ray and neutron scattering showed that the Guinier RG values were 6.95 nm for glycosylated IgG3 and were unchanged after deglycosylation, again indicating an elongated structure. The distance distribution function P(r) showed a maximum length of 25 to 28 nm and three distinct maxima. The molecular structure of IgG3 was determined using atomistic modeling based on molecular dynamics simulations of the IgG3 hinge and Monte Carlo simulations to identify physically realistic arrangements of the Fab and Fc regions. This resulted in libraries containing 135,135 and 73,905 glycosylated and deglycosylated IgG3 structures, respectively. Comparisons with the X-ray and neutron scattering curves gave 100 best-fit models for each form of IgG3 that accounted for the experimental scattering curves. These models revealed the first molecular structures for full-length IgG3. The structures exhibited relatively restricted Fab and Fc conformations joined by an extended semirigid hinge, which explains the potent effector functions of IgG3 relative to the other subclasses IgG1, IgG2, and IgG4.

Allosteric regulation accompanied by oligomeric state changes of Trypanosoma brucei GMP reductase through cystathionine-ß-synthase domain.

Guanosine 5'-monophosphate reductase (GMPR) is involved in the purine salvage pathway and is conserved throughout evolution. Nonetheless, the GMPR of Trypanosoma brucei (TbGMPR) includes a unique structure known as the cystathionine-ß-synthase (CBS) domain, though the role of this domain is not fully understood. Here, we show that guanine and adenine nucleotides exert positive and negative effects, respectively, on TbGMPR activity by binding allosterically to the CBS domain. The present structural analyses revealed that TbGMPR forms an octamer that shows a transition between relaxed and twisted conformations in the absence and presence of guanine nucleotides, respectively, whereas the TbGMPR octamer dissociates into two tetramers when ATP is available instead of guanine nucleotides. These findings demonstrate that the CBS domain plays a key role in the allosteric regulation of TbGMPR by facilitating the transition of its oligomeric state depending on ligand nucleotide availability.

Structure and mechanism of a Type III CRISPR defence DNA nuclease activated by cyclic oligoadenylate.

The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes. On binding invading RNA species, Type III CRISPR systems generate cyclic oligoadenylate (cOA) signalling molecules, potentiating a powerful immune response by activating downstream effector proteins, leading to viral clearance, cell dormancy or death. Here we describe the structure and mechanism of a cOA-activated CRISPR defence DNA endonuclease, CRISPR ancillary nuclease 1 (Can1). Can1 has a unique monomeric structure with two CRISPR associated Rossman fold (CARF) domains and two DNA nuclease-like domains. The crystal structure of the enzyme has been captured in the activated state, with a cyclic tetra-adenylate (cA4) molecule bound at the core of the protein. cA4 binding reorganises the structure to license a metal-dependent DNA nuclease activity specific for nicking of supercoiled DNA. DNA nicking by Can1 is predicted to slow down viral replication kinetics by leading to the collapse of DNA replication forks.

Structural insight into the human mitochondrial tRNA purine N1-methyltransferase and ribonuclease P complexes.

Mitochondrial tRNAs are transcribed as long polycistronic transcripts of precursor tRNAs and undergo posttranscriptional modifications such as endonucleolytic processing and methylation required for their correct structure and function. Among them, 5'-end processing and purine 9 N1-methylation of mitochondrial tRNA are catalyzed by two proteinaceous complexes with overlapping subunit composition. The Mg2+-dependent RNase P complex for 5'-end cleavage comprises the methyltransferase domain-containing protein tRNA methyltransferase 10C, mitochondrial RNase P subunit (TRMT10C/MRPP1), short-chain oxidoreductase hydroxysteroid 17ß-dehydrogenase 10 (HSD17B10/MRPP2), and metallonuclease KIAA0391/MRPP3. An MRPP1-MRPP2 subcomplex also catalyzes the formation of 1-methyladenosine/1-methylguanosine at position 9 using S-adenosyl-l-methionine as methyl donor. However, a lack of structural information has precluded insights into how these complexes methylate and process mitochondrial tRNA. Here, we used a combination of X-ray crystallography, interaction and activity assays, and small angle X-ray scattering (SAXS) to gain structural insight into the two tRNA modification complexes and their components. The MRPP1 N terminus is involved in tRNA binding and monomer-monomer self-interaction, whereas the C-terminal SPOUT fold contains key residues for S-adenosyl-l-methionine binding and N1-methylation. The entirety of MRPP1 interacts with MRPP2 to form the N1-methylation complex, whereas the MRPP1-MRPP2-MRPP3 RNase P complex only assembles in the presence of precursor tRNA. This study proposes low-resolution models of the MRPP1-MRPP2 and MRPP1-MRPP2-MRPP3 complexes that suggest the overall architecture, stoichiometry, and orientation of subunits and tRNA substrates.

Self-Assembly of Telechelic Tyrosine End-Capped PEO Star Polymers in Aqueous Solution.

We investigate the self-assembly of two telechelic star polymer-peptide conjugates based on poly(ethylene oxide) (PEO) four-arm star polymers capped with oligotyrosine. The conjugates were prepared via N-carboxy anhydride-mediated ring-opening polymerization from PEO star polymer macroinitiators. Self-assembly occurs above a critical aggregation concentration determined via fluorescence probe assays. Peptide conformation was examined using circular dichroism spectroscopy. The structure of self-assembled aggregates was probed using small-angle X-ray scattering and cryogenic transmission electron microscopy. In contrast to previous studies on linear telechelic PEO-oligotyrosine conjugates that show self-assembly into ß-sheet fibrils, the star architecture suppresses fibril formation and micelles are generally observed instead, a small population of fibrils only being observed upon pH adjustment. Hydrogelation is also suppressed by the polymer star architecture. These peptide-functionalized star polymer solutions are cytocompatible at sufficiently low concentration. These systems present tyrosine at high density and may be useful in the development of future enzyme or pH-responsive biomaterials.

Structural basis of Smoothened regulation by its extracellular domains.

Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzledclass G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked a top the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.

Crystal structure of the RNA-dependent RNA polymerase from influenza C virus.

Negative-sense RNA viruses, such as influenza, encode large, multidomain RNA-dependent RNA polymerases that can both transcribe and replicate the viral RNA genome. In influenza virus, the polymerase (FluPol) is composed of three polypeptides: PB1, PB2 and PA/P3. PB1 houses the polymerase active site, whereas PB2 and PA/P3 contain, respectively, cap-binding and endonuclease domains required for transcription initiation by cap-snatching. Replication occurs through de novo initiation and involves a complementary RNA intermediate. Currently available structures of the influenza A and B virus polymerases include promoter RNA (the 5' and 3' termini of viral genome segments), showing FluPol in transcription pre-initiation states. Here we report the structure of apo-FluPol from an influenza C virus, solved by X-ray crystallography to 3.9 Å, revealing a new 'closed' conformation. The apo-FluPol forms a compact particle with PB1 at its centre, capped on one face by PB2 and clamped between the two globular domains of P3. Notably, this structure is radically different from those of promoter-bound FluPols. The endonuclease domain of P3 and the domains within the carboxy-terminal two-thirds of PB2 are completely rearranged. The cap-binding site is occluded by PB2, resulting in a conformation that is incompatible with transcription initiation. Thus, our structure captures FluPol in a closed, transcription pre-activation state. This reveals the conformation of newly made apo-FluPol in an infected cell, but may also apply to FluPol in the context of a non-transcribing ribonucleoprotein complex. Comparison of the apo-FluPol structure with those of promoter-bound FluPols allows us to propose a mechanism for FluPol activation. Our study demonstrates the remarkable flexibility of influenza virus RNA polymerase, and aids our understanding of the mechanisms controlling transcription and genome replication.

Structural Characterisation of Non-Deamidated Acidic Variants of Erwinia chrysanthemi L-asparaginase Using Small-Angle X-ray Scattering and Ion-Mobility Mass Spectrometry.

PURPOSE: Erwinia chrysanthemi L-asparaginase (ErA) is an enzyme commonly used in the treatment regimen for Acute Lymphoblastic Leukaemia (ALL). Biopharmaceutical products such as ErA must be monitored for modifications such as deamidation, typically using ion-exchange chromatography (IEX). Analysis of clinical-grade ErA using native IEX resolves a number of enzymatically-active, acidic variants that were poorly characterised. METHODS: ErA IEX variants were isolated and fully characterised using capillary electrophoresis (cIEF), LC-MS and LC-MS/MS of proteolytic digests, and structural techniques including circular dichroism, small-angle X-ray scattering (SAXS) and ion-mobility mass spectrometry (IM-MS). RESULTS: LC-MS, MS/MS and cIEF demonstrated that all ErA isolates consist mainly of enzyme lacking primary-sequence modifications (such as deamidation). Both SAXS and IM-MS revealed a different conformational state in the most prominent acidic IEX peak. However, SAXS data also suggested conformational differences between the main peak and major acidic variant were minor, based on comparisons with crystal structures. CONCLUSIONS: IEX data for biopharmaceuticals such as ErA should be thoroughly characterised, as the most common modifications, such as deamidation, may be absent.

A structure-specific nucleic acid-binding domain conserved among DNA repair proteins.

SMARCAL1, a DNA remodeling protein fundamental to genome integrity during replication, is the only gene associated with the developmental disorder Schimke immuno-osseous dysplasia (SIOD). SMARCAL1-deficient cells show collapsed replication forks, S-phase cell cycle arrest, increased chromosomal breaks, hypersensitivity to genotoxic agents, and chromosomal instability. The SMARCAL1 catalytic domain (SMARCAL1(CD)) is composed of an SNF2-type double-stranded DNA motor ATPase fused to a HARP domain of unknown function. The mechanisms by which SMARCAL1 and other DNA translocases repair replication forks are poorly understood, in part because of a lack of structural information on the domains outside of the common ATPase motor. In the present work, we determined the crystal structure of the SMARCAL1 HARP domain and examined its conformation and assembly in solution by small angle X-ray scattering. We report that this domain is conserved with the DNA mismatch and damage recognition domains of MutS/MSH and NER helicase XPB, respectively, as well as with the putative DNA specificity motif of the T4 phage fork regression protein UvsW. Loss of UvsW fork regression activity by deletion of this domain was rescued by its replacement with HARP, establishing the importance of this domain in UvsW and demonstrating a functional complementarity between these structurally homologous domains. Mutation of predicted DNA-binding residues in HARP dramatically reduced fork binding and regression activities of SMARCAL1(CD). Thus, this work has uncovered a conserved substrate recognition domain in DNA repair enzymes that couples ATP-hydrolysis to remodeling of a variety of DNA structures, and provides insight into this domain's role in replication fork stability and genome integrity.

RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response.

The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining.

SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication.

SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like1) maintains genome integrity during DNA replication. Here we investigated its mechanism of action. We found that SMARCAL1 travels with elongating replication forks, and its absence leads to MUS81-dependent double-strand break formation. Binding to specific nucleic acid substrates activates SMARCAL1 activity in a reaction that requires its HARP2 (Hep-A-related protein 2) domain. Homology modeling indicates that the HARP domain is similar in structure to the DNA-binding domain of the PUR proteins. Limited proteolysis, small-angle X-ray scattering, and functional assays indicate that the core enzymatic unit consists of the HARP2 and ATPase domains that fold into a stable structure. Surprisingly, SMARCAL1 is capable of binding three-way and four-way Holliday junctions and model replication forks that lack a designed ssDNA region. Furthermore, SMARCAL1 remodels these DNA substrates by promoting branch migration and fork regression. SMARCAL1 mutations that cause Schimke immunoosseous dysplasia or that inactivate the HARP2 domain abrogate these activities. These results suggest that SMARCAL1 continuously surveys replication forks for damage. If damage is present, it remodels the fork to promote repair and restart. Failures in the process lead to activation of an alternative repair mechanism that depends on MUS81-catalyzed cleavage of the damaged fork.

Free state conformational sampling of the SAM-I riboswitch aptamer domain.

Riboswitches are highly structured elements residing in the 5' untranslated region of messenger RNAs that specifically bind cellular metabolites to alter gene expression. While there are many structures of ligand-bound riboswitches that reveal details of bimolecular recognition, their unliganded structures remain poorly characterized. Characterizing the molecular details of the unliganded state is crucial for understanding the riboswitch's mechanism of action because it is this state that actively interrogates the cellular environment and helps direct the regulatory outcome. To develop a detailed description of the ligand-free form of an S-adenosylmethionine binding riboswitch at the local and global levels, we have employed a series of biochemical, biophysical, and computational methods. Our data reveal that the ligand binding domain adopts an ensemble of states that minimizes the energy barrier between the free and bound states to establish an efficient decision making branchpoint in the regulatory process.

Comparison and functional implications of the 3D architectures of viral tRNA-like structures.

RNA viruses co-opt the host cell's biological machinery, and their infection strategies often depend on specific structures in the viral genomic RNA. Examples are tRNA-like structures (TLSs), found at the 3' end of certain plant viral RNAs, which can use the cell's aminoacyl tRNA-synthetases (AARSs) to drive addition of an amino acid to the 3' end of the viral RNA. TLSs are multifunctional RNAs involved in processes such as viral replication, translation, and viral RNA stability; these functions depend on their fold. Experimental result-based structural models of TLSs have been published. In this study, we further examine these structures using a combination of biophysical and biochemical approaches to explore the three-dimensional (3D) architectures of TLSs from the turnip yellow mosaic virus (TYMV), tobacco mosaic virus (TMV), and brome mosaic virus (BMV). We find that despite similar function, these RNAs are biophysically diverse: the TYMV TLS adopts a characteristic tRNA-like L shape, the BMV TLS has a large compact globular domain with several helical extensions, and the TMV TLS aggregates in solution. Both the TYMV and BMV TLS RNAs adopt structures with tight backbone packing and also with dynamic structural elements, suggesting complexities and subtleties that cannot be explained by simple tRNA mimicry. These results confirm some aspects of existing models and also indicate how these models can be improved. The biophysical characteristics of these TLSs show how these multifunctional RNAs might regulate various viral processes, including negative strand synthesis, and also allow comparison with other structured RNAs.

Structure of the SAM-II riboswitch bound to S-adenosylmethionine.

In bacteria, numerous genes harbor regulatory elements in the 5' untranslated regions of their mRNA, termed riboswitches, which control gene expression by binding small-molecule metabolites. These sequences influence the secondary and tertiary structure of the RNA in a ligand-dependent manner, thereby directing its transcription or translation. The crystal structure of an S-adenosylmethionine-responsive riboswitch found predominantly in proteobacteria, SAM-II, has been solved to reveal a second means by which RNA interacts with this important cellular metabolite. Notably, this is the first structure of a complete riboswitch containing all sequences associated with both the ligand binding aptamer domain and the regulatory expression platform. Chemical probing of this RNA in the absence and presence of ligand shows how the structure changes in response to S-adenosylmethionine to sequester the ribosomal binding site and affect translational gene regulation.