Correction: Boyd, P.S., et al. NMR Studies of Retroviral Genome Packaging. Viruses 2020, 12, 1115.

There was an error in the original article [...].

NMR Studies of Retroviral Genome Packaging.

Nearly all retroviruses selectively package two copies of their unspliced RNA genomes from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Over the past four decades, combinations of genetic experiments, phylogenetic analyses, nucleotide accessibility mapping, in silico RNA structure predictions, and biophysical experiments were employed to understand how retroviral genomes are selected for packaging. Genetic studies provided early clues regarding the protein and RNA elements required for packaging, and nucleotide accessibility mapping experiments provided insights into the secondary structures of functionally important elements in the genome. Three-dimensional structural determinants of packaging were primarily derived by nuclear magnetic resonance (NMR) spectroscopy. A key advantage of NMR, relative to other methods for determining biomolecular structure (such as X-ray crystallography), is that it is well suited for studies of conformationally dynamic and heterogeneous systems-a hallmark of the retrovirus packaging machinery. Here, we review advances in understanding of the structures, dynamics, and interactions of the proteins and RNA elements involved in retroviral genome selection and packaging that are facilitated by NMR.

Structural and Mechanistic Studies of the Rare Myristoylation Signal of the Feline Immunodeficiency Virus.

All retroviruses encode a Gag polyprotein containing an N-terminal matrix domain (MA) that anchors Gag to the plasma membrane and recruits envelope glycoproteins to virus assembly sites. Membrane binding by the Gag protein of HIV-1 and most other lentiviruses is dependent on N-terminal myristoylation of MA by host N-myristoyltransferase enzymes (NMTs), which recognize a six-residue "myristoylation signal" with consensus sequence: M1GXXX[ST]. For unknown reasons, the feline immunodeficiency virus (FIV), which infects both domestic and wild cats, encodes a non-consensus myristoylation sequence not utilized by its host or by other mammals (most commonly: M1GNGQG). To explore the evolutionary basis for this sequence, we compared the structure, dynamics, and myristoylation properties of native FIV MA with a mutant protein containing a consensus feline myristoylation motif (MANOS) and examined the impact of MA mutations on virus assembly and ability to support spreading infection. Unexpectedly, myristoylation efficiency of MANOS in Escherichia coli by co-expressed mammalian NMT was reduced by ~70% compared to the wild-type protein. NMR studies revealed that residues of the N-terminal myristoylation signal are fully exposed and mobile in the native protein but partially sequestered in the MANOS chimera, suggesting that the unusual FIV sequence is conserved to promote exposure and efficient myristoylation of the MA N terminus. In contrast, virus assembly studies indicate that the MANOS mutation does not affect virus assembly, but does prevent virus spread, in feline kidney cells. Our findings indicate that residues of the FIV myristoylation sequence play roles in replication beyond NMT recognition and Gag-membrane binding.

Assigning NMR spectra of RNA, peptides and small organic molecules using molecular network visualization software.

NMR assignment typically involves analysis of peaks across multiple NMR spectra. Chemical shifts of peaks are measured before being assigned to atoms using a variety of methods. These approaches quickly become complicated by overlap, ambiguity, and the complexity of correlating assignments among multiple spectra. Here we propose an alternative approach in which a network of linked peak-boxes is generated at the predicted positions of peaks across all spectra. These peak-boxes correlate known relationships and can be matched to the observed spectra. The method is illustrated with RNA, but a variety of molecular types should be readily tractable with this approach.

Synthesis of distal and proximal fleximer base analogues and evaluation in the nucleocapsid protein of HIV-1.

Anti-HIV-1 drug design has been notably challenging due to the virus' ability to mutate and develop immunity against commercially available drugs. The aims of this project were to develop a series of fleximer base analogues that not only possess inherent flexibility that can remain active when faced with binding site mutations, but also target a non-canonical, highly conserved target: the nucleocapsid protein of HIV (NC). The compounds were predicted by computational studies not to function via zinc ejection, which would endow them with significant advantages over non-specific and thus toxic zinc-ejectors. The target fleximer bases were synthesized using palladium-catalyzed cross-coupling techniques and subsequently tested against NC and HIV-1. The results of those studies are described herein.

Long-Range RNA Structural Information via a Paramagnetically Tagged Reporter Protein.

NMR has provided a wealth of structural and dynamical information for RNA molecules of up to ∼50 nucleotides, but its application to larger RNAs has been hampered in part by difficulties establishing global structural features. A potential solution involves measurement of NMR perturbations after site-specific paramagnetic labeling. Although the approach works well for proteins, the inability to place the label at specific sites has prevented its application to larger RNAs transcribed in vitro. Here, we present a strategy in which RNA loop residues are modified to promote binding to a paramagnetically tagged reporter protein. Lanthanide-induced pseudocontact shifts are demonstrated for a 232-nucleotide RNA bound to tagged derivatives of the spliceosomal U1A RNA-binding domain. Further, the method is validated with a 36-nucleotide RNA for which measured NMR values agreed with predictions based on the previously known protein and RNA structures. The ability to readily insert U1A binding sites into ubiquitous hairpin and/or loop structures should make this approach broadly applicable for the atomic-level study of large RNAs.

Accurate Measurement of Residual Dipolar Couplings in Large RNAs by Variable Flip Angle NMR.

NMR approaches using nucleotide-specific deuterium labeling schemes have enabled structural studies of biologically relevant RNAs of increasing size and complexity. Although local structure is well-determined using these methods, definition of global structural features, including relative orientations of independent helices, remains a challenge. Residual dipolar couplings, a potential source of orientation information, have not been obtainable for large RNAs due to poor sensitivity resulting from rapid heteronuclear signal decay. Here we report a novel multiple quantum NMR method for RDC determination that employs flip angle variation rather than a coupling evolution period. The accuracy of the method and its utility for establishing interhelical orientations are demonstrated for a 36-nucleotide RNA, for which comparative data could be obtained. Applied to a 78 kDa Rev response element from the HIV-1 virus, which has an effective rotational correlation time of ca. 160 ns, the method yields sensitivity gains of an order of magnitude or greater over existing approaches. Solution-state access to structural organization in RNAs of at least 230 nucleotides is now possible.

Structure of the 30 kDa HIV-1 RNA Dimerization Signal by a Hybrid Cryo-EM, NMR, and Molecular Dynamics Approach.

Cryoelectron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy are routinely used to determine structures of macromolecules with molecular weights over 65 and under 25 kDa, respectively. We combined these techniques to study a 30 kDa HIV-1 dimer initiation site RNA ([DIS]2; 47 nt/strand). A 9 Å cryo-EM map clearly shows major groove features of the double helix and a right-handed superhelical twist. Simulated cryo-EM maps generated from time-averaged molecular dynamics trajectories (10 ns) exhibited levels of detail similar to those in the experimental maps, suggesting internal structural flexibility limits the cryo-EM resolution. Simultaneous inclusion of the cryo-EM map and 2H-edited NMR-derived distance restraints during structure refinement generates a structure consistent with both datasets and supporting a flipped-out base within a conserved purine-rich bulge. Our findings demonstrate the power of combining global and local structural information from these techniques for structure determination of modest-sized RNAs.

Mechanism of Competitive Inhibition and Destabilization of Acidothermus cellulolyticus Endoglucanase 1 by Ionic Liquids.

The ability of ionic liquids (ILs) to solubilize cellulose has sparked interest in their use for enzymatic biomass processing. However, this potential is yet to be realized, primarily because ILs inactivate requisite cellulases by mechanisms that are yet to be identified. We used a combination of enzymology, circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular dynamics (MD) methods to investigate the molecular basis for the inactivation of the endocellulase 1 (E1) from Acidothermus cellulolyticus by the imidazolium IL 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). Enzymatic studies revealed that [BMIM][Cl] inactivates E1 in a biphasic manner that involves rapid, reversible inhibition, followed by slow, irreversible deactivation. Backbone NMR signals of the 40.5 kDa E1 were assigned by triple resonance NMR methods, enabling monitoring of residue-specific perturbations. 1H-15N NMR titration experiments revealed that [BMIM][Cl] binds reversibly to the E1 active site, indicating that reversible deactivation is due to competitive inhibition of substrate binding. Prolonged incubation with [BMIM][Cl] led to substantial global changes in the 1H-15N heteronuclear single quantum coherence NMR and CD spectra of E1 indicative of protein denaturation. Notably, weak interactions between [BMIM][Cl] and residues at the termini of several helices were also observed, which, together with MD simulations, suggest that E1 denaturation is promoted by [BMIM][Cl]-induced destabilization of helix capping structures. In addition to identifying determinants of E1 inactivation, our findings establish a molecular framework for engineering cellulases with improved IL compatibility.

NMRFx Processor: a cross-platform NMR data processing program.

NMRFx Processor is a new program for the processing of NMR data. Written in the Java programming language, NMRFx Processor is a cross-platform application and runs on Linux, Mac OS X and Windows operating systems. The application can be run in both a graphical user interface (GUI) mode and from the command line. Processing scripts are written in the Python programming language and executed so that the low-level Java commands are automatically run in parallel on computers with multiple cores or CPUs. Processing scripts can be generated automatically from the parameters of NMR experiments or interactively constructed in the GUI. A wide variety of processing operations are provided, including methods for processing of non-uniformly sampled datasets using iterative soft thresholding. The interactive GUI also enables the use of the program as an educational tool for teaching basic and advanced techniques in NMR data analysis.

Structural and Molecular Determinants of Membrane Binding by the HIV-1 Matrix Protein.

Assembly of HIV-1 particles is initiated by the trafficking of viral Gag polyproteins from the cytoplasm to the plasma membrane, where they co-localize and bud to form immature particles. Membrane targeting is mediated by the N-terminally myristoylated matrix (MA) domain of Gag and is dependent on the plasma membrane marker phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Recent studies revealed that PI(4,5)P2 molecules containing truncated acyl chains [tr-PI(4,5)P2] are capable of binding MA in an "extended lipid" conformation and promoting myristoyl exposure. Here we report that tr-PI(4,5)P2 molecules also readily bind to non-membrane proteins, including HIV-1 capsid, which prompted us to re-examine MA-PI(4,5)P2 interactions using native lipids and membrane mimetic liposomes and bicelles. Liposome binding trends observed using a recently developed NMR approach paralleled results of flotation assays, although the affinities measured under the equilibrium conditions of NMR experiments were significantly higher. Native PI(4,5)P2 enhanced MA binding to liposomes designed to mimic non-raft-like regions of the membrane, suggesting the possibility that binding of the protein to disordered domains may precede Gag association with, or nucleation of, rafts. Studies with bicelles revealed a subset of surface and myr-associated MA residues that are sensitive to native PI(4,5)P2, but cleft residues that interact with the 2'-acyl chains of tr-PI(4,5)P2 molecules in aqueous solution were insensitive to native PI(4,5)P2 in bicelles. Our findings call to question extended-lipid MA:membrane binding models, and instead support a model put forward from coarse-grained simulations indicating that binding is mediated predominantly by dynamic, electrostatic interactions between conserved basic residues of MA and multiple PI(4,5)P2 and phosphatidylserine molecules.

Conserved determinants of lentiviral genome dimerization.

BACKGROUND: Retroviruses selectively package two copies of their unspliced genomes by what appears to be a dimerization-dependent RNA packaging mechanism. Dimerization of human immunodeficiency virus Type-1 (HIV-1) genomes is initiated by "kissing" interactions between GC-rich palindromic loop residues of a conserved hairpin (DIS), and is indirectly promoted by long-range base pairing between residues overlapping the gag start codon (AUG) and an upstream Unique 5' element (U5). The DIS and U5:AUG structures are phylogenetically conserved among divergent retroviruses, suggesting conserved functions. However, some studies suggest that the DIS of HIV-2 does not participate in dimerization, and that U5:AUG pairing inhibits, rather than promotes, genome dimerization. We prepared RNAs corresponding to native and mutant forms of the 5' leaders of HIV-1 (NL4-3 strain), HIV-2 (ROD strain), and two divergent strains of simian immunodeficiency virus (SIV; cpz-TAN1 and -US strains), and probed for potential roles of the DIS and U5:AUG base pairing on intrinsic and NC-dependent dimerization by mutagenesis, gel electrophoresis, and NMR spectroscopy. RESULTS: Dimeric forms of the native HIV-2 and SIV leaders were only detectable using running buffers that contained Mg(2+), indicating that these dimers are more labile than that of the HIV-1 leader. Mutations designed to promote U5:AUG base pairing promoted dimerization of the HIV-2 and SIV RNAs, whereas mutations that prevented U5:AUG pairing inhibited dimerization. Chimeric HIV-2 and SIV leader RNAs containing the dimer-promoting loop of HIV-1 (DIS) exhibited HIV-1 leader-like dimerization properties, whereas an HIV-1NL4-3 mutant containing the SIVcpzTAN1 DIS loop behaved like the SIVcpzTAN1 leader. The cognate NC proteins exhibited varying abilities to promote dimerization of the retroviral leader RNAs, but none were able to convert labile dimers to non-labile dimers. CONCLUSIONS: The finding that U5:AUG formation promotes dimerization of the full-length HIV-1, HIV-2, SIVcpzUS, and SIVcpzTAN1 5' leaders suggests that these retroviruses utilize a common RNA structural switch mechanism to modulate function. Differences in native and NC-dependent dimerization propensity and lability are due to variations in the compositions of the DIS loop residues rather than other sequences within the leader RNAs. Although NC is a well-known RNA chaperone, its role in dimerization has the hallmarks of a classical riboswitch.

Structure-function analysis reveals that the Pseudomonas aeruginosa Tps4 two-partner secretion system is involved in CupB5 translocation.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, synonymous with cystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a model organism to study biofilms: a bacterial population embedded in an extracellular matrix that provide protection from environmental pressures and lead to persistence. A number of Chaperone-Usher Pathways, namely CupA-CupE, play key roles in these processes by assembling adhesive pili on the bacterial surface. One of these, encoded by the cupB operon, is unique as it contains a nonchaperone-usher gene product, CupB5. Two-partner secretion (TPS) systems are comprised of a C-terminal integral membrane ß-barrel pore with tandem N-terminal POTRA (POlypeptide TRansport Associated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). Using NMR we show that TpsB4 (LepB) interacts with CupB5 and its predicted cognate partner TpsA4 (LepA), an extracellular protease. Moreover, using cellular studies we confirm that TpsB4 can translocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observation that suggested the CupB3 P-usher secretes CupB5. In support of our findings we also demonstrate that tps4/cupB operons are coregulated by the RocS1 sensor suggesting P. aeruginosa has developed synergy between these systems. Furthermore, we have determined the solution-structure of the TpsB4-POTRA1 domain and together with restraints from NMR chemical shift mapping and in vivo mutational analysis we have calculated models for the entire TpsB4 periplasmic region in complex with both TpsA4 and CupB5 secretion motifs. The data highlight specific residues for TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for each POTRA domain.

Structural insight into host recognition by aggregative adherence fimbriae of enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) is a leading cause of acute and persistent diarrhea worldwide. A recently emerged Shiga-toxin-producing strain of EAEC resulted in significant mortality and morbidity due to progressive development of hemolytic-uremic syndrome. The attachment of EAEC to the human intestinal mucosa is mediated by aggregative adherence fimbria (AAF). Using X-ray crystallography and NMR structures, we present new atomic resolution insight into the structure of AAF variant I from the strain that caused the deadly outbreak in Germany in 2011, and AAF variant II from archetype strain 042, and propose a mechanism for AAF-mediated adhesion and biofilm formation. Our work shows that major subunits of AAF assemble into linear polymers by donor strand complementation where a single minor subunit is inserted at the tip of the polymer by accepting the donor strand from the terminal major subunit. Whereas the minor subunits of AAF have a distinct conserved structure, AAF major subunits display large structural differences, affecting the overall pilus architecture. These structures suggest a mechanism for AAF-mediated adhesion and biofilm formation. Binding experiments using wild type and mutant subunits (NMR and SPR) and bacteria (ELISA) revealed that despite the structural differences AAF recognize a common receptor, fibronectin, by employing clusters of basic residues at the junction between subunits in the pilus. We show that AAF-fibronectin attachment is based primarily on electrostatic interactions, a mechanism not reported previously for bacterial adhesion to biotic surfaces.

Structural insight into SUMO chain recognition and manipulation by the ubiquitin ligase RNF4.

The small ubiquitin-like modifier (SUMO) can form polymeric chains that are important signals in cellular processes such as meiosis, genome maintenance and stress response. The SUMO-targeted ubiquitin ligase RNF4 engages with SUMO chains on linked substrates and catalyses their ubiquitination, which targets substrates for proteasomal degradation. Here we use a segmental labelling approach combined with solution nuclear magnetic resonance (NMR) spectroscopy and biochemical characterization to reveal how RNF4 manipulates the conformation of the SUMO chain, thereby facilitating optimal delivery of the distal SUMO domain for ubiquitin transfer.

Conformational recognition of an intrinsically disordered protein.

There is a growing interest in understanding the properties of intrinsically disordered proteins (IDPs); however, the characterization of these states remains an open challenge. IDPs appear to have functional roles that diverge from those of folded proteins and revolve around their ability to act as hubs for protein-protein interactions. To gain a better understanding of the modes of binding of IDPs, we combined statistical mechanics, calorimetry, and NMR spectroscopy to investigate the recognition and binding of a fragment from the disordered protein Gab2 by the growth factor receptor-bound protein 2 (Grb2), a key interaction for normal cell signaling and cancer development. Structural ensemble refinement by NMR chemical shifts, thermodynamics measurements, and analysis of point mutations indicated that the population of preexisting bound conformations in the free-state ensemble of Gab2 is an essential determinant for recognition and binding by Grb2. A key role was found for transient polyproline II (PPII) structures and extended conformations. Our findings are likely to have very general implications for the biological behavior of IDPs in light of the evidence that a large fraction of these proteins possess a specific propensity to form PPII and to adopt conformations that are more extended than the typical random-coil states.

Structures of the compact helical core domains of feline calicivirus and murine norovirus VPg proteins.

We report the solution structures of the VPg proteins from feline calicivirus (FCV) and murine norovirus (MNV), which have been determined by nuclear magnetic resonance spectroscopy. In both cases, the core of the protein adopts a compact helical structure flanked by flexible N and C termini. Remarkably, while the core of FCV VPg contains a well-defined three-helix bundle, the MNV VPg core has just the first two of these secondary structure elements. In both cases, the VPg cores are stabilized by networks of hydrophobic and salt bridge interactions. The Tyr residue in VPg that is nucleotidylated by the viral NS7 polymerase (Y24 in FCV, Y26 in MNV) occurs in a conserved position within the first helix of the core. Intriguingly, given its structure, VPg would appear to be unable to bind to the viral polymerase so as to place this Tyr in the active site without a major conformation change to VPg or the polymerase. However, mutations that destabilized the VPg core either had no effect on or reduced both the ability of the protein to be nucleotidylated and virus infectivity and did not reveal a clear structure-activity relationship. The precise role of the calicivirus VPg core in virus replication remains to be determined, but knowledge of its structure will facilitate future investigations.

Molecular determinants of inactivation of the resuscitation promoting factor B from Mycobacterium tuberculosis.

Inactivation of revival of Mycobacterium tuberculosis from dormancy is one of the main goals of the WHO Global Plan to stop tuberculosis (TB) 2011-2015, given the huge reservoir of latently infected individuals. This process requires a group of secreted proteins, denoted as resuscitation-promoting factors (Rpfs). Of these, RpfB is the sole member indispensable for resuscitation in vivo. The first class of inhibitors of RpfB was identified among 2-nitrophenylthiocyanates. However, their inactivation mechanism is hitherto not known. To gain insight into the inactivation mechanism of one of the most promising RpfB inhibitors, 4-benzoyl-2-nitrophenyl thiocyanate, NPT7, we have performed replica exchange molecular dynamics (REMD) simulations, starting from the crystal structure of RpfB catalytic domain, derived in this study. We validated our results by resuscitation experiments of M. tuberculosis cultures. The atomic resolution crystal structure of RpfB catalytic domain identified the potential of the enzyme catalytic cleft to bind benzene rings. REMD simulations, 48 replicas, identified the key interactions for the binding of NPT7 to RpfB catalytic site. Of these, an important role is played by the thiocyanate group of NPT7. Consistently, we prove that the substitution of this group implies a complete loss of RpfB inactivation. Our results provide valuable information for modifications of NPT7 structure to enhance its binding affinity to RpfB, with the final aim of developing second-generation inhibitors of therapeutic interest in TB eradication strategy.

Microneme protein 5 regulates the activity of Toxoplasma subtilisin 1 by mimicking a subtilisin prodomain.

Toxoplasma gondii is the model parasite of the phylum Apicomplexa, which contains obligate intracellular parasites of medical and veterinary importance. Apicomplexans invade host cells by a multistep process involving the secretion of adhesive microneme protein (MIC) complexes. The subtilisin protease TgSUB1 trims several MICs on the parasite surface to activate gliding motility and host invasion. Although a previous study showed that expression of the secretory protein TgMIC5 suppresses TgSUB1 activity, the mechanism was unknown. Here, we solve the three-dimensional structure of TgMIC5 by nuclear magnetic resonance (NMR), revealing that it mimics a subtilisin prodomain including a flexible C-terminal peptide that may insert into the subtilisin active site. We show that TgMIC5 is an almost 50-fold more potent inhibitor of TgSUB1 activity than the small molecule inhibitor N-[N-(N-acetyl-L-leucyl)-L-leucyl]-L-norleucine (ALLN). Moreover, we demonstrate that TgMIC5 is retained on the parasite plasma membrane via its physical interaction with the membrane-anchored TgSUB1.

Galactose recognition by the apicomplexan parasite Toxoplasma gondii.

Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.