The Redox State Regulates the Conformation of Rv2466c to Activate the Antitubercular Prodrug TP053

Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin-fold with a Cys¹⁹-Pro²⁰-Trp²¹-Cys²² motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle x-ray scattering showed that the fully reduced form of Rv2466c adopts a “closed” compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an “open” conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical, and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily.     

Structural and Functional Characterization of the Mumps Virus Phosphoprotein

The phosphoprotein (P) is virally encoded by the Rhabdoviridae and Paramyxoviridae in the order Mononegavirales. P is a self-associated oligomer and forms complexes with the large viral polymerase protein (L), the nucleocapsid protein (N), and the assembled nucleocapsid. P from different viruses has shown structural diversities even though their essential functions are the same. We systematically mapped the domains in mumps virus (MuV) P and investigated their interactions with nucleocapsid-like particles (NLPs). Similar to other P proteins, MuV P contains N-terminal, central, and C-terminal domains with flexible linkers between neighboring domains. By pulldown assays, we discovered that in addition to the previously proposed nucleocapsid binding domain (residues 343 to 391), the N-terminal region of MuV P (residues 1 to 194) could also bind NLPs. Further analysis of binding kinetics was conducted using surface plasmon resonance. This is the first observation that both the N- and C-terminal regions of a negative-strand RNA virus P are involved in binding the nucleocapsid. In addition, we defined the oligomerization domain (P_OD) of MuV P as residues 213 to 277 and determined its crystal structure. The tetrameric MuV P_OD is formed by one pair of long parallel α-helices with another pair in opposite orientation. Unlike the parallel orientation of each α-helix in the tetramer of Sendai virus P_OD, this represents a novel orientation of a P_OD where both the N- and the C-terminal domains are at either end of the tetramer. This is consistent with the observation that both the N- and the C-terminal domains are involved in binding the nucleocapsid.     

Signature Protein of the PVC Superphylum

The phyla Planctomycetes, Verrucomicrobia, Chlamydiae, Lentisphaerae, and “Candidatus Omnitrophica (OP3)” comprise bacteria that share an ancestor but show highly diverse biological and ecological features. Together, they constitute the PVC superphylum. Using large-scale comparative genome sequence analysis, we identified a protein uniquely shared among all of the known members of the PVC superphylum. We provide evidence that this signature protein is expressed by representative members of the PVC superphylum. Its predicted structure, physicochemical characteristics, and overexpression in Escherichia coli and gel retardation assays with purified signature protein suggest a housekeeping function with unspecific DNA/RNA binding activity. Phylogenetic analysis demonstrated that the signature protein is a suitable phylogenetic marker for members of the PVC superphylum, and the screening of published metagenome data indicated the existence of additional PVC members. This study provides further evidence of a common evolutionary history of the PVC superphylum and presents a unique case in which a single protein serves as an evolutionary link among otherwise highly diverse members of major bacterial groups.     

Controlling RNA self-assembly to form filaments

Fundamental control over supra-molecular self-assembly for organization of matter on the nano-scale is a major objective of nanoscience and nanotechnology. ‘RNA tectonics’ is the design of modular RNA units, called tectoRNAs, that can be programmed to self-assemble into novel nano- and meso-scopic architectures of desired size and shape. We report the three-dimensional design of tectoRNAs incorporating modular 4-way junction (4WJ) motifs, hairpin loops and their cognate loop–receptors to create extended, programmable interaction interfaces. Specific and directional RNA–RNA interactions at these interfaces enable conformational, topological and orientational control of tectoRNA self-assembly. The interacting motifs are precisely positioned within the helical arms of the 4WJ to program assembly from only one helical stacking conformation of the 4WJ. TectoRNAs programmed to assemble with orientational compensation produce micrometer-scale RNA filaments through supra-molecular equilibrium polymerization. As visualized by transmission electron microscopy, these RNA filaments resemble actin filaments from the protein world. This work emphasizes the potential of RNA as a scaffold for designing and engineering new controllable biomaterials mimicking modern cytoskeletal proteins.     

Assembly Properties of Human Immunodeficiency Virus Type 1 Gag-Leucine Zipper Chimeras: Implications for Retrovirus Assembly

Expression of the retroviral Gag protein leads to formation of virus-like particles in mammalian cells. In vitro and in vivo experiments show that nucleic acid is also required for particle assembly. However, several studies have demonstrated that chimeric proteins in which the nucleocapsid domain of Gag is replaced by a leucine zipper motif can also assemble efficiently in mammalian cells. We have now analyzed assembly by chimeric proteins in which nucleocapsid of human immunodeficiency virus type 1 (HIV-1) Gag is replaced by either a dimerizing or a trimerizing zipper. Both proteins assemble well in human 293T cells; the released particles lack detectable RNA. The proteins can coassemble into particles together with full-length, wild-type Gag. We purified these proteins from bacterial lysates. These recombinant “Gag-Zipper” proteins are oligomeric in solution and do not assemble unless cofactors are added; either nucleic acid or inositol phosphates (IPs) can promote particle assembly. When mixed with one equivalent of IPs (which do not support assembly of wild-type Gag), the “dimerizing” Gag-Zipper protein misassembles into very small particles, while the “trimerizing” protein assembles correctly. However, addition of both IPs and nucleic acid leads to correct assembly of all three proteins; the “dimerizing” Gag-Zipper protein also assembles correctly if inositol hexakisphosphate is supplemented with other polyanions. We suggest that correct assembly requires both oligomeric association at the C terminus of Gag and neutralization of positive charges near its N terminus.Expression of a single retroviral protein, Gag, in mammalian cells is sufficient for assembly of virus-like particles (VLPs). RNA seems to play an essential role, however, in both the assembly and structure of VLPs. Thus, retrovirus particles always contain RNA; in the absence of genomic RNA, cellular mRNAs replace it in the virus particle (46). RNase treatment of immature murine leukemia virus disrupts the particles (37). Finally, nucleic acid is required for assembly in defined in vitro assembly systems (8, 9).The contribution of nucleic acid to the assembly and structure of retrovirus particles is not yet understood. As one approach to further understanding the role that nucleic acid binding plays in the assembly process, Zhang et al. (59) replaced the principal nucleic acid-binding domain of the HIV-1 Gag protein, nucleocapsid (NC), with a leucine zipper domain. This chimeric protein was able to assemble efficiently in mammalian cells as evidenced through immunoblotting of released VLPs. This observation was extended by Johnson et al. (28), who used Gag-leucine zipper (dimerizing) chimeras of Rous sarcoma virus and studied the morphologies of the resulting particles. The particles assembled from the chimeric proteins were similar, although not identical, to those formed by wild-type (WT) Gag. The fact that NC could be functionally replaced (with respect to particle assembly) with the dimerizing leucine zipper motif led these investigators to propose that the function of nucleic acid in assembly is to promote dimerization. Additional support for this hypothesis comes from the fact that the minimum length of nucleic acid needed to promote assembly is roughly enough to accommodate two molecules of Gag (30, 31).Further studies in which the NC domain of HIV-1 Gag has been replaced by leucine zipper motifs have been presented by Accola et al. (1). Interestingly, they found that a Gag-Zipper (Gag-Z) chimera containing a trimeric zipper motif also assembles efficiently. However, these VLPs, as well as those formed by a chimera containing a dimeric zipper motif, were not characterized morphologically.In the present work, we have extended the analysis of the assembly properties of these HIV-1 Gag-Z chimeras. This study includes the first analysis of recombinant Gag-Z proteins in vitro, as well as detailed characterization of the VLPs formed in mammalian cells. The in vitro assembly results suggest that Gag oligomerization alone is not sufficient to induce particle formation. We raise the possibility here that normal HIV-1 assembly requires neutralization of positive charges in matrix (MA) in addition to nucleic acid-induced oligomerization at the C terminus of the protein.     

Teneurin4 dimer structures reveal a calcium‐stabilized compact conformation supporting homomeric trans‐interactions

Establishment of correct synaptic connections is a crucial step during neural circuitry formation. The Teneurin family of neuronal transmembrane proteins promotes cell–cell adhesion via homophilic and heterophilic interactions, and is required for synaptic partner matching in the visual and hippocampal systems in vertebrates. It remains unclear how individual Teneurins form macromolecular cis‐ and trans‐synaptic protein complexes. Here, we present a 2.7 Å cryo‐EM structure of the dimeric ectodomain of human Teneurin4. The structure reveals a compact conformation of the dimer, stabilized by interactions mediated by the C‐rich, YD‐shell, and ABD domains. A 1.5 Å crystal structure of the C‐rich domain shows three conserved calcium binding sites, and thermal unfolding assays and SAXS‐based rigid‐body modeling demonstrate that the compactness and stability of Teneurin4 dimers are calcium‐dependent. Teneurin4 dimers form a more extended conformation in conditions that lack calcium. Cellular assays reveal that the compact cis‐dimer is compatible with homomeric trans‐interactions. Together, these findings support a role for teneurins as a scaffold for macromolecular complex assembly and the establishment of cis‐ and trans‐synaptic interactions to construct functional neuronal circuits.     

A Functional Antigenomic Promoter for the Paramyxovirus Simian Virus 5 Requires Proper Spacing between an Essential Internal Segment and the 3′ Terminus

A previous analysis of naturally occurring defective interfering (DI) RNA genomes of the prototypic paramyxovirus simian virus 5 (SV5) indicated that 113 bases at the 3′ terminus of the antigenome were sufficient to direct RNA encapsidation and replication. A nucleotide sequence alignment of the antigenomic 3′-terminal 113 bases of members of the Rubulavirus genus of the Paramyxoviridae family identified two regions of sequence identity: bases 1 to 19 at the 3′ terminus (conserved region I [CRI]) and a more distal region consisting of antigenome bases 73 to 90 (CRII) that was contained within the 3′ coding region of the L protein gene. To determine whether these regions of the antigenome were essential for SV5 RNA replication, a reverse genetics system was used to analyze the replication of copyback DI RNA analogs that contained a foreign gene (GL, encoding green fluorescence protein) flanked by 113 5′-terminal bases and various amounts of SV5 3′-terminal antigenomic sequences. Results from a deletion analysis showed that efficient encapsidation and replication of SV5-GL DI RNA analogs occurred when the 90 3′-terminal bases of the SV5 antigenomic RNA were retained, but replication was reduced ~5- to 14-fold in the case of truncated antigenomes that lacked the 3′-end CRII sequences. A chimeric copyback DI RNA containing the 3′-terminal 98 bases including the CRI and CRII sequences from the human parainfluenza virus type 2 (HPIV2) antigenome in place of the corresponding SV5 sequences was efficiently replicated by SV5 cDNA-derived components. However, replication was reduced ~20-fold for a truncated SV5-HPIV2 chimeric RNA that lacked the HPIV2 CRII sequences between antigenome bases 72 and 90. Progressive deletions of 6 to 18 bases in the region located between the SV5 antigenomic CRI and CRII segments (3′-end nucleotides 21 to 38) resulted in a ~25-fold decrease in SV5-GL RNA synthesis. Surprisingly, replication was restored to wild-type levels when these length alterations between CRI and CRII were corrected by replacing the deleted bases with nonviral sequences. Together, these data suggest that a functional SV5 antigenomic promoter requires proper spacing between an essential internal region and the 3′ terminus. A model is presented for the structure of the 3′ end of the SV5 antigenome which proposes that positioning of CRI and CRII along the same face of the helical nucleocapsid is an essential feature of a functional antigenomic promoter.     

A Refined Model of the Prototypical Salmonella SPI-1 T3SS Basal Body Reveals the Molecular Basis for Its Assembly

The T3SS injectisome is a syringe-shaped macromolecular assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed of a “basal body”, a lock-nut structure spanning both bacterial membranes, and a “needle” that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body assembly, and notably demonstrate a conserved oligomerization mechanism.     

The crystal structure of an ‘All Locked’ nucleic acid duplex

‘Locked nucleic acids’ (LNAs) are known to introduce enhanced bio- and thermostability into natural nucleic acids rendering them powerful tools for diagnostic and therapeutic applications. We present the 1.9 Å X-ray structure of an ‘all LNA’ duplex containing exclusively modified β-d-2′-O-4′C-methylene ribofuranose nucleotides. The helix illustrates a new type of nucleic acid geometry that contributes to the understanding of the enhanced thermostability of LNA duplexes. A notable decrease of several local and overall helical parameters like twist, roll and propeller twist influence the structure of the LNA helix and result in a widening of the major groove, a decrease in helical winding and an enlarged helical pitch. A detailed structural comparison to the previously solved RNA crystal structure with the corresponding base pair sequence underlines the differences in conformation. The surrounding water network of the RNA and the LNA helix shows a similar hydration pattern.     

The Crystal Structures of Yeast Get3 Suggest a Mechanism for Tail-Anchored Protein Membrane Insertion

Tail-anchored (TA) proteins represent a unique class of membrane proteins that contain a single C-terminal transmembrane helix. The post-translational insertion of the yeast TA proteins into the ER membrane requires the Golgi ER trafficking (GET) complex which contains Get1, Get2 and Get3. Get3 is an ATPase that recognizes and binds the C-terminal transmembrane domain (TMD) of the TA proteins. We have determined the crystal structures of Get3 from two yeast species, S. cerevisiae and D. hansenii, respectively. These high resolution crystal structures show that Get3 contains a nucleotide-binding domain and a “finger” domain for binding the TA protein TMD. A large hydrophobic groove on the finger domain of S. cerevisiae Get3 structure might represent the binding site for TMD of TA proteins. A hydrophobic helix from a symmetry-related Get3 molecule sits in the TMD-binding groove and mimics the TA binding scenario. Interestingly, the crystal structures of the Get3 dimers from S. cerevisiae and D. hansenii exhibit distinct conformations. The S. cerevisiae Get3 dimer structure does not contain nucleotides and maintains an “open” conformation, while the D. hansenii Get3 dimer structure binds ADP and stays in a “closed” conformation. We propose that the conformational changes to switch the Get3 between the open and closed conformations may facilitate the membrane insertions for TA proteins.     

Functional Analysis of a Rickettsial OmpA Homology Domain of Shigella flexneri IcsA

Shigella flexneri is a gram-negative bacterium that causes diarrhea and dysentery by invasion and spread through the colonic epithelium. Bacteria spread by assembling actin and other cytoskeletal proteins of the host into “actin tails” at the bacterial pole; actin tail assembly provides the force required to move bacteria through the cell cytoplasm and into adjacent cells. The 120-kDa S. flexneri outer membrane protein IcsA is essential for actin assembly. IcsA is anchored in the outer membrane by a carboxy-terminal domain (the β domain), such that the amino-terminal 706 amino acid residues (the α domain) are exposed on the exterior of the bacillus. The α domain is therefore likely to contain the domains that are important to interactions with host factors. We identify and characterize a domain of IcsA within the α domain that bears significant sequence similarity to two repeated domains of rickettsial OmpA, which has been implicated in rickettsial actin tail formation. Strains of S. flexneri and Escherichia coli that carry derivatives of IcsA containing deletions within this domain display loss of actin recruitment and increased accessibility to IcsA-specific antibody on the surface of intracytoplasmic bacteria. However, site-directed mutagenesis of charged residues within this domain results in actin assembly that is indistinguishable from that of the wild type, and in vitro competition of a polypeptide of this domain fused to glutathione S-transferase did not alter the motility of the wild-type construct. Taken together, our data suggest that the rickettsial homology domain of IcsA is required for the proper conformation of IcsA and that its disruption leads to loss of interactions of other IcsA domains within the amino terminus with host cytoskeletal proteins.     

Recognition of two distinct elements in the RNA substrate by the RNA-binding domain of the T. thermophilus DEAD box helicase Hera

DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for “heat-resistant RNA-binding ATPase”) contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA–protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall, the mode of RNA binding by Hera is consistent with a possible function as a general RNA chaperone.     

Global Stabilization of rRNA Structure by Ribosomal Proteins S4, S17, and S20

Ribosomal proteins stabilize the folded structure of the ribosomal RNA and enable the recruitment of further proteins to the complex. Quantitative hydroxyl radical footprinting was used to measure the extent to which three different primary assembly proteins, S4, S17, and S20, stabilize the three-dimensional structure of the Escherichia coli 16S 5′ domain. The stability of the complexes was perturbed by varying the concentration of MgCl₂. Each protein influences the stability of the ribosomal RNA tertiary interactions beyond its immediate binding site. S4 and S17 stabilize the entire 5′ domain, while S20 has a more local effect. Multistage folding of individual helices within the 5′ domain shows that each protein stabilizes a different ensemble of structural intermediates that include nonnative interactions at low Mg²⁺ concentration. We propose that the combined interactions of S4, S17, and S20 with different helical junctions bias the free-energy landscape toward a few RNA conformations that are competent to add the secondary assembly protein S16 in the next step of assembly.     

Crystal Structure of a Four-Layer Aggregate of Engineered TMV CP Implies the Importance of Terminal Residues for Oligomer Assembly

Background

Crystal structures of the tobacco mosaic virus (TMV) coat protein (CP) in its helical and disk conformations have previously been determined at the atomic level. For the helical structure, interactions of proteins and nucleic acids in the main chains were clearly observed; however, the conformation of residues at the C-terminus was flexible and disordered. For the four-layer aggregate disk structure, interactions of the main chain residues could only be observed through water–mediated hydrogen bonding with protein residues. In this study, the effects of the C-terminal peptides on the interactions of TMV CP were investigated by crystal structure determination.

Methodology/Principal Findings

The crystal structure of a genetically engineered TMV CP was resolved at 3.06 Å. For the genetically engineered TMV CP, a six-histidine (His) tag was introduced at the N-terminus, and the C-terminal residues 155 to 158 were truncated (N-His-TMV CP¹⁹). Overall, N-His-TMV CP¹⁹ protein self-assembled into the four-layer aggregate form. The conformations of residues Gln36, Thr59, Asp115 and Arg134 were carefully analyzed in the high radius and low radius regions of N-His-TMV CP¹⁹, which were found to be significantly different from those observed previously for the helical and four-layer aggregate forms. In addition, the aggregation of the N-His-TMV CP¹⁹ layers was found to primarily be mediated through direct hydrogen-bonding. Notably, this engineered protein also can package RNA effectively and assemble into an infectious virus particle.

Conclusion

The terminal sequence of amino acids influences the conformation and interactions of the four-layer aggregate. Direct protein–protein interactions are observed in the major overlap region when residues Gly155 to Thr158 at the C-terminus are truncated. This engineered TMV CP is reassembled by direct protein–protein interaction and maintains the normal function of the four-layer aggregate of TMV CP in the presence of RNA.