H-NS and Lsr2 are nucleoid-associated proteins from Gram-negative bacteria and Mycobacteria , respectively, that play an important role in the silencing of horizontally acquired foreign DNA that is more AT-rich than the resident genome. Despite the fact that Lsr2 and H-NS proteins are dissimilar in sequence and structure, they serve apparently similar functions and can functionally complement one another. The mechanism by which these xenogeneic silencers selectively target AT-rich DNA has been enigmatic. We performed high-resolution protein binding microarray analysis to simultaneously assess the binding preference of H-NS and Lsr2 for all possible 8-base sequences. Concurrently, we performed a detailed structure-function relationship analysis of their C-terminal DNA binding domains by NMR. Unexpectedly, we found that H-NS and Lsr2 use a common DNA binding mechanism where a short loop containing a “Q/RGR” motif selectively interacts with the DNA minor groove, where the highest affinity is for AT-rich sequences that lack A-tracts. Mutations of the Q/RGR motif abolished DNA binding activity. Netropsin, a DNA minor groove-binding molecule effectively outcompeted H-NS and Lsr2 for binding to AT-rich sequences. These results provide a unified molecular mechanism to explain findings related to xenogeneic silencing proteins, including their lack of apparent sequence specificity but preference for AT-rich sequences. Our findings also suggest that structural information contained within the DNA minor groove is deciphered by xenogeneic silencing proteins to distinguish genetic material that is self from nonself.

Mitophagy is a fundamental process that determines mitochondrial quality and homeostasis. Several mitophagy receptors, including the newly identified FUNDC1, mediate selective removal of damaged or superfluous mitochondria through their specific interaction with LC3. However, the precise mechanism by which this interaction is regulated to initiate mitophagy is not understood. Here, we report the solution structure of LC3 in complex with a peptide containing the FUNDC1 LC3-interacting region (LIR) motif. The structure reveals a noncanonical LC3-LIR binding conformation, in which the third LIR residue (Val20) is also inserted into the hydrophobic pocket of LC3, together with the conserved residues Tyr18 and Leu21. This enables Tyr18 to be positioned near Asp19 of LC3, and thus phosphorylation of Tyr18 significantly weakens the binding affinity due to electrostatic repulsion. Functional analysis revealed that mitochondrial targeting of the LIR-containing cytosolic portion of FUNDC1 is necessary and sufficient to initiate mitophagy when Tyr18 is unphosphorylated, even in the absence of mitochondrial fragmentation. Thus, we demonstrated that phosphorylation of Tyr18 of FUNDC1 serves as a molecular switch for mitophagy. This may represent a novel target for therapeutic intervention.

Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5ʹ-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. Here, we show that the dimeric 5′-leader contains more than two dozen NC binding sites with affinities ranging from 40 nM to 1.4 μM, and that all high-affinity sites (K_d ≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, Ψ^CES). The four initial binding sites with highest affinity reside near two symmetrically equivalent three-way junction structures. Unlike the other high-affinity sites, which bind NC with exothermic energetics, binding to these sites occurs endothermically due to concomitant unwinding of a weakly base-paired [UUUU]:[GGAG] helical element. Mutations that stabilize base pairing within this element eliminate NC binding to this site and severely impair RNA packaging into virus-like particles. NMR studies reveal that a recently discovered small-molecule inhibitor of HIV-1 RNA packaging that appears to function by stabilizing the structure of the leader binds directly to the [UUUU]:[GGAG] helix. Our findings suggest a sequential NC binding mechanism for Gag-genome assembly and identify a potential RNA Achilles’ heel to which HIV therapeutics may be targeted. Significance Understanding the molecular determinants of retroviral genome packaging is important for drug discovery and development of vectors for gene delivery. We show that the HIV-1 leader, which contains the RNA elements necessary for genome packaging, binds approximately two dozen copies of the cognate NC protein with affinities ranging from ∼40 nM to 1.4 µM. Binding to the four highest-affinity “initial” binding sites occurs with endothermic energetics attributed to NC-induced localized RNA melting. Mutations that stabilize these sites inhibit NC binding in vitro and RNA packaging in transfected cells. A small-molecule inhibitor of RNA packaging binds specifically to the initial NC binding sites and stabilizes the RNA structure. Our findings identify a potential RNA Achilles’ heel for HIV therapeutic development.

Bacterial xenogeneic silencing proteins selectively bind to and silence expression from many AT rich regions of the chromosome. They serve as master regulators of horizontally acquired DNA, including a large number of virulence genes. To date, three distinct families of xenogeneic silencers have been identified: H-NS of Proteobacteria, Lsr2 of the Actinomycetes, and MvaT of Pseudomonas sp. Although H-NS and Lsr2 family proteins are structurally different, they all recognize the AT-rich DNA minor groove through a common AT-hook-like motif, which is absent in the MvaT family. Thus, the DNA binding mechanism of MvaT has not been determined. Here, we report the characteristics of DNA sequences targeted by MvaT with protein binding microarrays, which indicates that MvaT prefers binding flexible DNA sequences with multiple TpA steps. We demonstrate that there are clear differences in sequence preferences between MvaT and the other two xenogeneic silencer families. We also determined the structure of the DNA-binding domain of MvaT in complex with a high affinity DNA dodecamer using solution NMR. This is the first experimental structure of a xenogeneic silencer in complex with DNA, which reveals that MvaT recognizes the AT-rich DNA both through base readout by an “AT-pincer” motif inserted into the minor groove and through shape readout by multiple lysine side chains interacting with the DNA sugar-phosphate backbone. Mutations of key MvaT residues for DNA binding confirm their importance with both in vitro and in vivo assays. This novel DNA binding mode enables MvaT to better tolerate GC-base pair interruptions in the binding site and less prefer A tract DNA when compared to H-NS and Lsr2. Comparison of MvaT with other bacterial xenogeneic silencers provides a clear picture that nature has evolved unique solutions for different bacterial genera to distinguish foreign from self DNA. During evolution, the bacteria frequently acquire new genes through horizontal transfer, in order to adapt new environments. However, foreign DNA sequences acquired are more likely to decrease rather than increase the fitness of the recipient bacteria. Therefore, many bacterial genera have evolved unique proteins to selectively repress the transcription of foreign genes. The opportunistic pathogen Pseudomonas aeruginosa is the principal cause of the morbidity and mortality in cystic fibrosis patients and is among the major causes of nosocomial infections. As a xenogeneic silencer, the MvaT protein of P. aeruginosa is a master regulator of horizontally acquired genes including many critical for drug resistance and virulence. Here, we have characterized the DNA sequences preferentially targeted by MvaT, and identified differences in sequence preferences between MvaT and other xenogeneic silencers. The high resolution structure of the DNA-binding domain of MvaT in complex with a high affinity DNA target reveals a novel AT-rich DNA minor groove recognition mechanism, which perfectly explains the characteristic of MvaT’s DNA sequence preferences. Comparison between MvaT and other bacterial xenogeneic silencers demonstrates how unique solutions have been employed by different bacterial genera in distinguishing foreign DNA from DNA of their own genome.

HIV-1 selectively packages two copies of its 5′-capped RNA genome (gRNA) during virus assembly, a process mediated by the nucleocapsid (NC) domain of the viral Gag polyprotein and encapsidation signals located within the dimeric 5′ leader of the viral RNA. Although residues within the leader that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency remain unknown. Here, we show that a previously characterized 159-nt region of the leader that possesses all elements required for RNA dimerization, high-affinity NC binding, and packaging in a noncompetitive RNA packaging assay (Ψ^CES) is unexpectedly poorly packaged when assayed in competition with the intact 5′ leader. Ψ^CES lacks a 5′-tandem hairpin element that sequesters the 5′ cap, suggesting that cap sequestration may be important for packaging. Consistent with this hypothesis, mutations within the intact leader that expose the cap without disrupting RNA structure or NC binding abrogated RNA packaging, and genetic addition of a 5′ ribozyme to Ψ^CES to enable cotranscriptional shedding of the 5′ cap promoted Ψ^CES-mediated RNA packaging to wild-type levels. Additional mutations that either block dimerization or eliminate subsets of NC binding sites substantially attenuated competitive packaging. Our studies indicate that packaging is achieved by a bipartite mechanism that requires both sequestration of the 5′ cap and exposure of NC binding sites that reside fully within the Ψ^CES region of the dimeric leader. We speculate that cap sequestration prevents irreversible capture by the cellular RNA processing and translation machinery, a mechanism likely employed by other viruses that package 5′-capped RNA genomes. Significance HIV-1 replication is critically dependent on the selective incorporation of viral RNA genomes into assembling virions. Although RNA elements that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency have until now remained unknown. The present studies show that genome selection is achieved by a bipartite mechanism that requires both dimerization-dependent exposure of packaging signals within the 5′ leader of the viral RNA and structural sequestration of the 5′ cap. Cap sequestration likely prevents capture by the cellular RNA processing and translation machinery, a mechanism that may help explain why some cellular RNA polymerase III transcripts that lack 5′ caps can parasitize virions. Cap sequestration may be commonly employed among RNA viruses that package 5′-capped genomes.