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 (Kd ≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, ΨCES). 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.

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.