The L1-ORF1p coiled coil enables formation of a tightly compacted nucleic acid-bound complex that is associated with retrotransposition

Abstract

Long interspersed nuclear element 1 (L1) parasitized most vertebrates and constitutes ∼20% of the human genome. It encodes ORF1p and ORF2p which form an L1-ribonucleoprotein (RNP) with their encoding transcript that is copied into genomic DNA (retrotransposition). ORF1p binds single-stranded nucleic acid (ssNA) and exhibits NA chaperone activity. All vertebrate ORF1ps contain a coiled coil (CC) domain and we previously showed that a CC-retrotransposition null mutant prevented formation of stably bound ORF1p complexes on ssNA. Here, we compared CC variants using our recently improved method that measures ORF1p binding to ssDNA at different forces. Bound proteins decrease ssDNA contour length and at low force, retrotransposition-competent ORF1ps (111p and m14p) exhibit two shortening phases: the first is rapid, coincident with ORF1p binding; the second is slower, consistent with formation of tightly compacted complexes by NA-bound ORF1p. In contrast, two retrotransposition-null CC variants (151p and m15p) did not attain the second tightly compacted state. The C-terminal half of the ORF1p trimer (not the CC) contains the residues that mediate NA-binding. Our demonstrating that the CC governs the ability of NA-bound retrotransposition-competent trimers to form tightly compacted complexes reveals the biochemical phenotype of these coiled coil mutants.

Figure 1.

ORF1p. (A) Annotated sequence of ORF1p showing conserved phosphorylation sites (red boxes), the 14 heptads of the CC (alternating green and yellow boxes with a stammer (stm) in heptad 6), the highly conserved non-canonical RNA recognition motif (RRM), and C-terminal domain (CTD) that contains sequences (notably R261, R262) involved in NA-binding and chaperone activity. The N terminal domain (NTD) and terminal 46 amino acids of the CTD are intrinsically disordered (see text). The insert shows the relevant part of the alignment of the CC variants and their % retro(transposition) activity relative to the 111 (L1Pa1) wild type protein (adapted from Figure 1 in ref. 12). The amino acids that differentiate the coiled coil variants from 111p are their ancestral counterparts in the resuscitated L1Pa5 family (32). (B) Depiction of L1RNP assembly, involvement in, and fate during retrotransposition. (C) Depiction of an ssNA tethered between two beads and its length Δx₀, before and after its initial Δx₊ⁱ and final Δx₊^f compaction.

Figure 2.

Binding of wild type and ORF1p variants to ssDNA at 5 pN. (A) When ssDNA is incubated with ORF1p at low force (5 pN), two phases of ssDNA compaction are observed: initial, rapid compaction (Δx₊ⁱ), followed by a slow, secondary compaction step (Δx₊^s). The curves were fit with a two-rate decaying exponential function to extract a rate and amplitude associated with both phases of ssDNA compaction. The absolute extension of bare ssDNA at 5 pN is ∼0.29 nm/nt. The total ssDNA extension changes seen for 111p and m14 asymptote to ∼-0.29 nm/nt, indicating that the DNA is almost fully compacted (purple dashed line) to near zero extension. (B) The four proteins exhibit similar initial compaction, but the magnitude of the secondary compaction is significantly reduced for complexes formed with the inactive variants. (C) The amplitudes of the secondary compaction events (Δx₊^s) are plotted as bar graphs for comparison. (D) The rates of secondary compaction (k₊^s) are similar for 111p, m14 and m15, however, we were unable to calculate k₊^s for 151p as secondary compaction was negligible.

Figure 3.

Dynamics of ORF1p-ssDNA compaction at low, fixed extension. (A) ssDNA previously incubated for 100 seconds at 30 pN with 30 nM 111p was held at a minimal, fixed extension (∼0.2 nm/nt) in protein-free buffer for 2 minutes. The protein–ssDNA complex was then stretched until reaching a tension of 75 pN (1), where it was held for 100 s (2). Tension on the strand was then released by reducing its extension to the initial value (3). (B) Incubation of the complexes formed by 111p in protein-free buffer at low extension (0.2 nm/nt) was repeated for 5, 15 or 30 min. The ssDNA extension for both the initial stretch (closed circles) and subsequent release (open circles) is inversely proportional to the initial incubation time. (C) Extension of the pre-formed ssDNA–ORF1p complex at 30 pN during stretching (normalized to the length of protein-free ssDNA) shows that the compaction of the active protein–DNA complexes (111p and m14) is greater than that of the inactive proteins (151p and m15). (D) Similarly, during release, the reduction of extension at 30 pN is greater for the active proteins than the inactive variants.

Figure 4.

Initial binding phases of active (111p, m14) and inactive (151p, m15) trimers at 30 pN. (A, B) The extension changes, Δx₊ⁱ and Δx₊^f, of ssDNA (absolute extension of bare ssDNA at 30 pN is ∼0.54 nm/nt) during incubation with wild type ORF1p and the three coiled coil variants (m14, m15 and 151p) show identical biphasic binding behavior, indicating that the proteins initially bind ssDNA in a similar manner at 30 pN. The initial rates of ssDNA compaction (k₊ⁱ), (C) and subsequent elongation (k₊^f), (D) due to ORF1p binding are equivalent for the four trimers.

Figure 5.

Dissociation phases of active (111p, m14) and inactive (151p, m15) trimers at 30 pN. (A) Representative 111p dissociation curve showing two phases of dissociation at 30 pN: an initial re-compaction (Δx₋ⁱ) followed by ssDNA elongation (Δx₋^f). (B) All ORF1p trimers eventually compact the DNA to the same extent during the initial dissociation phase. Bar graphs show the average amplitude of compaction from multiple (n ≥ 3) experiments with each variant. However, re-compaction of the ssDNA occurs approximately twice as fast with the inactive variants as it does with the active proteins (C). Final dissociation of the inactive ORF1 proteins is both more complete (D) and faster (E) than the active trimers. In contrast, the ssDNA binding dynamics of all three ORF1p variants (m14, m15 and 151p) are identical to those of the wild type at 30 pN (shown in Figure 4).