Open Reading Frame 1 Protein of the Human Long Interspersed Nuclear Element 1 Retrotransposon Binds Multiple Equivalents of Lead

Abstract

The human long interspersed nuclear element 1 (LINE1) has been implicated in numerous diseases and has been suggested to play a significant role in genetic evolution. Open reading frame 1 protein (ORF1p) is one of the two proteins encoded in this self-replicating mobile genetic element, both of which are essential for retro­transposition. The structure of the three-stranded coiled-coil domain of ORF1p was recently solved and showed the presence of tris-cysteine layers in the interior of the coiled-coil that could function as metal binding sites. Here, we demonstrate that ORF1p binds Pb­(II). We designed a model peptide, GRCS­L16CL23C, to mimic two of the ORF1p Cys3 layers and crystallized the peptide both as the apo-form and in the presence of Pb­(II). Structural comparison of the ORF1p with apo-(GRCS­L16CL23C)3 shows very similar Cys3 layers, preorganized for Pb­(II) binding. We propose that exposure to heavy metals, such as lead, could influence directly the structural parameters of ORF1p and thus impact the overall LINE1 retro­transposition frequency, directly relating heavy metal exposure to genetic modification.

Cu(I) Binding to Designed Proteins Reveals a Putative Copper Binding Site of the Human Line1 Retrotransposon Protein ORF1p

“…Although it has been reported that preformed ribonucleoprotein particles (RNPs) of ORF1p and L1RNA are insensitive to up to 10 mM EDTA, exposure to mercury, arsenic, lead, nickel, aluminum, and cadmium has been shown to increase retrotransposition. ,− These results suggest that ORF1p is stable when bound as RNP, but that when it is unbound from RNA, metal ions interfere with the normal function, likely by increasing the stability of the homotrimer through metal coordination to the tris­(cysteine) site in the coiled coil domain. This hypothesis is supported by other structural studies that show that Hg­(II), As­(III), and Pb­(II) can bind to similar cysteine environments in 3SCCs and our previous report on lead binding to ORF1p . Of particular significance is that the oligomerization rate is critical to the ORF1p activity and we have previously shown that the oligomerization of 3SCC peptides is altered by incorporating metal binding sites and exposing them to metal ions. , Additionally, a -position (Cys) 3 sites have been shown to be preorganized for heavy metal binding, with only minor structural changes to the Cys side chains .…”

“…20 We recently reported that L1ORF1p binds to lead and suggested that it could also be responsive to thiophilic metals, with important consequences on the thermodynamics and kinetics of homotrimer stability and resistance to cysteine oxidation. 21 In that previous report, we also performed a detailed structural comparison between the metal-free and Pb-bound forms of ORF1p and our de novo designed 3SCC model system. Therefore, the activity of ORF1p, and ultimately the retrotransposition properties of L1 in genomes, may be dependent on metal ion levels based on direct metal complexation.…”

“…This hypothesis is supported by other structural studies that show that Hg(II), 32 As(III), 29 and Pb(II) 22 can bind to similar cysteine environments in 3SCCs and our previous report on lead binding to ORF1p. 21 Of particular significance is that the oligomerization rate is critical to the ORF1p activity 56 and we have previously shown that the oligomerization of 3SCC peptides is altered by incorporating metal binding sites and exposing them to metal ions. 25,57 Additionally, a-position (Cys) 3 sites have been shown to be preorganized for heavy metal binding, with only minor structural changes to the Cys side chains.…”

“…We mapped the position of the Cys residues in the ORF1p sequence to the de novo designed 3SCC TRI protein family (Figure B,C) that has been shown to bind to a wide variety of transition metals with high affinity . We recently reported that L1ORF1p binds to lead and suggested that it could also be responsive to thiophilic metals, with important consequences on the thermodynamics and kinetics of homotrimer stability and resistance to cysteine oxidation . In that previous report, we also performed a detailed structural comparison between the metal-free and Pb-bound forms of ORF1p and our de novo designed 3SCC model system.…”

Human Retrotransposons and Effective Computational Detection Methods for Next-Generation Sequencing Data

“…On the other hand, ORF1, characterized by three distinct domains, encodes RNA-binding protein. Even though the precise function is not fully understood, a study suggests that the strongly conserved long coiled-coil (CC) domain of the N-terminal domain contains cysteine residue layers that enable ORF1 to bind to various metal ions [ 34 , 35 , 36 ]. Both ORF1 and ORF2 proteins generally show cis preference, producing a ribonucleoprotein (RNP) particle that enables L1 to maintain its ability despite numerous non-functional elements [ 37 ].…”