The challenge of ORF1p phosphorylation: Effects on L1 activity and its host

Abstract

L1 non-LTR retrotransposons are autonomously replicating genetic elements that profoundly affected their mammalian hosts having generated upwards of 40% or more of their genomes. Although deleterious, they remain active in most mammalian species, and thus the nature and consequences of the interaction between L1 and its host remain major issues for mammalian biology. We recently showed that L1 activity requires phosphorylation of one of its 2 encoded proteins, ORF1p, a nucleic acid chaperone and the major component of the L1RNP retrotransposition intermediate. Reversible protein phosphorylation, which is effected by interacting cascades of protein kinases, phosphatases, and ancillary proteins, is a mainstay in the regulation and coordination of many basic biological processes. Therefore, demonstrating phosphorylation-dependence of L1 activity substantially enlarged our knowledge of the scope of L1 / host interaction. However, developing a mechanistic understanding of what this means for L1 or its host is a formidable challenge, which we discuss here.

Keywords: L1 retrotransposon; Pin1; intrinsically disordered region; peptidyl prolyl isomerase 1; proline-directed protein kinase.

Figure 1.

Structural Features of ORF1p (A). The top panel illustrates the trimeric structure of the protein revealed by atomic force microscopy and the domains present in the monomer: N-terminal domain (NTD), coiled coil domain, RNA recognition domain (RRM), the C-terminal domain (CTD). The ovals that correspond to the NTD and the C-terminal half of the trimer cartoon are scaled to the their relative masses. The large red arrowheads indicate the amino-terminus of the protein that was expressed in E. coli for crystallization. (B) The arrows indicate the locations of phosphokinase target sites and the amino acids corresponding to the PDPK sites are highlighted in red, those comprising the RNP1 motif in purple, those making up the PDPK docking sites in gray, and those corresponding to the PKA target sites are underlined in black. C. The results generated by the DISOPRED, PSIPRED and DISPHOS predictions programs (see text). The green rectangles correspond to the DISPHOS predictions for phosphorylated serines, the red rectangles correspond to those found by mass spectroscopy on ORF1p expressed in HeLa cells.

Figure 2.

Structure of the ORF1p RRM The structure the C monomer RRM was displayed using MacPymol 1.7.6.3 based on the PDB file, 2yko. The upper left insert shows the amino acids missing from the structures of the A, B, and C monomers (black dashed line, red box c, Fig. 4) in the vicinity of T203 – T213. The lower right insert show the amino acid sequence encompassing the highly conserved PKA sites in the PDPK docking sites shown in Figure 1B.

Figure 3.

Top view of the C-terminal half of the ORF1p trimer. This structure (also based on the PDP file 2yko) shows a top view of the 3 trimers starting with the last heptad (LQWIEDY) of the coiled coil - as if the trimer was resected at the heavy line in the insert and then rotated toward the viewer. The hinge between the carboxy terminus of the coiled coil and the N-terminus of the RRM is in tan (see Fig. 2) and the RNP2 and RNP1 motifs are respectively in teal and deep purple for each monomer as also indicated on Figures 1 and 2. The PKA sites and surrounding amino acids are also colored the same for each monomer as indicated in the lower right insert of Figure 2. The black dashed lines show the location of the missing amino acids (starting at P204) in each monomer (also see upper left insert, Fig. 2) and red box c in Figure 4. Other than these features the major secondary structures of monomers A, B and C are colored differently. For each of the respective monomers these are: α helices, green, teal, and red; β sheets, magenta, red, and yellow; loops, tan, magenta and green. The amino acid positions indicated in magenta in the monomer B structure flank the amino acids (167–172, corresponding to mauve box a, Figure 4) that are missing from the X-ray structure of the RRM just 3’ of RNP2.

Figure 4.

Intrinsic disorder profile of ORF1p The profiles of intrinsically disordered sequence and potential protein binding surface were generated by DISOPRED3. The cartoon showing the monomer domains is the same as that in Figure 1A, annotated as follows: The gray box indicates the region of the protein that had been deleted prior to expression in E. coli. The mauve and red boxes indicate the amino acids missing from the X-ray structure in the 2yko PDB file – (a), positions 167–172, monomer B; (b), 191–193, and 190–194, monomers A and B respectively; (c), 204–210, 204–210, 203–212 monomers A, B, and C respectively; (d), 323–338, monomers A, B, and C.