Trimeric structure for an essential protein in L1 retrotransposition - Analytical Methods & Tools for Lyophilization

Trimeric structure for an essential protein in L1 retrotransposition
September 26, 2003 (received for review May 20, 2003)
Published online before print November 13, 2003
Sandra L. Martin * , Dan Branciforte *, David Keller , and David L. Bain
PNAS
*Department of Cell and Developmental Biology and Program in Molecular Biology, University of Colorado School of Medicine, 4200 East Ninth Avenue, Denver, CO 80262; Department of Chemistry, University of New Mexico, Albuquerque, NM 87131; and Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, CO 80262
Communicated by Clyde A. Hutchison III, University of North Carolina, Chapel Hill, NC,
Abstract
Two proteins are encoded by the mammalian retrotransposon long interspersed nuclear element 1 (LINE-1 or L1); both are essential for retrotransposition. The function of the protein encoded by the 5'-most ORF, ORF1p, is incompletely understood, although the ORF1p from mouse L1 is known to bind single-stranded nucleic acids and function as a nucleic acid chaperone. ORF1p self-associates by means of a long coiled-coil domain in the N-terminal region of the protein, and the basic, C-terminal region (C-1/3 domain) contains the nucleic acid binding activity. The full-length and C-1/3 domains of ORF1p were purified to near homogeneity then analyzed by gel filtration chromatography and analytical ultracentrifugation. Both proteins were structurally homogeneous and asymmetric in solution, with the full-length version forming a stable trimer and the C-1/3 domain remaining a monomer. Examination of the full-length protein by atomic force microscopy revealed an asymmetric dumbbell shape, congruent with the chromatography and ultracentrifugation results. These structural features are compatible with the nucleic acid binding and chaperone activities of L1 ORF1p and offer further insight into the functions of this unique protein during LINE-1 retrotransposition.
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Long interspersed nuclear element 1, known as LINE-1 or L1, is a highly successful retrotransposon of mammalian genomes, comprising 17% of the human genome and 19% of the mouse genome, and likely generating an additional 8?13% in the form of short interspersed nuclear elements (SINEs) and processed pseudogenes (1). L1 belongs to a larger group of mobile elements known as the non-LTR retrotransposons, which are widely distributed throughout eukaryotes (2).
Non-LTR retrotransposons replicate by reverse transcription of an RNA intermediate, employing a unique mechanism known as target-site primed reverse transcription, or TPRT. In mammalian L1s, two element-encoded proteins are required for retrotransposition: ORF2p, which provides the crucial enzymatic activities for TPRT, endonuclease, and reverse transcriptase (reviewed in ref. 3); and ORF1p, which is a single-stranded nucleic acid binding protein (4, 5) that also acts as a nucleic acid chaperone (6).
The calculated mass of the protein encoded by ORF1 in the retrotransposition-competent element, L1spa (7), is 42,921 Da, and a 43-kDa ORF1p is detected in extracts prepared from a subset of mouse cells by Western blotting. Immunocytochemistry with the same anti-ORF1p Ab reveals a punctate cytoplasmic staining pattern in the two embryonal carcinoma cell types, F9 and JC44, where ORF1p is detected by Western blotting (8), as well as in prepachytene germ cells of the testes (9) and ovary (10). p43 is coenriched with full-length L1 RNA in ribonucleoprotein particles that can be fractionated from F9 cells (11, 12). Taken together, these observations suggest that L1 RNA is bound with many molecules of ORF1p during the cytoplasmic phase of the L1 replication cycle, perhaps playing a structural role in protecting the RNA and organizing the replication intermediates. A second role for ORF1p likely occurs during the TPRT reaction, where the nucleic acid chaperone activity of this protein may facilitate the strand transfers that are required to prime reverse transcription as well as the reverse transcriptase reaction itself (6).
To fully understand the role(s) of ORF1p in L1 retrotransposition, detailed information about the structure of this unusual protein is required. Although mouse ORF1p is homologous to other mammalian ORF1ps, as well as the ORF1ps from LINE-like elements in teleost fish (3), there are no structures available for any of these proteins. Furthermore, the L1 ORF1ps lack conserved sequence motifs, except for a long coiled-coil domain in the N-terminal half of the protein. In the case of mouse ORF1p, the coiled-coil region is both necessary and sufficient for protein?protein interaction by either two-hybrid or GST pull-down assay (13). The only other structural feature known for the mouse ORF1p is that the C-terminal, basic domain of the protein is responsible for nucleic acid binding and annealing activity (13).
An increased understanding of ORF1p structure will provide significant insights into the role of this unique protein in L1 retrotransposition. Here, we use biochemical and biophysical approaches to demonstrate that ORF1p exists as a stable trimer in solution, thus forming a distinctive structure for a known nucleic acid chaperone and single-strand nucleic acid binding protein. Parallel studies of the isolated C-1/3 domain demonstrate that this region is quantitatively monomeric in solution, supporting previous results that the coiled-coil domain mediates ORF1p self-association. Hydrodynamic analysis of the ORF1p trimer suggests that it forms a highly anisotropic, elongated conformation in solution. Indeed, direct visualization of ORF1p by atomic force microscopy clearly reveals an elongated dumbbell-like structure. Taken together, these results suggest a structural basis for the nucleic acid chaperone activity of ORF1p during L1 retrotransposition.
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