Sequence and structure evolved separately in a ribosomal ubiquitin variant

Abstract

Encoded by a multigene family, ubiquitin is expressed in the form of three precursor proteins, two of which are fusions to the ribosomal subunits S27a and L40. Ubiquitin assists in ribosome biogenesis and also functions as a post-translational modifier after its release from S27a or L40. However, several species do not conserve the ribosomal ubiquitin domains. We report here the solution structure of a distant variant of ubiquitin, found at the N-terminus of S27a in Giardia lamblia, referred to as GlUb(S27a). Despite the considerable evolutionary distance that separates ubiquitin from GlUb(S27a), the structure of GlUb(S27a) is largely identical to that of ubiquitin. The variant domain remains attached to S27a and is part of the assembled holoribosome. Thus, conservation of tertiary structure suggests a role of this variant as a chaperone, while conservation of the primary structure--necessary for ubiquitin's function as a post-translational modifier--is no longer required. Based on these observations, we propose a model to explain the origin of the widespread ubiquitin superfold in eukaryotes.

Figure 1

Multiple sequence alignment of ubiquitin-S27a fusion proteins. Shown are the ubiquitin domains Ub_S27a (top) and the ribosomal domains S27a (bottom) of 15 different species. H. sapiens and S. cerevisiae express conserved ubiquitin, all other species depicted encode ubiquitin variants. The asterisks indicate a conserved putative zinc-binding motif in the S27a domain (Chan et al, 1995).

Figure 2

(A) Comparison of the primary and secondary structures between human ubiquitin and GlUb_S27a. Statistically, the sequence alignment between these two polypeptides lacks significant similarity. However, the color-coding, indicating hydrophobic (blue) and polar (red) side chains, reveals a homologous pattern in both proteins. Asterisks represent conserved core leucine residues. The secondary structures are shown at the top for ubiquitin (based on 1ubi.pdb) (Vijay-Kumar et al, 1987; Ramage et al, 1994) and at the bottom for GlUb_S27a (determined by NMR). (B) Solution structure of GlUb_S27a. Stereoview of an ensemble of 10 out of 50 CYANA-calculated structures superimposed by backbone atoms, representing the models with lowest energy (see table in Materials and methods). The α-helix is colored in red and the β-strands in blue. (C) Ribbon diagram, comparing the NMR structures of GlUb_S27a (top) and ubiquitin (bottom). Except for a relative tilt of the α-helix, both structures have similar size and conformation (ubiquitin based on 1ubi.pdb), consisting of five β-strands grasping a central α-helix. The strands are highly superimposable in both structures. The second α-helix is missing in GlUb_S27a due to a truncation of five residues. (D) 3D surface reconstructions of GlUb_S27a (left panels) and ubiquitin (right panels). The top panels show charged side chains on the surface of GlUb_S27a and ubiquitin. Conserved residues are R6, E15, R43, and E47 (circled in GlUb_S27a). Exposed hydrophobic side chains are colored in green (bottom panel). The hydrophobic surface ratio is increased by 51% in GlUb_S27a compared to ubiquitin.

Figure 3

(A) GlUb_S27a appears as a single protein in G. lamblia lysate, in size consistent with a fusion to S27a. Whole-cell G. lamblia lysate (20 μg protein content per lane) was analyzed with polyclonal anti-GlUb_S27a antibodies by immunoblot. The serum reacts with a single protein (left lane), close to the predicted size of the GlUb_S27a–S27a fusion (15.1 kDa). No signal is detected at the predicted size of the GlUb_S27a domain alone (7 kDa). Incubation of the immunoblot with recombinantly expressed GlUb_S27a (10 μg) competes with detection of the protein (right lane). Pretreatment of trophozoites with the proteasome inhibitor ZL₃VS (50 μM, 2 h) did not alter the results (not shown). Immunoblots were resolved by reducing SDS–PAGE (12%). Molecular weight markers are indicated at the left (in kDa). (B) GlUb_S27a is enriched in the high-molecular-weight fraction. Anti-GlUb_S27a immunoblot of fractionated G. lamblia lysate. 30 μg of proteins were loaded per lane. GlUb_S27a is enriched in the sedimented fraction of G. lamblia lysate after ultracentrifugation (100 000 g for 1 h), but is absent from the supernatant. Samples were resolved by reducing SDS–PAGE (11%). (C) GlUb_S27a is associated with RNA-rich fractions, consistent with ribosome association. G. lamblia lysate was fractionated on 10–40% sucrose gradients, divided into equal fractions by volume and analyzed by UV spectroscopy (top panel). Addition of Mg²⁺ stabilizes the main RNA peak and we used such treated samples for the subsequent analyses. The top 15 fractions and the solubilized pellet (P) were assayed by anti-GlUb_S27a immunoblots (bottom panel), using equal volumes of each fraction (20 μl). The presence of GlUb_S27a coincides with the RNA peak in fractions 9, 10, and 11. Samples were resolved by reducing SDS–PAGE (11%). (D) Fractions 9–11 contain large and small ribosomal subunits, indicative of the assembled holoribosome. The previously obtained fractions (C) were resolved by reducing SDS–PAGE (11%) and silver stained. A total of 22 bands were excised, trypsinized and analyzed by MS/MS. All but one peptides identified are of ribosomal origin (see Table I). (E) Large-scale preparation of fraction 9 identifies S27a at the same molecular weight as GlUb_S27a. 70 μg protein lysate of fraction 9 was resolved by reducing SDS–PAGE (11%), silver stained, and individual polypeptides were analyzed by MS/MS. Excised bands and proteins identified are indicated at the right, with theoretical molecular weight of the full-length proteins in parentheses. We found peptides representing 34.3% of the C-terminal S27a domain (bottom panel, in red). The corresponding silver-stained protein (indicated with red) runs at the same relative position compared to the molecular weight markers as does the GlUb_S27a domain in the immunoblot shown in panel A. In addition, electrophoretic mobility in comparison with neighboring ribosomal proteins is consistent with a combined weight of 15.1 kDa of the GlUb_S27a–S27a fusion (SWISSPROT designation Q7QPK7).

Figure 4

Evidence for ubiquitin-specific but not for GlUb_S27a-specific proteases in G. lamblia. 20 μg of G. lamblia lysate was incubated with 0.2 μg of electrophilic probes, in which HA-ubiquitin or 3 × Flag-GlUb_S27a were synthesized with a C-terminal vinylmethyl-ester trap (VME) (Borodovsky et al, 2002). Such traps react with substrate-specific cysteine proteases. The GlUb_S27a-based probe does not react with any proteins (left panel), whereas two dominant bands can be detected with a probe based on ubiquitin (right panel). Simultaneous addition of the cysteine-alkylating agent N-ethylmaleimide (5 mM) competes for binding to the ubiquitin trap. By immunoprecipitation and tandem-mass spectroscopy, the three polypeptides that react with ubiquitin-VME were identified as the putative deubiquitinating proteases Q7QR60 (93.1 kDa), Q7R2V7 (50.5 kDa), and Q7QV72 (49.9 kDa) (not shown). Samples were resolved by reducing SDS–PAGE (10%).

Figure 5

Model of the evolution of ubiquitin variants. We hypothesize that the irreversible fusion of ubiquitin to a larger protein exempts it from purifying selection and therefore allows for mutations at high rate. Ubiquitin is represented colored by surface charges; S27a and GlUb_S27a are colored in red. The structure of S27a has not been solved yet; we calculated a hypothetical model ab initio (Kim et al, 2004).