Structure, Stability, and Flexibility of Ribosomal Protein L14e from Sulfolobus solfataricus

Abstract

Ribosomal protein L14e is a component of the large ribosomal subunit in both archaea and eukaryotes. We report here a high resolution NMR solution structure of recombinant L14e and show that the N-terminal 57 residues adopt a classic SH3 fold. The protein contains a tight turn between strands 1 and 2 instead of the typical SH3 RT-loop, indicating that it is unlikely to interact with neighboring ribosomal proteins using the common SH3 site for proline-rich sequences. The remainder of the protein (39 residues) forms a largely extended chain with a short helix which packs onto the surface of the SH3 domain via hydrophobic interactions. It has the potential of adopting an alternative structure to expose a hydrophobic surface for protein-protein interactions in the ribosome without disruption of the SH3 fold. 15 N relaxation data demonstrate that the majority of the Cterminal chain is well defined on the SH3 surface. The globular protein unfolds reversibly with a T m of 102.8 °C at pH 7, making it one of the most stable SH3 domain proteins described to date. The structure of L14e is expected to serve as a model for other members of the L14e family, along with members of the COG2163 group, including L6e and L27e. Interestingly, the N-terminal sequence of L14e shows the greatest similarity of any Sulfolobus protein to the reported N-terminal sequence of Sac8b, a DNA-binding protein reported by Grote et al. (Biochim. Biophys. Acta 873, 405-413 (1986)). The likelihood that L14e and Sac8b are the same protein is discussed. KeywordsArchaea; Sulfolobus; ribosome; protein stabilityThe ribosome is the universal molecular machine which is responsible for translating mRNA into polypeptides and proteins. It is composed of about 80 proteins assembled onto ribosomal RNA to form a 70S particle in prokaryotes (eubacteria and archaea) and a larger 80S particle in eukaryotes. The intact ribosome can be separated into two subunits referred to as small and large: 30S and 50S in prokaryotes, and 40S and 60S in eukaryotes. Although the intact archaeal ribosome subunits are smaller than those in eukaryotes, the archaeal proteins are more similar to those in eukaryotes than eubacteria (1). The structure of the bacterial ribosome has been defined by crystal structures of the 30S and 50S subunits (2-4) and the intact 70S particle (5-8). Crystal structures of the archaeal 50S subunit from Haloarcula (9) and the intact yeast ribosome (10) have been published, along with cryomicroscopy re-constructions of the canine ribosome (11,12 L14e is known to be a protein component of the large ribosome subunit in eukaryotes and Archaea based on 2D gel electrophoresis and sequencing, but the structure and position in the ribosome have not been described. The L14e sequence is conserved in archaea and eukaryotes (including yeast, Drosophila, rat, and human), where the protein is sometimes referred to as L14 (1,(13)(14)(15)(16). L14e shows no homology to the bacterial L14p protein (unfortunately also sometimes referred to as L14) for which a structur...

Disordered Proteinaceous Machines

“…These observations suggest that almost all ribosomal proteins are likely to be intrinsically disordered in isolation but fold to a different degree upon the ribosome formation . This hypothesis is in agreement with the earlier experimental studies, which showed that many individual ribosomal proteins do not possess ordered structure in their nonbound forms or at least contain long disordered regions. ,− Further support to this idea came from the comprehensive bioinformatics analysis of 3411 ribosomal proteins from 32 species . This analysis revealed that the vast majority of ribosomal proteins are intrinsically disordered, and that intrinsic disorder is very important for various biological functions of these important RNA-binding proteins, being commonly used as means for the numerous interactions of any given ribosomal protein with its various binding partners of different nature, such as other ribosomal proteins, RNA, and proteins from the translational machinery.…”

Promiscuous behaviour of archaeal ribosomal proteins: Implications for eukaryotic ribosome evolution

“…Indeed, we found that L14e could be unambiguously fit to the remaining electron density located on the 50S subunit (Figure 4C). Archaeal L14e has an Src-homology 3 (SH3)-like β-barrel (49) and in the canonical position on the ribosome, L14e(1), is located at the back of the central protuberance adjacent to LX, where it interacts with the backbone of H41 and the tip of H25 (ES7L) (Figure 4C) (7,9,15,44). Analogously, the second L14e(2) interacts with the backbone of H58 and the tip of ES20L (Figure 4D).…”

The Archaeal Sac10b Protein Family: Conserved Proteins with Divergent Functions

“…The 8 kDa proteins contain two groups, the Sac8a and Sac8b proteins. Sac8b itself may be ribosomal protein L14e with an SH3-like structure [14]. The recently discovered protein Cren7 may be a Sac8a family protein because it is an abundant, conserved chromatin protein in Crenarchaea with many properties similar to those of Sac8a, for example unusual CD spectra, a histidine-and tryptophancontaining sequence, and an apparent molecular weight of 8.6 kDa in solution [15][16][17].…”