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, 430 (10), 1403-1416

Crystal Structure of Human Rpp20/Rpp25 Reveals Quaternary Level Adaptation of the Alba Scaffold as Structural Basis for Single-stranded RNA Binding

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Crystal Structure of Human Rpp20/Rpp25 Reveals Quaternary Level Adaptation of the Alba Scaffold as Structural Basis for Single-stranded RNA Binding

Clarence W Chan et al. J Mol Biol.

Abstract

Ribonuclease P (RNase P) catalyzes the removal of 5' leaders of tRNA precursors and its central catalytic RNA subunit is highly conserved across all domains of life. In eukaryotes, RNase P and RNase MRP, a closely related ribonucleoprotein enzyme, share several of the same protein subunits, contain a similar catalytic RNA core, and exhibit structural features that do not exist in their bacterial or archaeal counterparts. A unique feature of eukaryotic RNase P/MRP is the presence of two relatively long and unpaired internal loops within the P3 region of their RNA subunit bound by a heterodimeric protein complex, Rpp20/Rpp25. Here we present a crystal structure of the human Rpp20/Rpp25 heterodimer and we propose, using comparative structural analyses, that the evolutionary divergence of the single-stranded and helical nucleic acid binding specificities of eukaryotic Rpp20/Rpp25 and their related archaeal Alba chromatin protein dimers, respectively, originate primarily from quaternary level differences observed in their heterodimerization interface. Our work provides structural insights into how the archaeal Alba protein scaffold was adapted evolutionarily for incorporation into several functionally-independent eukaryotic ribonucleoprotein complexes.

Keywords: Alba; Ribonuclease P; Rpp20/Rpp25; molecular evolution; structure.

Conflict of interest statement

Conflict of Interest: None declared.

Figures

Figure 1
Figure 1. Binding of human Rpp20, Rpp25, and the Rpp20/Rpp25 heterodimer to the P3 stem loop
(a) Top: schematic diagram of the RNA component of human RNase P (H1 RNA). The position of the P3 loop is shown in black. Bottom: schematic diagram of the P3 stem loop. Electrophoretic mobility shift assay of in vitro transcribed human RNase P RNA P3 stem loop by recombinantly purified human (b) Rpp20, (c) Rpp25, and (d, e) the Rpp20/Rpp25 heterodimer. Arrows show the position of free P3 RNA, whereas asterisks show the position of gel shifted, protein bound P3. No significant differences were observed as a result of forming the Rpp20/Rpp25 heterodimer by (d) post purification mixing of Rpp20 to Rpp25 or by (e) co-purification of both subunits. Despite the disappearance of free P3 RNA, Rpp20 and Rpp25 alone do not produce a clear gel shift, whereas Rpp20/Rpp25 associates readily with the P3 RNA to produce a defined complex. Even at a modest stoichiometric excess of Rpp20/Rpp25, higher order oligomers or assemblies appear to form (crosses).
Figure 2
Figure 2. Crystal structure of the human Rpp20/Rpp25 heterodimer
(a) Cartoons showing the structure of the Rpp20/Rpp25 heterodimer in orthogonal views. Rpp20 and Rpp25 are shown in blue and green, respectively. Disordered regions are indicated by dotted lines. (b) Schematic diagram of the secondary structures of Rpp20 and Rpp25. The diagrams reveal a common βαβαββ fold (black) identical to that of their yeast homologues, Pop7 and Pop6, respectively, and of Alba proteins in general [21, 41, 42, 44, 56]. The core of Alba proteins consists of four beta strands (β14) forming a sheet through mostly hydrophobic interactions. Alpha helices α1 and α2 are roughly coplanar and point in generally the same direction as β14. In addition to the Alba fold, Rpp25 contains an additional short beta strand (β5) that lies coplanar to β14 and is present in yeast Pop6 as well. Disordered N- and C-terminal and internal loop regions in the crystal structure are shown as dotted lines in the secondary structure diagram [35].
Figure 3
Figure 3. Superposition of human Rpp20/Rpp25 and yeast MRP P3 RNA bound Pop6/Pop7
The superposition of the human Rpp20/Rpp25 heterodimer structure on the yeast MRP P3 RNA bound Pop6/Pop7 structure (PDB ID: 3IAB) [21] reveals minimal overall conformational differences between the RNA free and RNA bound structures of the homologous heterodimers. Despite exhibiting relatively low sequence similarity, Rpp20 and Pop7, as well as Rpp25 and Pop6, exhibit significant homology at the tertiary level. Notable structural differences between the two homologous pairs occur in terminal regions that do not facilitate protein-protein or RNA-RNA interactions [9], whereas the heterodimerization interface and RNA binding surface are both conserved. Regardless of whether RNA is present, the regions that make up the electropositive RNA binding surfaces align closely, even though there is divergence between the two surfaces at the sequence level. The long antiparallel beta strands β3 and β4 of all four polypeptides shown here are disordered to different extents in both crystal structures and represent one of the more flexible, but still conserved regions of these proteins. (a) Orthogonal views of the superposed structures and protein-RNA interaction. (b) End-on views of the superposed structures show that the P3 stem loop binds primarily to proximal regions of Rpp20/Pop7 and Rpp25/Pop6 and does not make contacts with distal regions of the beta strands β3 and β4 of either polypeptide.
Figure 4
Figure 4. Comparison of the heterodimerization interface of the human Rpp20/Rpp25 heterodimer, yeast Pop6/Pop7 heterodimer, an S. solfataricus Alba1/Alba2 heterodimer, and an S. solfataricus Alba1 homodimer reveals a conserved mechanism of association between their respective subunits
Although each protein pair associate in a generally similar fashion (top), a notable difference between the eukaryotic heterodimers (Rpp20/Rpp25 and Pop6/Pop7) and their archaeal counterparts is the relative orientation of the subunits. We quantified this difference by calculating the angle between vectors through the helical axis of α2 of each subunit, which we took to represent the general direction of each subunit given the apparent rigidity of the core of each protein. Whereas such a measure of inter-subunit orientation resulted in 106° and 101° for Rpp20/Rpp25 and Pop6/Pop7, respectively, the angles between the archaeal Alba heterodimer and homodimer were more obtuse at 128° and 126°, respectively. The structural basis for this variation in subunit orientation appears to originate from differences in the distribution of additional electrostatic and van der Waals interactions flanking the central hydrophobic interactions at the heterodimerization interface (top, boxes shaded in grey). Insets show a close-up view of the heterodimerization interfaces. Every heterodimerization interface consists of an extensive network of hydrophobic residues (colored in yellow) that are in turn flanked by additional electrostatic and van der Waals interactions (by side chains shown in stick representation). Together, these interactions enable the formation of a stable homo/heterodimer, consistent with other observations [9, 14, 15, 41]. Between the subunits of Rpp20/Rpp25 and Pop6/Pop7, the peripheral protein-protein interactions are fewer in number and less symmetrically distributed along the long edge of each subunit. In contrast, the corresponding peripheral protein-protein interactions between the archaeal proteins are far more extensive and more evenly distributed along the long edge of each subunit, thus creating a larger interaction surface and a more obtuse angle of association. The atomic coordinates of Pop6/Pop7, Alba1/Alba2, and Alba1/Alba1 correspond to PDB ID: 3IAB [21], 2BKY [41], and 1H0X [44], respectively.
Figure 5
Figure 5. Differences in the nucleic acid binding surfaces of the human Rpp20/Rpp25 heterodimer, yeast Pop6/Pop7 heterodimer, an S. shibitae Alba1/Alba1 heterodimer, and an A. pernix Alba2 homodimer
Electrostatic potential of the nucleic acid binding surface of (a) human Rpp20/Rpp25, (b) yeast Pop6/Pop7 (PDB ID: 3IAB) [21], (c) an archaeal homodimer of Alba2/Alba2 from A. pernix (PDB ID: 3U6Y) [42], and (d) an archaeal homodimer of Alba1/Alba1 from Sulfolobus shibatae (PDB ID: 3WBM) [56] calculated using APBS [36-38] and rendered with a range of +/- 8 kT/e. Whereas the crystal structure of Rpp20/Rpp25 was solved in the absence of nucleic acid, the crystal structures of Pop6/Pop7 [21, 42], A. pernix Alba2/Alba2 [42], and S. shibatae Alba1/Alba1 [56] were solved in complex with RNA, DNA, and RNA, respectively. The two archaeal Alba homodimers are purposefully shown here as representative examples to compare a DNA binding Alba dimer and a helical RNA binding Alba dimer. Rpp20/Rpp25 and Pop6/Pop7 contain a relatively curved and elongated electropositive RNA binding surface (represented by an arrow drawn in a lightly shaded boxed region), which bind to less structured, single-stranded RNAs rather than helical elements. In contrast, and in spite of the type of nucleic acid to which they bind, both archaeal Alba homodimers exhibit a more compact and flatter electropositive surface, which facilitates binding to nucleic acid helices. The distal end of β34 of each polypeptide is labeled to provide landmarks for the orientations shown of each dimer. The yellow shaded regions are drawn according to where the proteins contact RNA or DNA in the respective crystal structures, whereas the white shaded region depicted on Rpp20/Rpp25 is extrapolated from the crystal structure of Pop6/Pop7 bound to P3 RNA (PDB ID: 3IAB) [21].

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