Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly

Abstract

Biogenesis of the 12-subunit RNA polymerase II (Pol II) transcription complex requires so-called GPN-loop GTPases, but the function of these enzymes is unknown. Here we report the first crystal structure of a eukaryotic GPN-loop GTPase, the Saccharomyces cerevisiae enzyme Npa3 (a homolog of human GPN1, also called RPAP4, XAB1, and MBDin), and analyze its catalytic mechanism. The enzyme was trapped in a GDP-bound closed conformation and in a novel GTP analog-bound open conformation displaying a conserved hydrophobic pocket distant from the active site. We show that Npa3 has chaperone activity and interacts with hydrophobic peptide regions of Pol II subunits that form interfaces in the assembled Pol II complex. Biochemical results are consistent with a model that the hydrophobic pocket binds peptides and that this can allosterically stimulate GTPase activity and subsequent peptide release. These results suggest that GPN-loop GTPases are assembly chaperones for Pol II and other protein complexes.

FIG 1

Crystal structures of Npa3 in GDP-bound (closed) and GMPPCP-bound (open) forms. (A) Schematic representation of yeast (S. cerevisiae [S.c.]) Npa3 domain organization. The color code is used throughout the figures. (B) Amino acid sequence alignment of Npa3 from S. cerevisiae, with eukaryotic homologs from Schizosaccharomyces pombe (S.p.) and GPN1 from Homo sapiens (H.s.) and the archaeal homolog Pab0955 from Pyrococcus abyssi (P.a.). Secondary structure elements are indicated above the sequence (cylinders, α-helices; arrows, β-strands). Amino acid numbering above the sequence corresponds to Npa3 from S. cerevisiae. Motifs G1 to G5 and insertions 1 and 2 are marked with bars. (C) Ribbon representations of the closed (GDP-bound) (left) and open (GMPPCP-bound) (right) conformations. The G motifs and insertion regions are colored as described above for panel A. A fatty acid bound to the hydrophobic pocket that is opened in the Npa3-GMPPCP structure is shown as slate blue sticks. Missing residues are indicated with dashed lines.

FIG 2

Superposition of closed Npa3-GDP and open Npa3-GMPPCP structures. Magnesium ions are shown as blue (Npa3-GDP) or gray (Npa3-GMPPCP) spheres, water molecules are shown as red spheres, and hydrogen bonds are shown as dashed lines. (A) Peptide flip of D189 enables pocket opening and formation of a single helix from helices α8 and α9. (B) Conformational changes in insertion 2 and helix α7 facilitate the opening of an extended, hydrophobic pocket. (C) A set of residues in insertion 1 rearranges, including the GPN loop and a DIRD motif, leading to increased flexibility of this region in the GMPPCP-bound state. (D) Pocket opening allosterically alters the active site via the G4 motif. (E) Conformational changes in helix α7 are linked to the G3 motif and the GPN loop.

FIG 3

Nucleotide-binding pocket and active site. (A) Initial unbiased F_o-F_c difference electron density of GMPPCP and the magnesium ion, including coordinated water molecules contoured at 3σ (green mesh). The final Npa3-GMPPCP model is superimposed. Motifs G1 to G5 are color-coded as described in the legend to Fig. 1A. Water molecules are shown as red spheres, magnesium ions are shown as pink spheres, and hydrogen bonds are shown as dashed lines. (B and C) Nucleotide interaction network of Npa3 with GMPPCP (B) and GDP (C). Metal ion-ligand interactions are shown as solid black lines.

FIG 4

Catalytic mechanism. (A) GTPase activity of Npa3 mutants. Bars represent free orthophosphate concentrations after 40 min at 37°C. Kinetics are shown for wild-type Npa3 (Npa3wt) (gray) and the crystallized variant Npa3ΔCΔLoop (blue). (B) Schematic of the mechanism of GTP hydrolysis. The active site of Npa3-GMPPCP is shown. The GPN loop of monomer B is modeled by superpositioning of two Npa3-GMPPCP enzymes on the archaeal Pab0955 dimer (PDB accession number 1YRB) (18). The nucleophilic water (N) that attacks the γ-phosphate and the buttressing water (B) are superimposed from the Npa3-GDP structure. Hydrogen bonds are shown as dashed black lines, potential hydrogen bonds derived from dimer modeling are shown as dashed gray lines, water molecules are shown as red spheres, the magnesium ion is shown as a pink sphere, and metal ion-ligand interactions are shown as solid black lines.

FIG 5

Putative peptide-binding pocket and GTPase-stimulating chaperone activity. (A) Initial unbiased F_o-F_c difference electron density for lauric acid (slate blue sticks) bound to the putative peptide-binding pocket of Npa3, contoured at 2σ (green mesh). (B) Highly conserved surface of the putative peptide-binding pocket. Invariant residues are in green, conserved residues are in yellow, and variable residues are in gray. Insertion regions and G motifs are depicted. Lauric acid is shown as blue spheres. (C) Npa3 has chaperone-like activity in vitro. Npa3 suppresses thermally induced (43°C) aggregation of the general nonnative chaperone substrate protein citrate synthase (CS). Different amounts of wild-type Npa3 were added, as indicated on the right. (D) Chaperone-like activity of Npa3 is independent of added nucleotides under limiting conditions. A total of 1 mM GMPPCP, GTP, or GDP was added, and relative CS aggregation in the presence of 0.5× molar amounts of Npa3 was determined after 30 min. (E) GTPase activity of Npa3 is stimulated >4-fold in the presence of the nonnative chaperone-substrate protein citrate synthase. Free orthophosphate concentrations were determined after 40 min at 43°C (see Materials and Methods).

FIG 6

Npa3 binds Pol II-derived peptides located at subunit interfaces. (A) Box plot representation of a representative portion of the heat map describing the Npa3 peptide-binding landscape. Tested were a total of 1,139 15-mer Pol II-derived peptides, covering the complete sequence of the 12-subunit Pol II in the presence of GMPPCP, GTP, and GDP. Control experiments were performed without Npa3 and nucleotides to test the cross-reactivity of the anti-His antibody. Intensity distribution is shown on a logarithmic scale. Peptides with a signal intensity of <3.5 were defined as unbound peptides (gray area). (B) Location of Npa3-binding peptides in the assembled Pol II complex (PDB accession number 1WCM) (32). Npa3 binding to Pol II peptides is depicted in yellow (signal intensity of 3.5 to 3.75), orange (3.75 to 4), and red (>4), whereas unbound regions are in gray (<3.5). A schematic representation of the 12 Pol II subunits Rpb1 to Rpb12 in the folded Pol II complex is shown on the right. (C and D) Surface representations of Pol II subunits showing that Npa3 interacts with hydrophobic Pol II-derived peptides located at subunit interfaces (left). Peptides in Pol II subunit surfaces involved in interactions with other subunits are colored as described above for panel B. Numbers correspond to the peptide numbers from the array (see Table S1 in the supplemental material for a list of all peptides, Table S2 in the supplemental material for a list of all peptides that interact with Npa3, and Fig. S4 in the supplemental material for original peptide interaction analysis) and are color-coded according to the subunit from which they are derived, as shown on the left and in the schematic of Pol II subunit organization in panel B. Npa3-bound peptides from other subunits that interact with Rpb1 are shown as sticks. Residues involved in subunit interfaces are shown in black (middle). White solid lines highlight hydrophobic interface regions, bound by Npa3 (right). Dashed lines indicate where other Pol II subunits are positioned in the assembled complex.

FIG 7

Model for Pol II biogenesis. Whereas the “Npa3 cycle” drives cytoplasmic assembly of Pol II, the “Iwr1 cycle” drives Pol II nuclear import. In the Npa3 cycle, pocket opening of Npa3·GDP is induced by binding of hydrophobic regions of Pol II subunits that form interfaces in the assembled Pol II complex, thereby preventing misassembly (step 1). Pocket opening allosterically communicates with the active site, stimulates GDP displacement, and thereby facilitates GTP rebinding (step 2). GTP hydrolysis leads to the release of Pol II peptides, facilitating the formation of Pol II subunit interfaces and the assembly of Pol II in the cytoplasm (step 3). In the Iwr1 cycle, assembled Pol II is recognized by Iwr1, which provides an import adaptor for nuclear import via its nuclear localization sequence (NLS). Iwr1 is recycled with the use of its nuclear export signal (NES).