X-ray structure and activities of an essential Mononegavirales L-protein domain

Abstract

The L protein of mononegaviruses harbours all catalytic activities for genome replication and transcription. It contains six conserved domains (CR-I to -VI; Fig. 1a). CR-III has been linked to polymerase and polyadenylation activity, CR-V to mRNA capping and CR-VI to cap methylation. However, how these activities are choreographed is poorly understood. Here we present the 2.2-Å X-ray structure and activities of CR-VI+, a portion of human Metapneumovirus L consisting of CR-VI and the poorly conserved region at its C terminus, the +domain. The CR-VI domain has a methyltransferase fold, which besides the typical S-adenosylmethionine-binding site ((SAM)P) also contains a novel pocket ((NS)P) that can accommodate a nucleoside. CR-VI lacks an obvious cap-binding site, and the (SAM)P-adjoining site holding the nucleotides undergoing methylation ((SUB)P) is unusually narrow because of the overhanging +domain. CR-VI+ sequentially methylates caps at their 2'O and N7 positions, and also displays nucleotide triphosphatase activity.

Figure 1. The structure of the MTase domain of hMPV L.

(a) Domain organization of hMPV L, with at its C terminus the 46.5 kDa CR-VI+ domain (residues 1,599–2,005), comprising CR-VI (green), which contains the K-D-K-E motif typical for 2′O-MTases, and the +domain (red), carrying the K-K-G motif (blue). Boundaries of CR-I to -V are approximate. CR-III contains the G-D-N-Q signature motif for polymerase (RdRP) activity, and CR-V contains the HR motif for PRNTase activity. (b) Cartoon representation of the CR-VI+ crystal structure, from amino (N) to carboxy (C) terminus (no structure could be assigned to the first ∼18 residues). The +domain is shown in red, with K₁₉₉₁ and K₁₉₉₅ of the K-K-G motif in stick format. The CR-VI (MTase) domain is coloured purple (β-strands) and green (helices and loops), except for ^β1λ, ^β2λ and ^β4λ (the loop regions C terminal of β-strands 1, 2 and 4 that form ^SAMP; orange), and λ_1650–1666 (which disengages itself from the main CR-VI-fold to interact with the +domain; yellow). Nomenclature of helices and strands follows that used for other MTases (c). The pale-blue sticks show the K-D-K-E motif. A Zn-ion (silver sphere) is co-ordinated by H₁₇₆₆, H₁₇₉₈, C₁₈₀₂ and C₁₈₀₅. (c) Schematic representation of the secondary structure of a prototypical SAM-dependent MTase (top) and of the hMPV CR-VI domain (bottom). Helices are in green, strands in light purple and coils in blue, except for ^β1λ, ^β2λ and ^β4λ (orange) and λ_1650–1666 (yellow). CR-VI displays some deviations from the prototypical SAM-MTase fold, some of which it shares with other RNA-MTases, including the long N-terminal coil, a longer αD and an extra helix (αX) at the C terminus. αE is absent, whereas, atypical for viral MTases, αB is fully formed. CR-VI, moreover, has an unusual β-sheet; it lacks β3, but this is compensated for by the addition, at the other end of the sheet, of a new strand (β0), which glues the start of the N-terminal coil to the main structure. Also unusual is the fragmentation of αZ (resulting in the small z′-helix).

Figure 2. MTase activity of CR-VI+.

(a). The transfer of tritiated methyl groups from SAM molecules to a capped RNA substrate (GpppGGGACAAGU), containing the consensus start sequence of hMPV transcripts (in red), was monitored over time. The rather slow in vitro methylation suggests the reaction is impaired compared with the in vivo activity of intact L, regions of which may aid the methyl transfer (for example, by correctly positioning substrate RNAs to the MTase; see main text). The bars and error bars correspond to the mean values from three independent measurements and their s.d.’s, respectively. (b). Substrate specificity was determined as above, but using various synthetic RNA substrates, and allowing the reactions to proceed for 16 h. Substrates were compared with GpppGGGACAAGU (red and blue panels) and pppGGGACAAGU (green panel), for which the degree of methylation was set at 100% (#, marked bars). The red-shaded panel compares the degree of methylation of nine-nucleotide-long hMPV start sequences with different 5′-ends and methylation states (the lighter bars represent uncapped RNAs). The results indicate efficient methylation of RNAs that already carried a (cold) methyl group, either at their N7-guanine or 2′O-ribose position (confirming the occurrence of 2′O- and N7-methylation, respectively), and of uncapped RNAs (especially pppRNA). The pppRNA substrate is predominantly methylated at N₁, as substrates that were methylated beforehand at this nucleotide do not undergo substantial additional methylation (green panel). CR-VI+ prefers the hMPV start sequence over the short GpppACCCC sequence, and over RNA-start sequences of Dengue virus and SARS coronavirus, irrespective of their lengths (blue panel). A nine-nucleotide hMPV substrate, however, is much preferred over one with only five nucleotides, indicating that additional interactions take place between the protein and nucleotides 6–9. Consistently a 10-times lower K_D (dissociation constant) characterizes the interaction of CR-VI+ with the 9-mer, compared with that with the 5-mer. The K_Ds, measured in triplicate using a dot-blot assay and listed at the right of the diagram (±s.d.’s), also show that capped and uncapped hMPV sequences are bound with comparable affinities, and that 2′O-methylated substrates are preferred over unmethylated ones.

Figure 3. N7- and 2′O-methylation.

(a) Thin-layer chromatograms. Following CR-VI+-mediated methyl transfer from SAM onto GpppGGGACAAGU (in which G was [³²P]-labelled), nucleotides 2–9 were removed by nuclease P1 digestion, and the caps were separated by thin-layer chromatography (TLC). The controls (GpppG, GpppG_m, ^mGpppG and ^mGpppG_m, from left to right) were obtained with the same substrate, using MTases that specifically methylate caps at the N7 or 2′O positions (human N7- and vaccinia virus 2′O-MTase). The TLC experiment used 0.65 M LiCl as mobile phase, allowing a clear separation of ^mGpppG and ^mGpppG_m (top). The caps on the TLC plate were subsequently further resolved, this time using 0.45 M (NH₂)₂SO₄ as mobile phase for a better separation of GpppG and GpppG_m (bottom). GpppG_m appears first (after a 1-h incubation), ^mGpppG_m becomes prominent at a later stage, and ^mGpppG was not observed, indicating that 2′O-methylation of N₁ precedes N7-methylation of G. (b). The effect of point mutations on the MTase activities of CR-VI+, measured after a 16-h incubation period (by means of a filter-binding assay, as in Fig. 2), using GpppGGGACAAGU substrates that were methylated beforehand at N7 of G or 2′O of N1, to specifically monitor 2′O or N7-MTase activities, respectively. Mutants are listed against a yellow, green or red background, to indicate that the altered residue belongs to λ_1650–1666, the rest of the CR-VI domain, or the +domain, respectively. They are also grouped according to whether they change the K-D-K-E tetrad, ^SAMP, ^SUBP or ^NSP. The results highlight the importance of essential ^SUBP residues (such as the K-K-G lysines K₁₉₉₁, K₁₉₉₂ and K₁₉₉₅ and λ_1650–1666 residues H₁₆₅₉ and R₁₆₆₂) for both 2′O- and N7-methylation. All tetrad residues are crucial for 2′O-methylation, while D₁₇₇₉ in particular is important for N7-MTase activity. The bars and error bars correspond to the mean values from three independent measurements and their s.d.’s, respectively.

Figure 4. CR-VI+-binding pockets.

The cartoon representations show the +domain in red and CR-VI in green, with λ_1650–1666 in yellow. SAM (in ^SAMP), GTP (in ^SUBP) and adenosine (ADN; in ^NSP) are shown as sticks, with the C atoms coloured gold, slate and magenta, respectively. Hydrogens (in white) accentuate the methyl group of SAM. 2Fo-Fc electron density maps around the ligands are represented in grey mesh (contoured at 1σ). (a) The relative positions of the pockets in the protein. (b) Close-up of ^SUBP, which is defined by residues of the +domain (particularly the K-K-G motif), λ_1650–1666 and the CR-VI domain. Residues involved in ligand binding are shown as sticks. GTP is fitted in different orientations into the density in the PDB 4UCZ structure (main figure, and top figure to the right, where the guanosine ring is turned 180°) and in the PDB 4UCI structure (bottom right, where the ligand lays in the opposite direction), highlighting that the ligand can bind in different orientations within the spacious pocket. (c) ^SAMP and ^NSP containing a SAM and ADN ligand, respectively (PDB 4UCI, in which ^SUBP is also occupied). Residues lining the pockets are shown as sticks. The loops delineating ^SAMP (^β1λ, ^β2λ and ^β4λ) and the β-strands they originate from are shown in magenta. The dashed yellow lines show putative hydrogen bonds. (d). Superposition of three other CR-VI+ structures onto that in c, highlighting the flexibility of ^β1λ (especially E₁₆₉₇), ^β2λ and ^β4λ. The structure in blue (PDB 4UCK) contains SAM, whereas those in yellow (4UCL) and aquamarine (4UCJ) have empty ^SAMPs (this suggests that there is no strict correlation between ^SAMP occupancy and the position of ^β2λ). ^NSP is empty in the three superposed structures, which apparently affects the position of their ^β4λ loops and especially of the R₁₇₈₅ side group, which closes the pocket when occupied. All overlaid structures have empty ^SUBPs.

Figure 5. Comparison of the CR-VI+ domains of hMPV and VSV.

(a). Cartoon representations. The CR-VI domains are similar, and share the unusual, strand-0 containing β-sheet (purple), the rather large ^β2λ—indicated by arrow (1)—and the long N-terminal loop (2), which runs somewhat differently in VSV. The λ_1650–1666 peptide on which the +domain rests (yellow) also has a homologue in VSV. The Zn-finger, however, is not conserved, and α-helices B and Z are not fragmented. Helix αE, an element of the standard MTase topology (Fig. 1c), is present in VSV (3), as a result of which ^NSP may have disappeared. αX is at a different location (4), and is preceded by an extra helix (αx′(5)). E₁₈₃₃, expected to belong to the K-D-K-E tetrad from sequence alignments, is buried in the structure and does not reach the surface of the catalytic pocket (6), and the position normally taken by the K-D-K-E glutamate is occupied by T₁₈₃₁. The +domain of VSV is tilted, compared with that of hMPV, and more elaborate. Helices α⁺1, α⁺2, α⁺3, α⁺5 and α⁺6 are conserved, but the α⁺1–α⁺2 loop is replaced by an extra helix (α⁺1′ (7)). α⁺4 is absent, whereas α⁺5 is enlarged and immediately follows α⁺3 (8). The 2-residue loop connecting α⁺5 and α⁺6 in hMPV is replaced by a 34-residue coil carrying a small three-stranded β-sheet (9). Helix α⁺6 seems best conserved between the two +domains, although a K-K-G motif is not present in VSV. However, R₂₀₃₈, which is strictly conserved in Rhabdoviridae, takes the place of K₁₉₉₅ ((10) and alignment below). In VSV, the +domain is extended beyond α⁺6 with a 65-residue, partly helical, but mainly unstructured polypeptide (in grey (11)). Colour scheme and labelling are as in Fig. 1b. (b). Alignment of α⁺6-helices from Mononegavirales L proteins. K-K-G motif residues are highlighted in red; the arginine replacing the second lysine of the motif in Filoviridae and Rhabdoviridae is highlighted in magenta. Red letters indicate other (less strictly) conserved residues, except for the G that replaces the first lysine of the K-K-G motif in most Rhabdoviridae (blue).

Figure 6. RNA-binding site comparisons.

(a). Comparison of the RNA-binding sites in vaccinia virus cap-MTase (PDB 1AV6) and CR-VI+. The vaccinia virus MTase (white surface, left) has a narrow cap-binding pocket (in between the red arrows) and a large, open RNA-binding site (adjoining the SAH-containing ^SAMP). In CR-VI+ (coloured surface, middle) the cap-binding pocket is not present, whereas the RNA-binding site is narrowed (into ^SUBP) by the +domain overhang (in red). A structural superposition (obtained by aligning the K-D-K-E tetrads, right) shows that the GTP ligand in CR-VI+ is situated at a considerably greater distance from the tetrad than the first transcribed nucleotide (N₁) in the vaccinia virus MTase–RNA complex (shown in light pink). The 2′O atoms of the nucleotides are shown as transparent, red spheres. (b). ^SUBP conservation within the Mononegavirales order. The surface presentation on the left shows the basic (blue) and acidic (red) charge distribution on CR-VI+. The ligands are in yellow. The other cartoons show the surface of the hMPV CR-VI+ domain in the same orientation, but in white. Residues that are conserved in the hRSV, Measles, Ebola or Rabies virus homologues of CR-VI+ are coloured dark red (identical residues) or pink (similar residues), and cluster around ^SUBP and ^SAMP.

Figure 7. NTPase activity.

Autoradiographs of urea–PAGE gels show CR-VI+-mediated conversion of radiolabelled GTP to GDP and ATP to ADP, over time. The smaller autoradiograph shows the requirement of Mg⁺⁺ for the reaction (allowed to proceed for 1 h), and also shows efficient GDP generation by washed, dissolved CR-VI+ crystals (CR-VI+*), dispelling the possibility that the activity is due to contaminants. The diagram on the right (obtained by phosphorimage analysis following electrophoresis) further illustrates the requirement of Mg⁺⁺ and shows the effect of the metal-ion chelator EDTA on the GTPase reaction. The bars and error bars correspond to the mean values from three independent measurements and their s.d.’s, respectively.