Analysis of flavivirus NS5 methyltransferase cap binding

Abstract

The flavivirus 2'-O-nucleoside N-terminal RNA methyltransferase (MTase) enzyme is responsible for methylating the viral RNA cap structure. To increase our understanding of the mechanism of viral RNA cap binding we performed a detailed structural and biochemical characterization of the guanosine cap-binding pocket of the dengue (DEN) and yellow fever (YF) virus MTase enzymes. We solved an improved 2.1 A resolution crystal structure of DEN2 Mtase, new 1.5 A resolution crystal structures of the YF virus MTase domain in apo form, and a new 1.45 A structure in complex with guanosine triphosphate and RNA cap analog. Our structures clarify the previously reported DEN MTase structure, suggest novel protein-cap interactions, and provide a detailed view of guanine specificity. Furthermore, the structures of the DEN and YF proteins are essentially identical, indicating a large degree of structural conservation amongst the flavivirus MTases. Guanosine triphosphate analog competition assays and mutagenesis analysis, performed to analyze the biochemical characteristics of cap binding, determined that the major interaction points are (i) guanine ring via pi-pi stacking with Phe24, N1 hydrogen interaction with the Leu19 backbone carbonyl via a water bridge, and C2 amine interaction with Leu16 and Leu19 backbone carbonyls; (ii) ribose 2' hydroxyl interaction with Lys13 and Asn17; and (iii) alpha-phosphate interactions with Lys28 and Ser215. Based on our mutational and analog studies, the guanine ring and alpha-phosphate interactions provide most of the energy for cap binding, while the combination of the water bridge between the guanine N1 and Leu19 carbonyl and the hydrogen bonds between the C2 amine and Leu16/Leu19 carbonyl groups provide for specific guanine recognition. A detailed model of how the flavivirus MTase protein binds RNA cap structures is presented.

Figure 1. Structural conservation of flavivirus NS5 MTase domain

a) Surface representation of the yellow fever virus NS5 MTase domain showing 2F_o − F_c electron density maps contoured at 1.9σ for the Ado-Hcy coproduct that co-purified with the protein (lower left), the soaked in GTP molecule (right), and the secondary phosphate binding site (upper left). b) Maximum likelihood multiple structure superposition of the dengue virus (magenta) and yellow fever virus (yellow) MTase domains. The Ado-Hcy coproduct and the phenylalanine residue in the GTP binding site are shown in sticks. Structural differences are largely limited to small movements of the helix adjacent to the GTP binding site (•).

Figure 2. Structural details of the cap binding site and ligand complexes in the citrate (a–c) and MPD (d–f) crystal forms

2F_o − F_c maps contoured at 1.8σ are shown for the apo (a,d) and the bound GTP ligand (b,e) structures. Composite 2500K simulated-anneal omit maps are shown for the non-methylated GpppA cap analog in the citrate crystal form (c) and for the N7 methylated GpppA cap analog in the MPD crystal form (f). The view in panel c is from the top of the GpppA binding site to show the stacking interactions involving Phe 24 and the guanosine and adenosine from the cap analog. “W” denotes the bridging water molecule. Residues described in the text are labeled in panels a and e.

Figure 3. Determination of Dengue, West Nile, and Yellow fever virus MTase GTP-Bodipy binding characteristics

A) Determination of K_d for GTP-B binding to Dengue, Yellow Fever, and West Nile virus MTase proteins. B) Determination of GTP K_d as a function of GTP-Bodipy displacement C) Comparison of DEN MTase K_d for GTP-Bodipy and ATP-Bodipy. Curve fits and K_d values were determined with the KaleidaGraph software package as described in the Data Analysis section. D) Determination of GTP and ATP K_i values. GTP-Bodipy displacement assays were performed as described in the text.

Figure 4. Conservation of binding site residues amongst flavivirus family members

Black background denotes non-similar substitutions, and the shaded rectangles bring attention to residues where increased variability is observed. Numbering for YF is noted and is used throughout the manuscript to describe all MTase residues. Residues with side chains within hydrogen bonding distance of GTP are marked (*). Residues with backbone carbonyl groups interacting with the conserved water and the guanine ring are marked (@).

Figure 5. Flatland model for interactions between flavivirus MTase cap-binding pocket and the GTP cap structure

A line structure of GTP is surrounded by MTase residues is presented, with types of interactions and relative strengths indicated within the figure. Numbering for the residues is representative of the YF MTase.