In this study we performed a detailed biochemical and structural analysis of the guanosine cap binding characteristics of the DEN and YF virus NS5 MTase domains. We present new high-resolution structures of the YF MTase domain complexed with GTP and cap analog, a higher resolution DEN MTase structure, and define many of the critical protein and GTP components of guanosine cap binding. Based on the combination of our structural and biochemical analysis, we present a detailed scheme for the binding of the guanosine triphosphate portion of the cap structure to the flavivirus MTase enzyme (). The relative binding affinities for most of the critical interactions observed in the structures are shown. Our data provides the first highly detailed description of the correlates of guanosine cap binding by the flavivirus MTase enzymes. Our studies have indicated a much higher affinity for guanosine cap binding than had previously been recognized, identified several new protein-cap interactions, and demonstrate a detailed mechanism of guanine recognition.
Flatland model for interactions between flavivirus MTase cap-binding pocket and the GTP cap structure
Using our new fluorescence polarization based assay, we determined that the Kd
of the flavivirus MTase for GTP-Bodipy is approximately 100 nM, and that the Ki
for both GTP and cap analog is in a similar range. The Kd
value of the flavivirus MTase domain for cap analog is somewhat higher that the 2 nM cap-binding affinity of the cellular cap-binding eIF4E protein for cap analog as determined by stopped-flow fluorescence titration experiments 24
. However, eIF4E stacks the cap guanine ring between two tryptophane residues, which likely provides more stabilization that the single phenylalanine residue in the flavivirus MTase and accounts for the increased affinity observed with eIF4E. In light of the high affinity of eIF4E for guanosine cap, the 100 nM Kd
value determined using our assay seems more reasonable than the 58 μM Kd
that was previously determined 5
We performed a comprehensive mutational analysis of the DEN MTase enzyme to determine the contribution of individual amino acids to guanosine cap binding. shows residues within a 10 Å sphere of the cap binding pocket. Conserved residues within hydrogen bonding distance of GTP in our crystal structures noted and were tested for their contribution to GTP interaction. Phe24 and Lys28 have been previously suggested as major interaction points 5; 20
. By far the most dramatic effect was seen with the Phe24Ala mutant, indicating that π–π interaction between the guanosine base and the phenylalanine side chain is essential for binding. However, mutation of Phe24 to Tyr did not significantly alter binding. The Phe residue is invariant across the flavivirus family, even though a single uracil to adenine transversion mutation in the second position of the codon would change the residue from Phe to Tyr. This suggests that the Phe residue is functionally conserved, but there may be an additional role for the residue outside of cap binding that requires the residue to remain phenylalanine rather than a tyrosine. Experiments mutating Phe24 to Tyr in infectious virus assays may shed light on why this residue is highly conserved.
Ser215 appears to coordinate the cap α-phosphate in combination with Lys28. The combination of Lys28 and Ser215 interacting with the α-phosphate appears to provide a great deal of stability to the bound cap, as the α-phosphate is well resolved in our structures whereas the β and γ phosphates are not. Mutational and analog competition analyses support this observation, as the Lys28Ala mutation binds GTP-Bodipy weakly and guanosine is a very poor competitor. We also observe a minor effect on binding when we mutate Arg213, even though there is only very weak electron density for the residue. This residue appears structurally poised to influence γ-phosphate binding. Arg213 is also in a region that could be involved in interaction with the 5′ adenosine ring structure of the first viral nucleotide. The position of the viral RNA or 5′ RNA adenosine has not been observed as of this report, but we have performed preliminary computer modeling of a capped viral RNA into the YF MTase structure and observed that the molecular distances are possible for this interaction. Additionally, if the 5′ adenosine base is stabilized between Arg213 and Arg56, this would position the ribose 2′ hydroxyl near AdoMet, increasing the efficiency and specificity of the 2′-O-methyltransferase reaction.
We observed electron density in the positively charged region across from the cap binding site in structures soaked with GTP () or cap analog (data not shown). We were able to model a triphosphate from GTP into the resolved density, although density for the ribose and guanine on the GTP was not observed. This density could represent a binding site for the triphosphate between the first and second nucleotides in the genomic RNA, with the adenosine residue bound somewhere on the face of the MTase between the AdoHcy and GTP binding sites. Consistent with this, the Arg213/Arg56 pocket at the back of this face could provide hydrogen bonding interactions with the adenosine base. Alternatively, this second phosphate binding site could be involved in binding the cap guanosine residue for the guanine N7 methylation reaction because we can fairly easily place the guanosine N7 atom within a few ångstroms of the Ado-Hcy sulfur after modeling a triphosphate into the observed density. As the methylation of the flavivirus cap occurs sequentially with the guanine N7 being methylated before the ribose 2′ hydroxyl, the observed triphosphate density could indicate the path for the first methylation event.
The α-phosphate is a major interaction point between the protein and cap structure. One possibility for the strength of this α-phosphate: Lys28: Ser215 interaction is that it may represent the as yet unidentified guanylyltransferase activity. Egloff et al. previously showed that α-32
P GTP can be covalently crosslinked to DEN MTase in a Mg2+
independent manner 5; 20
, which could represent the protein-GMP adduct. Such a role may explain the strength of interaction that is observed between the α-phosphate and the protein, whereas the β- and γ-phosphates appear more weakly associated with the protein. Additionally, in high ionic strength crystallization conditions (1.6M sodium citrate () or 0.4M ammonium sulfate/1.2 M lithium sulfate 20
) the cap analog appears to fold back on itself, leaving the α-phosphate exposed for nucleophilic attack or adduct formation. This orientation has been suggested to be involved in guanylyltransferase activity 20
, and Lys28 appears to be well positioned to act as a nucleophile. Non-UV based α-32
P-GTP labeling experiments examining the formation of DEN MTase-GMP adducts using established guanylyltransferase assays 25
did not detect adduct formation with either the wildtype or K28A DEN MTase proteins (Aaron Shatkin, personal communication). Additionally, the stacked orientation mentioned above was not observed in our lower strength ionic MPD conditions with the same crystal packing lattice () or that of the MVEV MTase 12
, raising questions about the physiological relevance of this stacked conformation. Further studies are needed to determine if the stacked conformation indeed plays a role in flavivirus guanylyltransferase activity.
The tight cap binding by MTase (Kd
~100 nM) may represent a mechanism to sequester the RNA cap structure from cellular eIF4E or ribosomes until the positive-strand genomic RNA has become long enough to prevent polyprotein translation from interfering with continued RNA replication. This model would require the guanosine cap to be released from its binding site either by a directed release mechanism (i.e. conformational change) or by folding of an RNA structure that triggers the release of the cap from the MTase. Flavivirus MTase specifically binds GTP (or cap), but binds the structurally similar ATP very poorly. It was initially postulated that the mechanism for guanine specificity lay in the interaction of the guanine C2 amine group with the backbone carbonyls of Leu16 and Leu19 5
. While this interaction does provide a small contribution to binding, our analog studies show that the C2 moiety is not the sole factor in guanine specificity. The observation that ITP, which lacks the C2 amine group but possess a hydrogen bond capable hydrogen at N1, can strongly compete with GTP-Bodipy binding indicates that there is at least a second contact between the guanine ring and the MTase. Were the C2 amine position the only selective interaction point, ITP would bind as weakly as ATP. We originally hypothesized that the Lys21 sidechain could interact with the C6 carbonyl group on guanine. However, our mutational analysis indicated that the Lys21 sidechain does not play a role in the observed specificity. Surprisingly, a structurally conserved water molecule is found between the guanine N1 hydrogen and the Leu19 backbone carbonyl in each of our GTP/cap soaked structures derived from completely different crystallization conditions. The presence of this bridging water molecule, which is within hydrogen bonding distance of the guanine N1 hydrogen and the Leu19 backbone carbonyl oxygen, was not observed in previously published lower resolution MTase-GTP structures 5; 9; 10; 11; 12; 13; 14
, but is present in a 1.8 Å Den MTase-cap analog structure (PDB Code 2P41) 10
. The presence of the water molecule in three different crystallization conditions with two different flavivirus MTase proteins suggests that the coordinated water is not a crystallization artifact and appears to represent an important interaction. The combination of the N1 water-bridge and C2 amine interactions with MTase backbone interactions appear to contribute almost equal amounts of binding energy based on our competition data, and the combination of the two interactions appear to confer specific recognition of guanine largely independent of side-chain composition. These two interactions increase our understanding of guanine binding by flavivirus MTase proteins. This recognition mode is considerably different from that observed in other RNA cap binding proteins (vaccinia VP39, cellular CBP20 and eIF4E) which bind guanosine caps between two aromatic sidechains 24; 26; 27
. Water-mediated nucleoside recognition has been reported previously with calf purine-nucleoside phosphorylase 28
, although in that case the water interaction was observed between the C6 carbonyl and an Asn sidechain.
The results of the experiments described here further our understanding of the architecture of the MTase guanosine cap-binding site. The flavivirus MTase is a new and potentially valuable antiviral target to develop drugs against flavivirus replication. Drugs that interfere with influenza virus RNA cap binding have been previously described 29
, indicating that viral cap binding is a viable drug target. Our increased understanding of how the flavivirus MTase binds RNA cap structures will aid in the rational development of highly potent and specific drugs for the treatment of flavivirus infections.