On 2016 Apr 22, Toby Gibson commented:

Dr. Gack, Thank you for taking the time to comment. One interesting way in which NS3 could potentially regulate 14-3-3 epsilon would be to bind across the top of the groove. Such a binding mode could act as a gatekeeper for phosphopeptide entry/exit into the deep binding pocket. This presupposes that the interaction is direct and strong enough and for this, bacterially expressed proteins need to be assessed in vitro for their dissociation constant.

Incidentally, regarding the work that you are building upon (Liu et al., 2012, PMID:22607805), there was no attempt to identify a candidate motif. But again, RIG-I is also unlikely to have a 14-3-3 phosphopeptide binding mode. RIG-I is almost entirely structured with a few short interdomain linkers. I don’t see good candidates for canonical 14-3-3 binding motifs in RIG-I. So, in this whole system, in my opinion there are no strong clues toward elucidating the molecular details of how 14-3-3 epsilon might be interacting. Assuming that this interaction also validates in vitro, there is some useful structural biology to be done.

On 2016 Apr 19, Michaela Gack commented:

Dr. Gibson’s comment about our manuscript solely stated that the viral RxEP motif identified in the Dengue NS3 protein does not fulfill all of the criteria of conventional 14-3-3-binding motifs found in cellular 14-3-3-interacting proteins. The main conclusions of our study, that the NS3 protein of Dengue virus binds to 14-3-3epsilon and thereby antagonizes RIG-I translocation to mitochondria and type I interferon induction, were never questioned by Dr. Gibson.

Using mapping experiments and site-directed mutagenesis, we identified that the 64-RxEP-67 motif in the Dengue NS3 protein is essential for 14-3-3epsilon binding. Mutation of the central Glu66 (E66) residue to positively-charged Lys66 (K66) markedly reduced the binding of NS3 to 14-3-3epsilon. Additional mutation of Arg64 to Lys64 (termed ‘NS3(KIKP)’ mutant) led to a nearly complete loss of binding to 14-3-3epsilon. These data together with the similarity of the viral RxEP motif with the cellular 14-3-3-binding motif Rxx(pS/pT)xP, where pS/pT indicates a phosphorylated Ser/Thr residue, suggested that the negatively charged E66 serves as a phosphomimetic residue. Although our study indicated that the RxEP motif is required for 14-3-3epsilon binding, our data also suggested that additional as-yet-unidentified residues in the Dengue NS3 protein are important for 14-3-3epsilon interaction, as introduction of the RxEP motif into the NS3 protein of Yellow Fever virus, which is unable to bind 14-3-3epsilon, did not result in gain of 14-3-3epsilon binding. Thus, the full characteristics of the binding mode of Dengue NS3 and 14-3-3epsilon and their binding affinity remain to be determined in future studies, as well as additional residues in NS3 and 14-3-3epsilon that are engaged in the interaction.

On 2016 Apr 08, Toby Gibson commented:

Is there an RxEP motif in Dengue NS3 protein?

This comment addresses the plausibility that the reported RxEP motif in Dengue NS3 is a genuine 14-3-3 binding motif and as such a target for rational design of therapeutics. It does not address other aspects of this published work, in particular that the NS3 protease plays a role in antagonising the cellular relocation and antiviral activity of RIG-I.

Viral mimicry of cellular protein motifs is very common and frequently provides a useful insight into the cellular mechanisms that are being disrupted (Davey et al., 2011). Given our interest in these interactions, I was keen to read this new viral motif paper. Given the current importance of flaviviral infections in human health, it then seemed desirable to post here the explanation as to why RxEP cannot be a phosphomimetic for 14-3-3 proteins. It is my hope that this comment will be of value to the Dengue research field e.g. in how follow up experiments might be planned.

There are several molecular structural issues relevant to 14-3-3 / phosphopeptide interactions:

Proline is disallowed at the +1 position. There is no ambiguity about this. Phosphopeptide arrays probed by (yeast) 14-3-3 clearly show that Pro is disallowed at +1 (Panni et al., 2011). The structural explanation is that there is no physical space for proline because the backbone peptide bond NH group hydrogen bonds to the 14-3-3 protein (residue Asn176 in the epsilon isoform) as part of local geometry that orients the adjacent phosphorylated side chain (See e.g. Fig. 1D, Molzan et al., 2012). Biologically, rejection of proline at +1 is crucial for separate readout of sites phosphorylated by basophilic kinases (PKA, CAMK etc.) vis-a-vis proline-directed kinases (MAPKs, CDKs etc.) as discussed by Panni et al. (2011).

Arginine is typically present at either positions -3 or -4 in many well-studied phosphopeptides binding to 14-3-3 proteins. Presence of Arginine at position -2 is also sometimes observed and structures show that the residue orients to H-bond with the phosphate group. Therefore Arg at the -2 position is not an issue in the present context.

14-3-3 phosphomimetics. We already know that aspartic acid cannot function as a mimetic (de Chiara et al., 2009). This means that the proposed RLDP motif in WNV NS3 can’t work. I am unaware whether a glutamic acid mimetic has previously been evaluated in vitro for its binding affinity. The reason why it may not be possible for any 14-3-3 phosphomimetic is that the PO3- group is complemented by three H-bond donor ligands: in epsilon they are Arg57, Arg130, Tyr131 (Fig. 1D, Molzan et al., 2012). The single negative charge and planar geometry of the carboxyl group rule out an equivalent interaction for glutamate.

The well-studied 14-3-3-binding phosphomotifs are overwhelmingly, perhaps exclusively, found in regions of intrinsically disordered protein (IDP). As the authors note, the motif postulated in NS3 is on the protein surface but is very well folded. Structurally-embedded motif candidates require a high standard of proof. It is essential that the affinity of the interaction between purified NS3 and 14-3-3 be measured in vitro. In our recent motif discovery guidelines paper, we have explained why a short linear motif is never reliably assigned unless in vitro binding assays using purified components have been undertaken in addition to the in-cell experiments (Gibson et al., 2015).

In my view the experiments reported here also do not rule out indirect interaction modes. The most direct experiment, in Fig. 1G, used HEK293T cell extracts for the GST-NS3 bead attachment and this might allow bridging proteins to be complexed with NS3 that can secondarily bind the added 14-3-3.

To recap, the data presented in the paper here indicate that NS3 and 14-3-3 epsilon associate within the same macromolecular complex. In my view, whether the interaction may be direct or indirect has not been unambiguously determined.

I wish to conclude with two straightforwardly testable predictions:

1. A synthetic peptide encompassing the viral RxEP sequence will bind 14-3-3 epsilon with an affinity that is millimolar or weaker when measured by ITC or an equivalent solution-based assay.

2. If NS3 directly and convincingly binds 14-3-3 epsilon at micromolar or nanomolar affinity, when measured by ITC or equivalent, it will do so by an interface that does not involve phosphomimicry.

References

Davey NE, Travé G, Gibson TJ. How viruses hijack cell regulation. Trends Biochem Sci. 2011 Mar;36(3):159-69.

de Chiara C, Menon RP, Strom M, Gibson TJ, Pastore A. Phosphorylation of S776 and 14-3-3 binding modulate ataxin-1 interaction with splicing factors. PLoS One. 2009 Dec 23;4(12):e8372.

Gibson TJ, Dinkel H, Van Roey K, Diella F. Experimental detection of short regulatory motifs in eukaryotic proteins: tips for good practice as well as for bad. Cell Commun Signal. 2015 Nov 18;13:42

Molzan M, Weyand M, Rose R, Ottmann C. Structural insights of the MLF1/14-3-3 interaction. FEBS J. 2012 Feb;279(4):563-71.

Panni S, Montecchi-Palazzi L, Kiemer L, Cabibbo A, Paoluzi S, Santonico E, Landgraf C, Volkmer-Engert R, Bachi A, Castagnoli L, Cesareni G. Combining peptide recognition specificity and context information for the prediction of the 14-3-3-mediated interactome in S. cerevisiae and H. sapiens. Proteomics. 2011 Jan;11(1):128-43.

On 2016 Apr 08, Toby Gibson commented:

Is there an RxEP motif in Dengue NS3 protein?

This comment addresses the plausibility that the reported RxEP motif in Dengue NS3 is a genuine 14-3-3 binding motif and as such a target for rational design of therapeutics. It does not address other aspects of this published work, in particular that the NS3 protease plays a role in antagonising the cellular relocation and antiviral activity of RIG-I.

Viral mimicry of cellular protein motifs is very common and frequently provides a useful insight into the cellular mechanisms that are being disrupted (Davey et al., 2011). Given our interest in these interactions, I was keen to read this new viral motif paper. Given the current importance of flaviviral infections in human health, it then seemed desirable to post here the explanation as to why RxEP cannot be a phosphomimetic for 14-3-3 proteins. It is my hope that this comment will be of value to the Dengue research field e.g. in how follow up experiments might be planned.

There are several molecular structural issues relevant to 14-3-3 / phosphopeptide interactions:

Proline is disallowed at the +1 position. There is no ambiguity about this. Phosphopeptide arrays probed by (yeast) 14-3-3 clearly show that Pro is disallowed at +1 (Panni et al., 2011). The structural explanation is that there is no physical space for proline because the backbone peptide bond NH group hydrogen bonds to the 14-3-3 protein (residue Asn176 in the epsilon isoform) as part of local geometry that orients the adjacent phosphorylated side chain (See e.g. Fig. 1D, Molzan et al., 2012). Biologically, rejection of proline at +1 is crucial for separate readout of sites phosphorylated by basophilic kinases (PKA, CAMK etc.) vis-a-vis proline-directed kinases (MAPKs, CDKs etc.) as discussed by Panni et al. (2011).

Arginine is typically present at either positions -3 or -4 in many well-studied phosphopeptides binding to 14-3-3 proteins. Presence of Arginine at position -2 is also sometimes observed and structures show that the residue orients to H-bond with the phosphate group. Therefore Arg at the -2 position is not an issue in the present context.

14-3-3 phosphomimetics. We already know that aspartic acid cannot function as a mimetic (de Chiara et al., 2009). This means that the proposed RLDP motif in WNV NS3 can’t work. I am unaware whether a glutamic acid mimetic has previously been evaluated in vitro for its binding affinity. The reason why it may not be possible for any 14-3-3 phosphomimetic is that the PO3- group is complemented by three H-bond donor ligands: in epsilon they are Arg57, Arg130, Tyr131 (Fig. 1D, Molzan et al., 2012). The single negative charge and planar geometry of the carboxyl group rule out an equivalent interaction for glutamate.

The well-studied 14-3-3-binding phosphomotifs are overwhelmingly, perhaps exclusively, found in regions of intrinsically disordered protein (IDP). As the authors note, the motif postulated in NS3 is on the protein surface but is very well folded. Structurally-embedded motif candidates require a high standard of proof. It is essential that the affinity of the interaction between purified NS3 and 14-3-3 be measured in vitro. In our recent motif discovery guidelines paper, we have explained why a short linear motif is never reliably assigned unless in vitro binding assays using purified components have been undertaken in addition to the in-cell experiments (Gibson et al., 2015).

In my view the experiments reported here also do not rule out indirect interaction modes. The most direct experiment, in Fig. 1G, used HEK293T cell extracts for the GST-NS3 bead attachment and this might allow bridging proteins to be complexed with NS3 that can secondarily bind the added 14-3-3.

To recap, the data presented in the paper here indicate that NS3 and 14-3-3 epsilon associate within the same macromolecular complex. In my view, whether the interaction may be direct or indirect has not been unambiguously determined.

I wish to conclude with two straightforwardly testable predictions:

1. A synthetic peptide encompassing the viral RxEP sequence will bind 14-3-3 epsilon with an affinity that is millimolar or weaker when measured by ITC or an equivalent solution-based assay.

2. If NS3 directly and convincingly binds 14-3-3 epsilon at micromolar or nanomolar affinity, when measured by ITC or equivalent, it will do so by an interface that does not involve phosphomimicry.

References

Davey NE, Travé G, Gibson TJ. How viruses hijack cell regulation. Trends Biochem Sci. 2011 Mar;36(3):159-69.

de Chiara C, Menon RP, Strom M, Gibson TJ, Pastore A. Phosphorylation of S776 and 14-3-3 binding modulate ataxin-1 interaction with splicing factors. PLoS One. 2009 Dec 23;4(12):e8372.

Gibson TJ, Dinkel H, Van Roey K, Diella F. Experimental detection of short regulatory motifs in eukaryotic proteins: tips for good practice as well as for bad. Cell Commun Signal. 2015 Nov 18;13:42

Molzan M, Weyand M, Rose R, Ottmann C. Structural insights of the MLF1/14-3-3 interaction. FEBS J. 2012 Feb;279(4):563-71.

Panni S, Montecchi-Palazzi L, Kiemer L, Cabibbo A, Paoluzi S, Santonico E, Landgraf C, Volkmer-Engert R, Bachi A, Castagnoli L, Cesareni G. Combining peptide recognition specificity and context information for the prediction of the 14-3-3-mediated interactome in S. cerevisiae and H. sapiens. Proteomics. 2011 Jan;11(1):128-43.

On 2016 Apr 19, Michaela Gack commented:

Dr. Gibson’s comment about our manuscript solely stated that the viral RxEP motif identified in the Dengue NS3 protein does not fulfill all of the criteria of conventional 14-3-3-binding motifs found in cellular 14-3-3-interacting proteins. The main conclusions of our study, that the NS3 protein of Dengue virus binds to 14-3-3epsilon and thereby antagonizes RIG-I translocation to mitochondria and type I interferon induction, were never questioned by Dr. Gibson.

Using mapping experiments and site-directed mutagenesis, we identified that the 64-RxEP-67 motif in the Dengue NS3 protein is essential for 14-3-3epsilon binding. Mutation of the central Glu66 (E66) residue to positively-charged Lys66 (K66) markedly reduced the binding of NS3 to 14-3-3epsilon. Additional mutation of Arg64 to Lys64 (termed ‘NS3(KIKP)’ mutant) led to a nearly complete loss of binding to 14-3-3epsilon. These data together with the similarity of the viral RxEP motif with the cellular 14-3-3-binding motif Rxx(pS/pT)xP, where pS/pT indicates a phosphorylated Ser/Thr residue, suggested that the negatively charged E66 serves as a phosphomimetic residue. Although our study indicated that the RxEP motif is required for 14-3-3epsilon binding, our data also suggested that additional as-yet-unidentified residues in the Dengue NS3 protein are important for 14-3-3epsilon interaction, as introduction of the RxEP motif into the NS3 protein of Yellow Fever virus, which is unable to bind 14-3-3epsilon, did not result in gain of 14-3-3epsilon binding. Thus, the full characteristics of the binding mode of Dengue NS3 and 14-3-3epsilon and their binding affinity remain to be determined in future studies, as well as additional residues in NS3 and 14-3-3epsilon that are engaged in the interaction.