The Measles, Nipah and Hendra viruses (MeV, NiV, HeV) are severe human pathogens members of the Paramyxovirinae sub-family within the family of Paramyxoviridae. MeV, NiV and HeV have a single stranded, non-segmented, RNA genome that is enveloped by the nucleoprotein (N) in a helical nucleocapsid. The RNA- dependent RNA polymerase (RdRp) is composed of the large (L) protein and the phosphoprotein (P). The N protein consists of a structured N-terminal domain (NCORE) that binds the genome, and a C-terminal disordered domain (NTAIL) exposed at nucleocapsid surface. In the three viruses, NTAIL binds to the C- terminal X domain (PXD) of the P protein leading the recruitment of the polymerase complex on the genome. The NTAIL domain contain a short, order-prone molecular recognition element (alpha-MoRE) that, upon the binding with XD, undergoes a disorder-to-order transition. The regions of NTAIL flanking the alpha-MoRe remains disordered also in the bound form and they are called "fuzzy" regions. Recent studies showed that the fuzzy region in MeV affects, decreasing, both the binding affinity between NTAIL and PXD and the folding rate of the alpha-MoRE after the binding.
NTAIL represents an extremely attractive target for the perspective of developing pharmaceutical strategies able to inhibit its interaction with PXD to interrupt the life cycle of the viruses. This research project aims to perform a characterization of the folding upon binding mechanism between NTAIL and XD and to unveil the role of disorder in this process. The informations obtained by these studies could be used to design a pharmacophore able to inhibit the interaction between PXD and NTAIL.
This scientific project aims to study the interaction mechanism between NTAIL and PXD analysing one of the interesting feature of the NTAIL protein: the presence of a disordered region flanking the MoRE that does not undergo a disorder-to-order transition upon binding with PXD. This fuzzy region, influencing the binding reaction of the protein, represents an opportunity to pinpoint a region not directly involved in the binding reaction to design antiviral strategy in order to inhibit the interaction between NTAIL and PXD.
In a recent work, a dependence of the binding affinity and the MoRE folding rate of MeV PXD-NTAIL by the fuzzy region was observed. In this project we propose to analyse how the fuzzy region affects the binding affinity and the alphaMoRe folding upon binding in the three viruses, performing kinetics study of the binding reaction between PXD and a series of variants of NTAIL. To perform kinetic binding experiments we will use Hi-Tech PTJ-64 capacitor-discharge Temperature-jump apparatus (Hi-Tech, Salisbury, UK).
So, first of all, I plan to produce progressively truncated variants of NTAIL and to perform kinetic binding experiments with a tryptophan variant of PXD (since we take advantage of the change in tryptophan fluorescence upon the binding to follow the kinetic of the interaction). What is expected from these experiments is a change of the kinetics of binding due to the truncation of the disordered region. After all the truncation variants binding reactions will be explored, site-directed mutants in the segments that will result particularly involved in regulating the binding and folding of the alpha-MoRE will be produced to asses the role of each single amminoacid in the regulation. The same kind of experiments will be performed also using artificial NTAIL variants in order to understand the role of the sequence, rather than disorder, in the effect of the disordered region close to the MoRE. The sequence of the disordered region will not be just a shuffled version of the wild-type sequence, but will be designed in order to be as much different as possible from the wild-type however conserving the same "amount" of disorder. Each artificial sequence will be analyzed using MultAlin tool (http://multalin.toulouse.inra.fr/multalin/multalin.html) and IUPred (http://iupred.enzim.hu/) to ensure a very low sequence identity with wild-type but similar disorder propensity.
Data arising from these experiments will be useful to better understand in which way disordered regions near the molecular recognition elements, in the three viruses, influence the binding affinity between PXD and NTAIL. This will also give the opportunity to increase the chances to study antiviral strategies targeting regions directly and indirectly involved in the binding reaction.