Demonstration of a Folding after Binding Mechanism in the Recognition between the Measles Virus N_TAIL and X Domains

Abstract

In the past decade, a wealth of experimental data has demonstrated that a large fraction of proteins, while functional, are intrinsically disordered at physiological conditions. Many intrinsically disordered proteins (IDPs) undergo a disorder-to-order transition upon binding to their biological targets, a phenomenon known as induced folding. Induced folding may occur through two extreme mechanisms, namely conformational selection and folding after binding. Although the pre-existence of ordered structures in IDPs is a prerequisite for conformational selection, it does not necessarily commit to this latter mechanism, and kinetic studies are needed to discriminate between the two possible scenarios. So far, relatively few studies have addressed this issue from an experimental perspective. Here, we analyze the interaction kinetics between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the viral phosphoprotein. Data reveal that NTAIL recognizes XD by first forming a weak encounter complex in a disordered conformation, which is subsequently locked-in by a folding step; i.e., binding precedes folding. The implications of our kinetic results, in the context of previously reported equilibrium data, are discussed. These results contribute to enhancing our understanding of the molecular mechanisms by which IDPs recognize their partners and represent a paradigmatic example of the need of kinetic methods to discriminate between reaction mechanisms.

Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength

“…Binding reactions were all found to be enthalpy-driven, with ΔH values in the same order of magnitude and ranging from -10.9 to -14.5 kcal/mol ( Fig 3 ). The estimates for the model parameters of the wt N TAIL /XD pair were found to be in very good agreement with those recently reported [46]. The estimates for binding parameters of variants R489Q, A492T and D493G yielded equilibrium dissociation constant (K D ) very close to that observed for wt N TAIL , indicating that these substitutions poorly affect the interaction ( Fig 3 ).…”

“…The n values for the A492T/XD and D493G/XD binding pairs were found to deviate from unit, a behaviour that is not unusual and that has been already observed with single-site tryptophan variants [46] and that may arise from relatively poorly defined baselines. In light of all the numerous previous studies [18,19,27,30,31,32,33,34,35,36,37] showing that N TAIL and XD form a 1:1 complex, these deviations were not taken to be significant.…”

“…Even if their molecular mass is ~16 kDa, they all migrate on a denaturing gel with an apparent molecular mass of approximately 20 kDa ( Fig 2B, inset ). This aberrant electrophoretic migration has been systematically observed for all N TAIL variants reported so far [26,30,31,32,46] including wt N TAIL [19]. This anomalous migration is frequently observed in IDPs and is due to a high content in acidic residues [51] and/or a large extension in solution [43].…”

“…In the case of NiV [42], Hendra virus [88], SeV [38,89] and MeV [27,30,44,89] the C-terminal domain of P (XD) is structurally conserved and consists of a bundle of 3 α-helices that are structurally analogous, and that dynamically binds to a α-MoRE located near the C-terminus of N TAIL ([90,91], see [92] for reviews). This N TAIL -XD interaction is commonly characterized by a rather low affinity (K D within the 3–50 μM range, [39,46,89] and this work). In Rubulavirus members, the C-terminal region of P spans in solution a structural continuum ranging from stable triple α-helical bundles to largely disordered, with crystal packing stabilizing the folded form [93,94].…”

“…The α-MoRE of N TAIL is partly preconfigured as an α-helix prior to binding to XD [31,32,35,37,45] and adopts an equilibrium between a fully unfolded form and four partly helical conformers [37]. In spite of this partial pre-configuration, N TAIL folds according to a “folding after binding mechanism” [45,46]. Previous mutational studies showed that Box2 is poorly evolvable in terms of its binding abilities towards XD, in that amino acid substitutions therein introduced lead to a dramatic drop in the binding strength, as judged from a protein complementation assay (PCA) based on split-GFP reassembly (gfp-PCA) [47].…”