Fuzzy regions in an intrinsically disordered protein impair protein–protein interactions

Abstract

Despite the partial disorder-to-order transition that intrinsically disordered proteins often undergo upon binding to their partners, a considerable amount of residual disorder may be retained in the bound form, resulting in a fuzzy complex. Fuzzy regions flanking molecular recognition elements may enable partner fishing through non-specific, transient contacts, thereby facilitating binding, but may also disfavor binding through various mechanisms. So far, few computational or experimental studies have addressed the effect of fuzzy appendages on partner recognition by intrinsically disordered proteins. In order to shed light onto this issue, we used the interaction between the intrinsically disordered C-terminal domain of the measles virus (MeV) nucleoprotein (N TAIL ) and the X domain (XD) of the viral phosphoprotein as model system. After binding to XD, the N-terminal region of N TAIL remains conspicuously disordered, with a-helical folding taking place only within a short molecular recognition element. To study the effect of the N-terminal fuzzy region on N TAIL /XD binding, we generated N-terminal truncation variants of N TAIL , and assessed their binding abilities towards XD. The results revealed that binding increases with shortening of the N-terminal fuzzy region, with this also being observed with hsp70 (another MeV N TAIL binding partner), and for the homologous N TAIL /XD pairs from the Nipah and Hendra viruses. Finally, similar results were obtained when the MeV N TAIL fuzzy region was replaced with a highly dissimilar artificial disordered sequence, supporting a sequence-independent inhibitory effect of the fuzzy region.

InSiDDe: A Server for Designing Artificial Disordered Proteins

“…The rationale for setting these Dt boundaries was that 0.5 is the IUPred threshold above which a sequence is considered as disordered and 1.0 is the highest IUPred disorder score [12]. IUPred was chosen because we had already successfully used it to analyze the artificial N TAIL sequence designed and experimentally characterized in our previous study [11] and because it could be run in local mode on our server. However, this choice does not exclude that other programs or meta-programs could have been used.…”

“…A BLAST search in the non-redundant protein sequence database retrieved no sequence. To functionally test 100-0.6, we used the measles N TAIL -XD interacting system already used in our previous work [11]. To that end, 100-0.6 was appended to the N -terminal end of N TAIL MoRE to yield a sequence referred to as 100-0.6-MoRE.…”

“…Among these IDRs, the measles virus (MeV) N TAIL is made of an N-terminal fuzzy region (amino acids 401 to 485) followed by a MoRE (amino acids 486 to 502). We showed that the fuzzy appendage acts as a natural dampener of the N TAIL /XD interaction, with gradual shortening of the fuzzy appendage leading to increased interaction strength [11]. To determine if the dampening effect was sequence-dependent or not, we designed an artificial fuzzy appendage (amino acids 401 to 481) with a low sequence identity with the wild-type (wt) sequence and a higher disorder score as assessed using IUPred [12].…”

“…To determine if the dampening effect was sequence-dependent or not, we designed an artificial fuzzy appendage (amino acids 401 to 481) with a low sequence identity with the wild-type (wt) sequence and a higher disorder score as assessed using IUPred [12]. When the wt and the artificial fuzzy appendages were swapped within full length N TAIL , the artificial N TAIL protein was successfully expressed in E. coli and its fuzzy region was found to inhibit the interaction with XD in a way very similar to that of wtN TAIL [11]. In addition, the gradual N-terminal shortening of the artificial region led to the same pattern as observed with wtN TAIL , that is, the interaction with XD increased with the shortening.…”

“…In addition, the gradual N-terminal shortening of the artificial region led to the same pattern as observed with wtN TAIL , that is, the interaction with XD increased with the shortening. These results allowed us to conclude that the effect of N TAIL fuzzy appendage on XD binding mainly depended on the length of the fuzzy appendage and not on a specific sequence [11]. …”