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. 2010 Jul;19(7):1376-85.
doi: 10.1002/pro.418.

Functional dissection of an intrinsically disordered protein: understanding the roles of different domains of Knr4 protein in protein-protein interactions

Adilia Dagkessamanskaia  1 , Fabien Durand, Vladimir N Uversky, Matteo Binda, Frédéric Lopez, Karim El Azzouzi, Jean Marie Francois, Hélène Martin-Yken
Affiliations

Functional dissection of an intrinsically disordered protein: understanding the roles of different domains of Knr4 protein in protein-protein interactions

Adilia Dagkessamanskaia et al. Protein Sci. 2010 Jul.

Abstract

Knr4, recently characterized as an intrinsically disordered Saccharomyces cerevisiae protein, participates in cell wall formation and cell cycle regulation. It is constituted of a functional central globular core flanked by a poorly structured N-terminal and large natively unfolded C-terminal domains. Up to now, about 30 different proteins have been reported to physically interact with Knr4. Here, we used an in vivo two-hybrid system approach and an in vitro surface plasmon resonance (BIAcore) technique to compare the interaction level of different Knr4 deletion variants with given protein partners. We demonstrate the indispensability of the N-terminal domain of Knr4 for the interactions. On the other hand, presence of the unstructured C-terminal domain has a negative effect on the interaction strength. In protein interactions networks, the most highly connected proteins or "hubs" are significantly enriched in unstructured regions, and among them the transient hub proteins contain the largest and most highly flexible regions. The results presented here of our analysis of Knr4 protein suggest that these large disordered regions are not always involved in promoting the protein-protein interactions of hub proteins, but in some cases, might rather inhibit them. We propose that this type of regions could prevent unspecific protein interactions, or ensure the correct timing of occurrence of transient interactions, which may be of crucial importance for different signaling and regulation processes.

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Figures

Figure 1
Figure 1
Interaction between different Knr4 parts and Tid3 in the two-hybrid system. A: Schematic representation of the two-hybrid constructions expressing different parts of Knr4 fused to the Gal4 DNA binding domain. B: Transformants of pJ69-4A strain each carrying pOAD-Tid3 plasmid and pOBD2 plasmid with or without Knr4 deleted variants were grown on SD-Trp-Leu solid media at 30°C for 1 day. Next day, replica was made on two plates: new SD-Trp-Leu and on SD-Ade. Presented photograph was taken after 72 h of growth. C: β-galactosidase activity measured in the cell extracts from the strains expressing AD-Tid3 fusion protein (Tid3 fused to the activating domain of Gal4) and different Knr4 deleted variants fused to the BD of Gal4. The results (triplicates) were normalized to protein concentrations and expressed in nanomoles of O-nitrophenyl-β-d-galactopyranoside converted/min/mg proteins. The upper bar corresponds to the negative control strain with only activating (AD) and DNA binding (BD) domains of Gal4 expressed.
Figure 2
Figure 2
Computational evaluation of the intrinsic disorder propensity of Knr4 and its N- and C-terminal domains. A: Composition profiling of Knr41–505 (red bars), Knr41–79 (green bars), Knr480–340 (yellow bars), and Knr4341–505 (blue bars) compared with a set of ordered proteins from PDB. Data for a set of disordered proteins from DisProt are shown by black bars. The bar for a given amino acid represents the fractional difference in composition between a given protein (or set of proteins) and the set of ordered proteins. The fractional difference is calculated as (CX − Cordered)/Cordered, where CX is the amount of the amino acid in a given protein/set, and Corder is the corresponding amount in the set of ordered proteins. Residues are ordered by their disorder propensity. Negative values indicate residues less abundant in the given protein/set than in the set of ordered proteins, positive indicates residues more abundant in the given protein/set than in the set of ordered proteins. B: Distribution of the PONDR®-FIT score over the sequences of Knr4. In PONDR plot, segments with scores above 0.5 correspond to disordered regions, whereas those below 0.5 correspond to ordered regions. Red and blue bars represent the respective positions of the alpha-helical and beta-structural elements predicted by the NPS (network protein sequence analysis) consensus secondary structure server, which runs the input sequence against several different secondary structure prediction tools and generates a consensus secondary structure out of them.
Figure 3
Figure 3
Interaction between different Knr4 parts and Tys1 in the two-hybrid system. A: Transformants of pJ69-4A strain each carrying pOAD-Tid3 plasmid and pOBD2 plasmid with or without Knr4 deleted variants were grown on SD-Trp-Leu solid media at 30°C for 1 day. Next day, replica was made on two plates: new SD-Trp-Leu and on SD-Ade. The presented photograph was taken after 72 h of growth. B: β-galactosidase activity measured in the cell extracts from the strains expressing AD-Tid3 fusion protein (Tid3 fused to the activating domain of Gal4) and different Knr4 deleted variants fused to the BD of Gal4. The results (triplicates) were normalized to protein concentrations and expressed in nanomoles of O-nitrophenyl-β-d-galactopyranoside converted/min/mg proteins. The upper bar corresponds to the negative control strain with only activating (AD) and DNA binding (BD) domains of Gal4 expressed.
Figure 4
Figure 4
Effect of the N- and C-terminal domains of Knr4 protein on its in vitro protein binding capacity. Purified Knr4 protein fragments 1–340, 1–505, and 80–340 were fixed on three channels of a CM5 BIAcore sensorchip. Solutions containing different concentrations (500 nM, 1 μM, and 2 μM) of purified Tys1 protein were flowed over the chip and the binding kinetics were measured and quantified versus the chip forth channel used as a negative control. Sensorgrams are expressed in resonance units (RU) as a function of time in seconds. Resonance units on the Y axis represent Tys1 binding to Knr4 fragments.
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