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On the role of residue phosphorylation in 14-3-3 partners: AANAT as a case study

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ABSTRACT

Twenty years ago, a novel concept in protein structural biology was discovered: the intrinsically disordered regions (IDRs). These regions remain largely unstructured under native conditions and the more are studied, more properties are attributed to them. Possibly, one of the most important is their ability to conform a new type of protein-protein interaction. Besides the classical domain-to-domain interactions, IDRs follow a ‘fly-casting’ model including ‘induced folding’. Unfortunately, it is only possible to experimentally explore initial and final states. However, the complete movie of conformational changes of protein regions and their characterization can be addressed by in silico experiments. Here, we simulate the binding of two proteins to describe how the phosphorylation of a single residue modulates the entire process. 14-3-3 protein family is considered a master regulator of phosphorylated proteins and from a modern point-of-view, protein phosphorylation is a three component system, with writers (kinases), erasers (phosphatases) and readers. This later biological role is attributed to the 14-3-3 protein family. Our molecular dynamics results show that phosphorylation of the key residue Thr31 in a partner of 14-3-3, the aralkylamine N-acetyltransferase, releases the fly-casting mechanism during binding. On the other hand, the non-phosphorylation of the same residue traps the proteins, systematically and repeatedly driving the simulations into wrong protein-protein conformations.

No MeSH data available.


Full molecular dynamics process from initial structures, through pseudo-native to native complex.(a) Glu87 and Arg89 distance to groups 1 and 2 in 14-3-3ζ (see the methods section). The asterisk means simulation was performed with Thr31 in AANAT in its phosphorylated state (b) Normalized solvent accessible surface area (SASA) of the full protein-protein complex. (c) RMSD Cα to native structure. Sections marked as I, II or III in the plots correspond to first harmonic potential applied (I), free simulation (II) and second harmonic potential applied (III).
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f2: Full molecular dynamics process from initial structures, through pseudo-native to native complex.(a) Glu87 and Arg89 distance to groups 1 and 2 in 14-3-3ζ (see the methods section). The asterisk means simulation was performed with Thr31 in AANAT in its phosphorylated state (b) Normalized solvent accessible surface area (SASA) of the full protein-protein complex. (c) RMSD Cα to native structure. Sections marked as I, II or III in the plots correspond to first harmonic potential applied (I), free simulation (II) and second harmonic potential applied (III).

Mentions: In this work, we compared results from unphosphorylated and phosphorylated AANAT during binding to 14-3-3ζ protein. As we previously described, there are ‘anchor’ amino acids in the globular part of AANAT that make the first contact to 14-3-3ζ. Their mutation to Ala impairs binding even if AANAT is phosphorylated4 (see Fig. 1). In this previous study4, we theoretically proposed that anchor amino acids provide steric constraints that help to stabilize a native-like intermediate. However, this was never experimentally proved. With this information we performed restrained dynamics for 10 ns of simulation time (Fig. 2 region I) applying a harmonic potential between anchor residues in AANAT and their partners in 14-3-3ζ (see details in the methods section). In this way we forced the formation of a pseudo-native complex. After that, we performed 90 ns of unrestrained dynamics (Fig. 2 region II) allowing for the system to freely evolve. In the final region (Fig. 2 region III), we applied another harmonic potential to guide the proteins to their x-ray solved structure (PDB ID: 1IB1). Figure 3 shows representative snapshots of the three different regions in our simulations, from the initial system where proteins are at 60 Å apart, to the final complex at only RMSD(Cα) = 3.53 Å to the crystallized structure.


On the role of residue phosphorylation in 14-3-3 partners: AANAT as a case study
Full molecular dynamics process from initial structures, through pseudo-native to native complex.(a) Glu87 and Arg89 distance to groups 1 and 2 in 14-3-3ζ (see the methods section). The asterisk means simulation was performed with Thr31 in AANAT in its phosphorylated state (b) Normalized solvent accessible surface area (SASA) of the full protein-protein complex. (c) RMSD Cα to native structure. Sections marked as I, II or III in the plots correspond to first harmonic potential applied (I), free simulation (II) and second harmonic potential applied (III).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384239&req=5

f2: Full molecular dynamics process from initial structures, through pseudo-native to native complex.(a) Glu87 and Arg89 distance to groups 1 and 2 in 14-3-3ζ (see the methods section). The asterisk means simulation was performed with Thr31 in AANAT in its phosphorylated state (b) Normalized solvent accessible surface area (SASA) of the full protein-protein complex. (c) RMSD Cα to native structure. Sections marked as I, II or III in the plots correspond to first harmonic potential applied (I), free simulation (II) and second harmonic potential applied (III).
Mentions: In this work, we compared results from unphosphorylated and phosphorylated AANAT during binding to 14-3-3ζ protein. As we previously described, there are ‘anchor’ amino acids in the globular part of AANAT that make the first contact to 14-3-3ζ. Their mutation to Ala impairs binding even if AANAT is phosphorylated4 (see Fig. 1). In this previous study4, we theoretically proposed that anchor amino acids provide steric constraints that help to stabilize a native-like intermediate. However, this was never experimentally proved. With this information we performed restrained dynamics for 10 ns of simulation time (Fig. 2 region I) applying a harmonic potential between anchor residues in AANAT and their partners in 14-3-3ζ (see details in the methods section). In this way we forced the formation of a pseudo-native complex. After that, we performed 90 ns of unrestrained dynamics (Fig. 2 region II) allowing for the system to freely evolve. In the final region (Fig. 2 region III), we applied another harmonic potential to guide the proteins to their x-ray solved structure (PDB ID: 1IB1). Figure 3 shows representative snapshots of the three different regions in our simulations, from the initial system where proteins are at 60 Å apart, to the final complex at only RMSD(Cα) = 3.53 Å to the crystallized structure.

View Article: PubMed Central - PubMed

ABSTRACT

Twenty years ago, a novel concept in protein structural biology was discovered: the intrinsically disordered regions (IDRs). These regions remain largely unstructured under native conditions and the more are studied, more properties are attributed to them. Possibly, one of the most important is their ability to conform a new type of protein-protein interaction. Besides the classical domain-to-domain interactions, IDRs follow a ‘fly-casting’ model including ‘induced folding’. Unfortunately, it is only possible to experimentally explore initial and final states. However, the complete movie of conformational changes of protein regions and their characterization can be addressed by in silico experiments. Here, we simulate the binding of two proteins to describe how the phosphorylation of a single residue modulates the entire process. 14-3-3 protein family is considered a master regulator of phosphorylated proteins and from a modern point-of-view, protein phosphorylation is a three component system, with writers (kinases), erasers (phosphatases) and readers. This later biological role is attributed to the 14-3-3 protein family. Our molecular dynamics results show that phosphorylation of the key residue Thr31 in a partner of 14-3-3, the aralkylamine N-acetyltransferase, releases the fly-casting mechanism during binding. On the other hand, the non-phosphorylation of the same residue traps the proteins, systematically and repeatedly driving the simulations into wrong protein-protein conformations.

No MeSH data available.