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Protein-protein interactions in clathrin vesicular assembly: radial distribution of evolutionary constraints in interfaces.

Gadkari RA, Srinivasan N - PLoS ONE (2012)

Bottom Line: In the present study, using the method developed by us earlier, we predict the protein-protein interface residues in clathrin assembly, taking guidance from the available low-resolution structures.The conservation status of these interfaces when investigated across eukaryotes, revealed a radial distribution of evolutionary constraints, i.e., if the members of the clathrin vesicular assembly can be imagined to be arranged in spherical manner, the cargo being at the center and clathrins being at the periphery, the detailed phylogenetic analysis of these members of the assembly indicated high-residue variation in the members of the assembly closer to the cargo while high conservation was noted in clathrins and in other proteins at the periphery of the vesicle.This points to the strategy adopted by the nature to package diverse proteins but transport them through a highly conserved mechanism.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India. rupali@mbu.iisc.ernet.in

ABSTRACT
In eukaryotic organisms clathrin-coated vesicles are instrumental in the processes of endocytosis as well as intracellular protein trafficking. Hence, it is important to understand how these vesicles have evolved across eukaryotes, to carry cargo molecules of varied shapes and sizes. The intricate nature and functional diversity of the vesicles are maintained by numerous interacting protein partners of the vesicle system. However, to delineate functionally important residues participating in protein-protein interactions of the assembly is a daunting task as there are no high-resolution structures of the intact assembly available. The two cryoEM structures closely representing intact assembly were determined at very low resolution and provide positions of Cα atoms alone. In the present study, using the method developed by us earlier, we predict the protein-protein interface residues in clathrin assembly, taking guidance from the available low-resolution structures. The conservation status of these interfaces when investigated across eukaryotes, revealed a radial distribution of evolutionary constraints, i.e., if the members of the clathrin vesicular assembly can be imagined to be arranged in spherical manner, the cargo being at the center and clathrins being at the periphery, the detailed phylogenetic analysis of these members of the assembly indicated high-residue variation in the members of the assembly closer to the cargo while high conservation was noted in clathrins and in other proteins at the periphery of the vesicle. This points to the strategy adopted by the nature to package diverse proteins but transport them through a highly conserved mechanism.

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Structure of Clathrin coat (PDB ID : 1xi4).Shown in the figure is the structure of clathrin coat, visualized in 3D using PyMOL software [53]. The structural model was generated by superimposing high resolution structural data over the low resolution cryoEM electron density by Fotin A and coworkers [26]. The model was provided at a resolution equivalent to 8 Å and it provides Cα atom positions only. Shown in the figure are the clathrin chains with the Cα atoms represented as spheres. The light chains of clathrin are seen as slender sticks in the figure while others occupying most of the space are the heavy chains.
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pone-0031445-g002: Structure of Clathrin coat (PDB ID : 1xi4).Shown in the figure is the structure of clathrin coat, visualized in 3D using PyMOL software [53]. The structural model was generated by superimposing high resolution structural data over the low resolution cryoEM electron density by Fotin A and coworkers [26]. The model was provided at a resolution equivalent to 8 Å and it provides Cα atom positions only. Shown in the figure are the clathrin chains with the Cα atoms represented as spheres. The light chains of clathrin are seen as slender sticks in the figure while others occupying most of the space are the heavy chains.

Mentions: Recognition of protein-protein interaction interface in case of clathrin cage was a twofold problem; a] The structures available for the clathrin cage provide positions of only Cα atoms and hence recognition of interface was a non-trivial task and b] To further add to the complexity, the structural models comprise eighteen polypeptide chains (as shown in the Figure 2) and hence, were difficult to process for computing solvent accessible surface area of every residue. We have developed a method which can recognize the protein-protein interaction interfaces solely from Cα positions in low resolution structures of big assemblies such as CCV [28]. However, prior to applying this method, in order to circumvent the second problem mentioned above, we identified near neighbors for every chain in the complex structures using distance criterion; if the distance between two Cα residues from different chains is less than or equal to 5 Å then the chains possessing the residues are termed as near neighbors. The complex structures (PDb IDs 1xi4 & 1xi5) were then divided into smaller sub-complexes that were treated as independent structures to recognize interface residues. These sub-complexes are listed in the Table 2. Subsequently, the interaction interfaces were recognized using the protocol as described in Methods section and are listed in the Table 3 and Table 4. As can be clearly seen in the tables, all the heavy chains in the structures contribute differently although there is an overlap in terms of the interacting residues. The Figure 3 shows interface residues recognized in case of G chain of 1xi4 and as is clear from the picture, our method has indeed identified the interface residues specifically from Cα positions available. When the interface predicted in clathrin coat was compared with that of clathrin coat with auxilin peptide bound to it, it was clearly seen that auxilin chains were bound to the terminal domain of clathrin heavy chain (Figure 3) (Table 4). Thus, it clearly reconfirmed the known fact that terminal domain of clathrin interacts with other non-clathrin components while the interactions between clathrin chains are restricted to the leg region of the chain [26], [27].


Protein-protein interactions in clathrin vesicular assembly: radial distribution of evolutionary constraints in interfaces.

Gadkari RA, Srinivasan N - PLoS ONE (2012)

Structure of Clathrin coat (PDB ID : 1xi4).Shown in the figure is the structure of clathrin coat, visualized in 3D using PyMOL software [53]. The structural model was generated by superimposing high resolution structural data over the low resolution cryoEM electron density by Fotin A and coworkers [26]. The model was provided at a resolution equivalent to 8 Å and it provides Cα atom positions only. Shown in the figure are the clathrin chains with the Cα atoms represented as spheres. The light chains of clathrin are seen as slender sticks in the figure while others occupying most of the space are the heavy chains.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285160&req=5

pone-0031445-g002: Structure of Clathrin coat (PDB ID : 1xi4).Shown in the figure is the structure of clathrin coat, visualized in 3D using PyMOL software [53]. The structural model was generated by superimposing high resolution structural data over the low resolution cryoEM electron density by Fotin A and coworkers [26]. The model was provided at a resolution equivalent to 8 Å and it provides Cα atom positions only. Shown in the figure are the clathrin chains with the Cα atoms represented as spheres. The light chains of clathrin are seen as slender sticks in the figure while others occupying most of the space are the heavy chains.
Mentions: Recognition of protein-protein interaction interface in case of clathrin cage was a twofold problem; a] The structures available for the clathrin cage provide positions of only Cα atoms and hence recognition of interface was a non-trivial task and b] To further add to the complexity, the structural models comprise eighteen polypeptide chains (as shown in the Figure 2) and hence, were difficult to process for computing solvent accessible surface area of every residue. We have developed a method which can recognize the protein-protein interaction interfaces solely from Cα positions in low resolution structures of big assemblies such as CCV [28]. However, prior to applying this method, in order to circumvent the second problem mentioned above, we identified near neighbors for every chain in the complex structures using distance criterion; if the distance between two Cα residues from different chains is less than or equal to 5 Å then the chains possessing the residues are termed as near neighbors. The complex structures (PDb IDs 1xi4 & 1xi5) were then divided into smaller sub-complexes that were treated as independent structures to recognize interface residues. These sub-complexes are listed in the Table 2. Subsequently, the interaction interfaces were recognized using the protocol as described in Methods section and are listed in the Table 3 and Table 4. As can be clearly seen in the tables, all the heavy chains in the structures contribute differently although there is an overlap in terms of the interacting residues. The Figure 3 shows interface residues recognized in case of G chain of 1xi4 and as is clear from the picture, our method has indeed identified the interface residues specifically from Cα positions available. When the interface predicted in clathrin coat was compared with that of clathrin coat with auxilin peptide bound to it, it was clearly seen that auxilin chains were bound to the terminal domain of clathrin heavy chain (Figure 3) (Table 4). Thus, it clearly reconfirmed the known fact that terminal domain of clathrin interacts with other non-clathrin components while the interactions between clathrin chains are restricted to the leg region of the chain [26], [27].

Bottom Line: In the present study, using the method developed by us earlier, we predict the protein-protein interface residues in clathrin assembly, taking guidance from the available low-resolution structures.The conservation status of these interfaces when investigated across eukaryotes, revealed a radial distribution of evolutionary constraints, i.e., if the members of the clathrin vesicular assembly can be imagined to be arranged in spherical manner, the cargo being at the center and clathrins being at the periphery, the detailed phylogenetic analysis of these members of the assembly indicated high-residue variation in the members of the assembly closer to the cargo while high conservation was noted in clathrins and in other proteins at the periphery of the vesicle.This points to the strategy adopted by the nature to package diverse proteins but transport them through a highly conserved mechanism.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India. rupali@mbu.iisc.ernet.in

ABSTRACT
In eukaryotic organisms clathrin-coated vesicles are instrumental in the processes of endocytosis as well as intracellular protein trafficking. Hence, it is important to understand how these vesicles have evolved across eukaryotes, to carry cargo molecules of varied shapes and sizes. The intricate nature and functional diversity of the vesicles are maintained by numerous interacting protein partners of the vesicle system. However, to delineate functionally important residues participating in protein-protein interactions of the assembly is a daunting task as there are no high-resolution structures of the intact assembly available. The two cryoEM structures closely representing intact assembly were determined at very low resolution and provide positions of Cα atoms alone. In the present study, using the method developed by us earlier, we predict the protein-protein interface residues in clathrin assembly, taking guidance from the available low-resolution structures. The conservation status of these interfaces when investigated across eukaryotes, revealed a radial distribution of evolutionary constraints, i.e., if the members of the clathrin vesicular assembly can be imagined to be arranged in spherical manner, the cargo being at the center and clathrins being at the periphery, the detailed phylogenetic analysis of these members of the assembly indicated high-residue variation in the members of the assembly closer to the cargo while high conservation was noted in clathrins and in other proteins at the periphery of the vesicle. This points to the strategy adopted by the nature to package diverse proteins but transport them through a highly conserved mechanism.

Show MeSH
Related in: MedlinePlus