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

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.

Figure 1. Components of Clathrin coated vesicles.

a] The basic functional unit of clathrin cage is clathrin triskelion. The triskelion consists of three clathrin heavy chains (dark green) interacting non-covalently with clathrin light chains (shown in light green). b] The clathrin chains surrounding the cargo polymerize to form a hexagonal barrel inside which the cargo is transported from one place to another safely. c] When the cargo is to be transported from one place to another it starts getting accumulated at the membrane (cargo shown as red spheres & membrane as black horizontal line), bound to its receptor (shown in purple). The cargo receptors recruit adaptor proteins (the heterotetramers in orange), which in turn employ clathins (in green). With the help of accessory proteins recruited by clathrins the plasma membrane invaginates and the clathrin coated vesicle is clipped off subsequently.

Figure 2. Structure of Clathrin coat (PDB ID : 1xi4).

Shown in the figure is the structure of clathrin coat, visualized in 3D using PyMOL software . The structural model was generated by superimposing high resolution structural data over the low resolution cryoEM electron density by Fotin A and coworkers . 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.

Figure 3. Interface residues of clathrin heavy chain.

The figure provides closer view of one of the heavy chains in the structure of clathrin coat (shown in figure 2) and its residues interacting with various components of the vesicular assembly. The clathrin heavy chain is shown in purple and clathrin light chain in yellow. The red spheres depict the residues of heavy chain interacting with other clathrin chains (either light chains or other heavy chains), pink spheres are the residues in interaction with auxillin peptide (an accessory protein) while orange spheres are the residues interacting with adaptor protein chain and the residues forming interface with amphiphysin peptide (another example of accessory protein) are in cyan.

Figure 4. Structure of adaptor protein 2.

The figure is a collage of three different structures available of the components of AP2 complex, generated, using PyMOL software , to provide an overall view of the entire AP2 complex. a] Structure of appendage domain of B chain (in cyan) with interface residues interacting with clathrin heavy chain peptide (shown in red); Towards this the interface residues on AP2 chain B in PDB structure 1c9i were mapped on to and highlighted in the structure of entire appendage domain (PDB id 2vi8). b] Structure of appendage domain of A chain of AP2 (shown in green) with the residues interacting with one of the accessory proteins arrestin shown in pink (PDB id. 1kyd). c] Structure of core AP2 (PDB id. 2vgl) with B chain in cyan, A chain in green, M chain in magenta and S chain in yellow while the residues in the interactions with the other chains in the structure are highlighted in either orange or blue.

Figure 5. Conservation status of interface residues of clathrin vesicle assembly components.

Residue conservation scores were calculated using Consurf (as described in “Methods” section). The relative measure of the evolutionary conservation at every position in the subunit was averaged for the interface residues and non interface surface exposed residues. The figure provides comparative picture of the conservation scores for the interface residues and non-interface surface exposed residues of clathrin heavy chains (shown in “a” panel), chains of adaptor protein 1 complex (b panel) and the chains of adaptor protein 2 complex (shown in “c” panel).

Figure 6. Phylogenetic tree topology comparison-1.

The figure provides comparative picture of phylogenetic trees of the functionally quivalent chains of the two adaptor protein complexes namely AP2B (2vglB) and AP1B (1w63B). The phylogenetic trees were constructed using PHYML programme, using maximum likelihood method (as described in Methods section).

Figure 7. Phylogenetic tree topology comparison-2.

Comparative picture of phylogenetic trees between functionally equivalent pair of subunits from the two adaptor protein complexes, AP2M (2vglM) and AP1M (1w63M). The trees were constructed as mentioned in the legend to Figure 6 and in the merthods section.

Figure 8. Correlation between genetic distance matrices of a pair of protein families.

To investigate the correlated evolution of the adaptor protein chains with clathrin heavy chain the genetic distance matrices were generated for the orthologous sequences of every chain and compared to that of clathrin heavy chain. The comparison of the two matrices was expressed as Pearson correlation coefficient value computed. The figure summarizes the comparison of the Pearson correlation coefficients obtained for all the subunits of adaptor proteins when compared with clathrin heavy chain (as listed on X-axis).

Figure 9. Distribution of evolutionary constraints in the clathrin coated vesicle assembly in the form of a cartoon.

If clathrin coated assembly can be imagined as a sphere, with cargo being at the center while clathrin heavy chain were being at the periphery, then the figure provides view of this assembly as a transverse section of this sphere. Different components (the subunits of the complexes) of the assembly are labeled appropriately in the figure. The shaded background depicts the observed pattern in evolutionary constraints, dark depicting maximum variation in sequence (least constraint), as observed towards centre of the assembly, while the lighter shades indicate less sequence divergence (maximum constraint) as seen more towards the periphery.