Cryo-EM of multiple cage architectures reveals a universal mode of clathrin self assembly

Clathrin forms diverse lattice and cage structures that change size and shape rapidly in response to the needs of eukaryotic cells during clathrin-mediated endocytosis and intracellular trafficking. We present the cryo-EM structure and molecular model of assembled porcine clathrin, providing insights into interactions that stabilize key elements of the clathrin lattice, namely, between adjacent heavy chains, at the light chain–heavy chain interface and within the trimerization domain. Furthermore, we report cryo-EM maps for five different clathrin cage architectures. Fitting structural models to three of these maps shows that their assembly requires only a limited range of triskelion leg conformations, yet inherent flexibility is required to maintain contacts. Analysis of the protein–protein interfaces shows remarkable conservation of contact sites despite architectural variation. These data reveal a universal mode of clathrin assembly that allows variable cage architecture and adaptation of coated vesicle size and shape during clathrin-mediated vesicular trafficking or endocytosis

Weak molecular interactions in clathrin-mediated endocytosis

Clathrin-mediated endocytosis is a process by which specific molecules are internalized from the cell periphery for delivery to early endosomes. The key stages in this step-wise process, from the starting point of cargo recognition, to the later stage of assembly of the clathrin coat, are dependent on weak interactions between a large network of proteins. This review discusses the structural and functional data that have improved our knowledge and understanding of the main weak molecular interactions implicated in clathrin-mediated endocytosis, with a particular focus on the two key proteins: AP2 and clathrin

Biophysical analysis of binding interactions between clathrin and its adaptor proteins

Clathrin-mediated endocytosis (CME) plays a central role in fundamental processes such as synaptic vesicle recycling, receptor recycling, signalling and development. CME begins with clathrin assembly on the plasma membrane, facilitated by adaptor proteins. This process forms an endocytic vesicle that allows transport of cargo into the cell, and is followed by clathrin disassembly through the action of different adaptor/accessory proteins. A large number of different adaptor and accessory proteins are recruited during CME, in a spatially and temporally ordered manner. Although our understanding is growing as to the roles of individual adaptor proteins, we still do not understand the way in which some adaptors interact with clathrin or the molecular details of their interactions with one another in the presence of clathrin. Clathrin adaptor proteins contain short, linear clathrin-binding motifs, which form the basis of their interaction with the four distinct sites on the clathrin N-terminal domain (TD). An adaptor protein with tighter binding or more numerous clathrin binding sequences could displace one with weaker or fewer binding elements. This raises the question of whether adaptor proteins compete for binding to clathrin or whether they can bind simultaneously. Using certain biochemical and biophysical techniques in vitro and purified WT and mutant adaptor proteins, I have shown the complex ‘multiple TD linking effect’ of epsin 1 via the cooperative action of its two clathrin box motifs and unstructured region. Using the newly developed SPR/IAC (2-injection) method, I explored competition between five purified structurally and functionally diverse adaptor proteins when simultaneously binding to clathrin TD. I have shown how the complex structure of epsin 1 causes competition with β-arrestin 1 for clathrin TD binding. Such competition is observed between espin 1 and auxilin 1 as well, which reveals information about the mechanism of disassembly. However, β2-adaptin and auxilin 1 demonstrate no such competition