The self-assembly of clathrin into lattices relies on the ability of heavy chain legs to form a three-legged pinwheel structure. We investigated the role of light chains in clathrin trimerization by challenging recombinant hub (plus and minus light chain) with an anionic detergent. The binding of light chain increases the amount of detergent needed to induce detrimerization, suggesting light chains reinforced hub trimers. We also show that light chain C-terminal residues are important for enhancing the in vitro assembly of hub at low pH. We assessed how much the C-terminus of light chain contributed to the stability of the trimerization domain by adding full-length and truncated light chains to trimer-defective hub mutants, C1573S and C1573A. Adding full-length LCb to C1573S caused some retrimerization, but little activity was restored, suggesting the majority of oligomeric C1573S was nonnative. A larger percentage of monomeric C1573A could be retrimerized into an assembly-competent form by adding intact LCb. We also discovered that C-terminally deleted light chains produced a heterogeneous population of hubs that were smaller than native hubs, but were assembly active. We propose a model showing how light chains reinforce the puckered clathrin triskelion. Finally, the ability of light chains to retrimerize C1573A hub suggests that the structural role of light chain may be conserved in yeast and mammals.
SummaryHuntington's disease is a genetic neurological disorder that is triggered by the dissociation of the huntingtin protein (htt) from its obligate interaction partner Huntingtin-interacting protein 1 (HIP1). The release of htt permits HIP-protein interactor (HIPPI) to bind to its recognition site on HIP1 to form a HIPPI/HIP1 complex that recruits Procaspase-8 to begin the process of apoptosis. The interaction module between HIPPI and HIP1 was predicted to resemble a death-effector domain (DED). Our 2.8 Å crystal structure of the HIP1 371-481 sub-fragment that includes F432 and K474 important for HIPPI binding is not a DED, but is a partially opened coiled-coil. The HIP1 371-481 model reveals a basic surface we hypothesize is suitable for binding HIPPI. There is an opened region next to the putative HIPPI site that is highly negatively charged. The acidic residues in this region are highly conserved in HIP1 and a related protein, HIP1R from different organisms, but are not conserved in the yeast homolog of HIP1, sla2p. We have modeled ∼85% of the coiled-coil domain by joining our new HIP1 371-481 structure to the HIP1 482-586 model (PDB code: 2NO2). Finally, the middle of this coiled-coil domain may be intrinsically flexible and suggests a new interaction model where HIPPI binds to a "U" shaped HIP1 molecule.
The interaction between HIP family proteins (HIP1 and HIP12/1R) and clathrin is fundamental to endocytosis. We used circular dichroism (CD) to study the stability of a HIP1 sub-fragment (aa468-530) that is splayed open. CD thermal melts show HIP1 468-530 is only stable at low temperatures, but this HIP1 fragment contains a structural unit that does not melt out even at 83°C. We then created HIP1 mutants to probe our hypothesis that a short hydrophobic path in the opened region is the binding site for clathrin light chain. We found that the binding of hub/LCb was sensitive to mutating two distantly separated basic residues (K474 and K494). The basic patches marked by K474 and K494 are conserved in HIP12/1R. The lack of conservation in sla2p (S. cerevisiae), HIP1 from D. melanogaster and HIP1 homolog ZK370.3 from C. elegans implies the binding of HIP1 and HIP1 homologs to clathrin light chain may be different in these organisms.
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