Clatherin Assembly Lymphoid Myeloid Leukemia Protein (CALM)
Created by Soo Hyun Ahn
Phosphatidylinositol-binding clathrin assembly protein (pdb ID=1HFA) (alternative name: Clathrin assembly lymphoid myeloid leukemia protein (CALM)) from Rattus norvegicus is an assembly protein recruiting clathrin and adaptor proteins to cell membranes at sites of coated-pit formation and clathrin-vesicle assembly. For example, CALM protein is involved in AP-2-dependent clathrin-mediated endocytosis at the neuromuscular junction (2). AP-2 binds to the terminal domain region of the clathrin molecule. A binding of 1-phosphatidylinositol occurs where clathrin selectively and non-covalently interacts with phosphatidlinositol. The molecular weight of CALM is 32823.80 Da, and its isoelectric point (pI) is 9.07.
CALM consists of subunit A and one ligand. The ligand component of CALM is phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). Its ligand provides a binding site for AP-180 and clathrin, serving to tether clathrin to the membrane. The binding site of CALM is uncommon. Compared to typical ligand-binding sites of a pocket or groove of protein, CALM has its binding site on the surface, with the phosphates rested on the tips of the side chains of three lysines and a histidine. The cluster of lysines and a histidine forms a positively charged patch, which is ideal for phosphate-binding protein (1).
The secondary structure of CALM is 59% alpha-helical, with 11 helices and 171 residues. The clathrin-box motif is mostly located in a helix. The α1-α2 loop regions appear on the three protein familes including CALM/AP-180 family and some other proteins with the PtdIns(4,5)P2-binding motif, and epsin family. CALM is structurally similar to the epsin NH2-terminal domain but epsin does not contain PtdIns(4,5)P2-binding site (1).
A triskelion-shaped clathrin(pdb ID=1xi4) is composed of a trimer of heavy-chain subunits of 1,675 residues, each binding a single light chain subunit in the hub domain, which covers residue 1074-1675. The heavy chains of clathrin are involved in its interactions with the light chains, with other clathrin molecules, and with adopter proteins. Three C-terminal heavy chains of clathrin cover residues 1615-1675 containing Pro-Lys rich motifs. A distal leg domain of clathrin covers residues ~523-1073 and are involved in a cage assembly. A terminal domain of clathrin covers residues 1-~479 and functions in AP-2 binding (3). The residues 1210-1516 in the hub domain mediate spontaneous clathrin heavy-chain polymerization as hub fragment folds into an elongated coil of α-helices. The crystal structure of these residues reveals that crossing antiparallel α-helices and a continuous hydrophobic core maintained by aromatic and bulky hydrophobic amino-acid side chains contribute to the rigidity of the protein (9).
For the first comparison protein, synapse-enriched clathrin adaptor protein (pdb ID=1HX8) (AP-180) has 81% identical sequencing identity with CALM. The result of DALI (Z=36.5, rmsd=1.1) and protein Blast (E value=6.89e-125) searches verify that this protein has both primary and tertiary similarities to CALM (5). Its superimposition of two proteins are shown.
As clathrin accessory proteins, both proteins proceed the formation of clathrin-coated vesicles. Both AP-180 and CALM have an epsin N-terminal homology domain that binds to phosphorylated membrane inositol lipids. Through their C-terminal motifs, both proteins interact with other clathrin and AP-2 complex. While both proteins are found in synapses, distributions are different. AP-180 is more heavily located in presynaptic, whereas CALM is located nonselectively in pre and postsynaptic profile.
CALM was identified and named due to its homology to AP-180 and to reflect its involvement in (10;11) chromosomal translocations. This rare chromosomal translocation is found in lymphoid, as well as, myeloid acute leukemias, therefore was named CALM (clathrin assembly lymphoid myeloid leukemia gene) (6).
The distribution of proline, alanine, and charged amino acids suggest a three-domain structure for AP-180. The first trypsin cut occurs at Lys-304 serving the covalent linkage between clathrin binding domain and the acidic central domain. These first 304 residues that bind to clathrin are mostly basic. The next cut occurs at Lys-744 freeing the basic C-terminal domain. (8) For the second comparison protein, epsin(pdb ID=1H0A) shares the N-terminal domain of CALM with a lysine-rich motif (Z=15.5, rmsd=1.1) (5).
The epsin family can be recognized by the presence of a N-terminal lipid-binding ENTH (epsin N-terminal homology) domain and a clathrin domain. The ENTH domain has the first 150 residues that are homologous to the N-terminal of AP180 and CALM, except the lipid-binding residues are different. Epsin, similar to AP-180, can recruit and promote clathrin polymerization on the monolayer but the clathrin is less uniformly polymerized and recruitment of clathrin to liposomes by epsin is less efficient (10).