Structural basis of the cytoplasmic tail of adhesion molecule CD43 and its binding to ERM proteins.

CD43/leukosialin/sialophorin is the major adhesion molecule in most hematopoietic cells and belongs to the sialomucin superfamily. In leukocyte emigration and activation, the exclusion of CD43 from the immunological synapse is an essential step. While the exclusion requires binding of the cytoplasmic region to ERM (ezrin/radixin/moesin) proteins, the detailed specific nature of the interaction between CD43 and ERM proteins is obscure. We have characterized the conformational properties of the CD43 cytoplasmic region, consisting of 124 amino acid residues, by hydrodynamic and spectroscopic measurements. Sedimentation equilibrium and velocity studies of ultracentrifugation revealed that the CD43 cytoplasmic peptide exists in a monomeric and extended form in solution. The crystal structure of the complex between the radixin FERM (4.1 and ERM) domain and the CD43 juxtamembrane region peptide reveals that the nonpolar region of the peptide binds subdomain C of the FERM domain. CD43 lacks the Motif-1 sequence for FERM binding found in the FERM-intercellular adhesion molecule-2 complex but possesses two conserved leucine residues that dock into the hydrophobic pocket of subdomain C without forming a 3(10)-helix. The FERM-binding site on CD43 is overlapped with the functional nuclear localization signal sequence. Our structure suggests that regulation of ERM binding may be coupled with regulated intramembrane proteolysis of CD43 followed by the nuclear transfer of the cytoplasmic peptide.

Structural basis of PSGL-1 binding to ERM proteins.

P-selectin glycoprotein ligand-1 (PSGL-1), an adhesion molecule with O-glycosylated extracellular sialomucins, is involved in leukocyte inflammatory responses. On activation, ezrin-radixin-moesin (ERM) proteins mediate the redistribution of PSGL-1 on polarized cell surfaces to facilitate binding to target molecules. ERM proteins recognize a short binding motif, Motif-1, conserved in cytoplasmic tails of adhesion molecules, whereas PSGL-1 lacks Motif-1 residues important for binding to ERM proteins. The crystal structure of the complex between the radixin FERM domain and a PSGL-1 juxtamembrane peptide reveals that the peptide binds the groove of FERM subdomain C by forming a beta-strand associated with strand beta5C, followed by a loop flipped out towards the solvent. The Motif-1 3(10) helix present in the FERM-ICAM-2 complex is absent in PSGL-1 given the absence of a critical Motif-1 alanine residue, and PSGL-1 reduces its contact area with subdomain C. Non-conserved positions are occupied by large residues Met9 and His8, which stabilize peptide conformation and enhance groove binding. Non-conserved residues play an important role in compensating for loss of binding energy resulting from the absence of conserved residues important for binding.

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tails of adhesion molecules CD43 and PSGL-1.

Radixin is a member of the ERM proteins that cross-link plasma membranes and actin filaments. The FERM domains located in the N-terminal regions of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. Here, crystals of the radixin FERM domain bound to the cytoplasmic peptides of two adhesion molecules, CD43 and PSGL-1, have been obtained. Crystals of the radixin FERM domain bound to CD43 belong to space group P4(3)22, with unit-cell parameters a = b = 68.72, c = 201.39 A, and contain one complex in the crystallographic asymmetric unit. Crystals of the radixin FERM domain bound to PSGL-1 belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 80.74, b = 85.73, c = 117.75 A, and contain two complexes in the crystallographic asymmetric unit. Intensity data sets were collected to a resolution of 2.9 A for the FERM-CD43 complex and 2.8 A for the FERM-PSGL-1 complex.