Structural Insights into Calmodulin-regulated L-selectin Ectodomain Shedding

Abstract

Background: Calmodulin inhibits the proteolysis of L-selectin's extracellular domains through an unknown mechanism. Results: Calmodulin binds the juxtamembrane and predicted membrane-spanning regions of L-selectin in a calcium-dependent manner. Conclusion: Binding of calmodulin to the cytoplasmic/transmembrane domain of L-selectin enacts a conformational change in the extracellular domains preventing cleavage. Significance: Elucidating the mechanisms of L-selectin shedding is critical to understanding leukocyte trafficking.

A Disintegrin and Metalloprotease (ADAM) 10 and ADAM17 Are Major Sheddases of T Cell Immunoglobulin and Mucin Domain 3 (Tim-3)

“…This might influence accessibility of Tim-3 or the cleavage site for ADAM17, respectively. It has been proposed that inhibition of L-selectin shedding by calmodulin is mediated by such a process, in which calmodulin binds to amino acids of the intracellular and transmembrane domain, thereby changing the conformation of the stalk region and reducing the accessibility for ADAM proteases (37). Whether such a mechanism might also contribute to Tim-3 shedding has to be investigated in future experiments.…”

“…CaM binds to the membrane-proximal region of the cytoplasmic domain plus a small region of the membrane-spanning domain of L-selectin (37). To analyze if the CaM-binding site of Tim-3 is also located in the intracellular domain of Tim-3, we used the AP-Tim-3 cDNA lacking the coding sequence of the complete intracellular domain from Lys 225 to Gln 301 (AP-Tim-3⌬ICD) and the deletion variant AP-Tim-3⌬W226-Gln 301 (⌬ICDϩ1), which differs from AP-Tim-3⌬ICD by the addition of the first intracellular residue Lys 225 .…”

Structural Characterization of the Interaction of Human Lactoferrin with Calmodulin

“…More than any other type of amino acid in these patches, the methionine side chains, in particular their terminal ε-methyl groups, are directly involved in target binding [34], [35], [55], [61], [62], and their presence has been shown through mutant-based studies to be essential [63], [64]. Using CaM 13 C, 1 H-labeled on the methionine ε-methyl group but otherwise isotopically 2 H and 12 C, [( 1 H/ 13 C-methyl-Met)/ 2 H/ 15 N CaM], the high number of methionine residues in CaM (9 out of 148 amino acid residues) as well as their location in the hydrophobic pockets was exploited to serve as probes for defining the binding interface of apo-LF on Ca 2+ -CaM [34], [35]. Agreeing with the 1 H( 15 N) CSPs observed in the TROSY-HSQC spectrum of apo-Lf-bound Ca 2+ -CaM, the methyl groups of methionines found in the N-terminal lobe of CaM (Met-36 and Met-51) experienced little change in chemical shift upon the addition of apo-Lf (Figure 3C).…”

“…The backbone chemical shift assignments of free or Lf-bound CaM were completed using either uniformly 13 C/ 15 N- or 2 H/ 13 C/ 15 N-labeled CaM prepared in M9 media containing 0.5 g/L 15 NH 4 Cl, 4 g/L [ 13 C 6 ]glucose, in H 2 O or 99.9% 2 H 2 O, respectively. A methionine methyl-labeled version of otherwise fully deuterated CaM, ( 1 H/ 13 C-methyl-Met)/ 2 H/ 15 N CaM, was created by supplementing 100 mg/L 1 H-α,ε- 13 C-ε- 2 H-methionine to 99.9% 2 H 2 O M9 media containing 0.5 g/L 15 NH 4 Cl and 4 g/L [ 2 H 7 ]glucose one hour prior to induction with IPTG [34], [35]. Finally, uniformly 2 H/ 15 N-labeled CaM was prepared in 99.9% 2 H 2 O M9 media with 0.5 g/L 15 NH 4 Cl and 4 g/L 2 H 6 -glucose.…”

Over-expression of a human CD62L ecto-domain and a potential role of RNA pseudoknot structures in recombinant protein expression

“…While the structures of E- and P-selectins have been determined with bound carbohydrates [4], such a structure has not been determined for L-selectin, and consequently the structural mechanism of L-selectin binding to its carbohydrate ligand remain unresolved. Furthermore, the only L-selectin structures available are those corresponding to truncated lectin and EGF domain constructs [5, 6], leaving the role of the two sushi domains undefined.…”