Solution Conformation of the Antibody-Bound Tyrosine Phosphorylation Site of the Nicotinic Acetylcholine Receptor β-Subunit in Its Phosphorylated and Nonphosphorylated States

Abstract

Phosphorylation of the acetylcholine receptor (AChR) seems to be responsible for triggering several effects including its desensitization and aggregation at the postsynaptic membrane and probably initiates a signal transduction pathway at the postsynaptic membrane. To study the structural and functional role of the tyrosine phosphorylation site of the AChR beta-subunit and contribute to the in-depth understanding of the structural basis of the ion channel function, we synthesized four peptides containing the phosphorylated and nonphosphorylated sequences (380-391) of the human and Torpedo AChR beta-subunits and studied their interaction with a monoclonal antibody (mAb 148) that is known to bind to this region and that is capable of blocking ion channel function. All four peptides were efficient inhibitors of mAb 148 binding to AChR, although the nonphosphorylated human peptide was considerably less effective than the three others. We then investigated the conformation acquired by all four peptides in their antibody-bound state, which possibly illustrates the local conformation of the corresponding sites on the intact AChR molecule. The phosphorylated human and Torpedo peptides adopted a distorted 3(10) helix conformation. The nonphosphorylated Torpedo peptide, which is also an efficient inhibitor, was also folded. In contrast, the nonphosphorylated human peptide (a less efficient inhibitor) presented an extended structure. It is concluded that the phosphorylation of the AChR at its beta-subunit Tyr site leads to a significant change in its conformation, which may affect several functions of the AChR.

Conformational Changes in Protein Loops and Helices Induced by Post-Translational Phosphorylation

“…Zhou and Abagyan calculated the binding energy of phosphotyrosine-containing peptides to SH2 and PTB domains [17]. Several molecular dynamics [18–30] and homology modeling [27,31,32] studies have been reported involving phosphorylated proteins [18–20,24,25,31–33] or peptides [21–23,26,27,34]. A Car-Parinello study has been reported [35], as well as a creative application of docking algorithms to investigate conformational changes upon phosphorylation of the N-terminal tail in phenylalanine hydroxylase [36].…”

Conformational Changes in Protein Loops and Helices Induced by Post-Translational Phosphorylation

“…Zhou and Abagyan calculated the binding energy of phosphotyrosine-containing peptides to SH2 and PTB domains [17]. Several molecular dynamics [18][19][20][21][22][23][24][25][26][27][28][29][30] and homology modeling [27,31,32] studies have been reported involving phosphorylated proteins [18][19][20]24,25,[31][32][33] or peptides [21][22][23]26,27,34]. A Car-Parinello study has been reported [35], as well as a creative application of docking algorithms to investigate conformational changes upon phosphorylation of the N-terminal tail in phenylalanine hydroxylase [36].…”

“…After phosphorylating a protein in silico, we need to explore the new energy landscape and identify the new global energy minimum, if in fact it is significantly different than that of the unphosphorylated protein. In principle, molecular dynamics could be used for this purpose, and in some cases, this type of strategy has been successful, in studies by ourselves and others [18][19][20][21][22][23][24][25][26][27][28][29][30]. However, the timescales for converting from the nonphospho to the phosphorylated form are unknown, and in fact are not physically meaningful because the in silico phosphorylation is alchemical, i.e., we do not attempt to mimic a kinase actually performing the phosphorylation.…”

Monoclonal Antibodies to Esophageal Cancer–Related Gene2 Protein

“…(13)(14)(15)(16) Although the mechanism of this modification is unknown, at least two possibilities exist: re-arrangement of disulfide bonds (17) and phosphorylation. (18,19) ECRG2 belongs to the kazal family which contains the conservative pattern x(8)-C-x(6)-C-x(7)-C-x(6)-Yx(3)-C-x(2,3)-C-x(17)-C, and these conservative cysteines are involved in three disulfide bonds. (20) The last cysteine located on c-terminal sequences was synthesized as immunogen for generating MAbs, so this cysteine may be involved in the formation of disulfide bond loop.…”

“…Another possibility for the alteration of MAb epitopes involves phosphorylated modification which will regulate nearly every aspect of cell life including protein folding, solubility, antigenicity, half-life, localization, and cell-to-cell interactions. (18,19) Bio-information analysis predicted that there is a serine phosphorylation site ser73 located on c-terminal of ECRG2 peptide, and these findings raised the possibility that the differential use of common phosphorylation sites in or near MAb epitopes or occupation of unique phosphorylation sites might contribute to the alteration of MAb reactivity.…”