Binding sites involved in the interaction of actin with the N‐terminal region of dystrophin

Abstract

Two actinmbinding sites have been identified on human dystrophin by proton NMR spectroscopy of synthetic peptides corresponding to delined regions of the polypeptidcsequencc. These are Act&Binding Site I (ABSI ) located at residues 17-26 and Actin-Binding Site 2 (AEtS2) in the region oi residues 125-156. Using defined fragments of the actin amino acid sequence. ABSI has been shown to bind to actin in the region represented by residues 83-1 I7 and ABS2 to the C-terminal region represented by residues 350-375. These dystrophin-binding sites lie on the exposed domain in the uctin filament.

Deletion analysis of the dystrophin‐actin binding domain

“…Three sites previously proposed to interact with actin are underlined and designated ABS1, ABS2, and ABS3. ABS1 (amino acids [18][19][20][21][22][23][24][25][26][27] and ABS2 (amino acids 131-148) are putative sites of interaction between actin and dystrophin identified by NMR spectroscopy experiments with synthetic dystrophin peptides [13,14]. ABS3 (amino acids 91 117) is highly conserved strech of amino acids shown to be important for the actin binding activity of ABP 120, Bcr-Abl, and ~-actinin [20][21][22][23].…”

“…Together these results indicate that neither ABS2 nor ABS3 is required for dytrophin binding to actin. It therefore seemed possible that the binding activity of DYS90 was due to the remaining site, ABS 1, which has been suggested as a potential binding site by Levine et al [13,14]. To test this hypothesis, we constructed a fusion protein (DYSd90) containing the first 90 amino acids of dystrophin but deleted for 4 amino acids, KTFT, the amino acids in ABSl proposed to be involved in actin interactions [14], and tested the ability of this protein to bind actin in the solid-phase binding assay.…”

“…Underlined are the three putative sites of interaction between dystrophin and actin based on experimental results with human dystrophin or with similarities to other actin binding proteins. ABS1 and ABS2 were identified by Levine et al [13,14] using NMR spectroscopy to identify interactions between synthetic dystrophin peptides and actin. ABS3 is a highly conserved stretch of amino acids shown to be important for actin binding of ABP-120, Bcr-Abl, and a-actinin [20][21][22][23].…”

“…It has also been shown that fusion proteins containing at least the N-terminal 233 amino acids of dystrophin bind actin in in vitro pelleting assays, and actin-stress fibers in situ when expressed in non-muscle cells [ 1 I, 121. In addition, two putative actin binding sites within the N-terminus have been identified by NMR spectroscopy experiments using synthetic dystrophin peptides [13,14]. To delineate further the actin binding domains of dystrophin, we have expressed and isolated various bacterial fusion proteins containing deletions of the N-terminal domain of dystrophin and have tested their ability to bind actin in vitro.…”

Purification of dystrophin‐related protein (utrophin) from lung and its identification in pulmonary artery endothelial cells

“…The homology of the amino acid sequence between DRP and dystrophin is high for the two domains which have important physiological functions [2,12,24]: (1) the amino-terminal domain which contains the actin-binding site [2,[25][26][27]; and (2) the cysteine-rich and carboxylterminal domains which are involved in the interaction with the sarcolemmal glycoprotein complex in skeletal muscle [28,29]. On the other hand, the sequence homology between these two proteins is relatively low for the rod domain which consists of repeats of triple helix [2,24].…”