Fine mapping of the alpha-actinin binding site within cysteine-rich protein.
The cysteine-rich proteins (CRPs) are a family of highly conserved LIM (an acronym derived from the three gene products lin-11, isl-1 and mec-3) domain proteins that have been implicated in muscle differentiation. All CRP family members characterized so far have been shown to interact with the filamentous actin cross-linker alpha-actinin. The region of CRP required for this interaction has previously been broadly mapped to the molecule's N-terminal half. Here we report that the alpha-actinin-binding region of CRP, which we have mapped by using a combination of blot overlay and Western immunoblot techniques, is confined to an 18-residue sequence occurring within the protein's N-terminal glycine-rich repeat. A site-directed mutagenesis analysis of the binding region has revealed the critical importance of a single lysine residue (lysine 65 in human CRP1). Alterations at this site lead to a 10-fold decrease in alpha-actinin binding in comparison with wild-type CRP. The critical lysine residue localizes within a short alpha-helix, raising the possibility that mutagenesis-induced alterations in alpha-actinin-binding capacity might be attributed to the disruption of a key structural element.
Department of Biology and Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112-5550, USA.
B D
BD
Harper
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MC
Beckerle
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Pomiès
D000185:Actinin / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism*; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D060588:LIM Domain Proteins; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
C486177:Csrp1 protein, mouse; D060588:LIM Domain Proteins; D009124:Muscle Proteins; D009687:Nuclear Proteins; D000185:Actinin; D003545:Cysteine; D005998:Glycine