Fine mapping of the alpha-actinin binding site within cysteine-rich protein

The cysteine-rich proteins (CRPs) are a family of highly conserved LIM tan acronym derived from the three gene products lin-11, isl-1 and mec-3) domai n proteins that have been implicated in muscle differentiation. All CRP fam ily members characterized so far have been shown to interact with the filam entous actin crosslinker a-actinin. The region of CRP required for this int eraction has previously been broadly mapped to the molecule's N-terminal ha lf. Here we report that the alpha-actinin-binding region of CRP, which we h ave mapped by using a combination of blot overlay and Western immunoblot te chniques, is confined to an 18-residue sequence occurring within the protei n'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-f old 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 c apacity might be attributed to the disruption of a key structural element.