FIGURE 1.

Probing oligopeptide arrays revealed Lasp-1 binding motifs in Krp1. a, oligopeptide membranes that were arrays of decamer oligopeptides spanning the entire length of Krp1, modulating by +1 amino acid at a time, were probed with recombinant His₆-Lasp-1. Two binding sites in the region of the C-terminal domain were identified (panel i), and these oligopeptides did not give a positive result on control membranes where no His₆-Lasp-1 had been applied (panel ii). These two motifs corresponded to Tyr³¹⁷–Thr³²⁷ (KLBP1) and Thr⁵⁶³–Asp⁵⁷⁴ (KLBP2). b, each motif was scanned by mutating each residue in turn to alanine. This demonstrated that the residues involved in Lasp-1 binding were Tyr³¹⁷, Glu³²¹, Glu³²³, Cys³²⁴, Tyr³²⁵, and Leu³²⁶ in KLBP1 and Asp⁵⁶⁷, Ile⁵⁶⁸, Trp⁵⁶⁹, Tyr⁵⁷¹, Glu⁵⁷², Asp⁵⁷³, and Asp⁵⁷⁴ in KLBP2. Mutation of Lys⁵⁷⁰ to alanine in KLBP2 consistently gave an increase in signal from Lasp-1 binding in replicate blots. WT, wild type. c, mutation of any of the 4 residues following Lys⁵⁷⁰ in KLBP2 resulted in an almost complete loss of Lasp-1 binding (all mutations are noted in bold). However, any or all of residues 571–574 could be removed without effect if Lys⁵⁷⁰ was mutated to alanine.