MODIFICATION BY SITE-DIRECTED MUTAGENESIS OF THE SPECIFICITY OF ERYTHRINA-CORALLODENDRON LECTIN FOR GALACTOSE DERIVATIVES WITH BULKY SUBSTITUENTS AT C-2

Examination of the three-dimensional structure of Erythrina coralloden dron lectin (ECorL) in complex with a ligand (lactose), the first of i ts kind for a Gal/GalNAc-specific lectin [(1991) Science 254, 862 866] , revealed the presence of a hydrophobic cavity, surrounded by Tyr108 and Pro134-Trp135, which can accommodate bulky substituents such as ac etamido or dansylamido (NDns) at C-2 of the lectin-bound galactose. Co mparison of the primary sequence of ECorL with that of soybean aggluti nin, specific for galactose and its C-2 substituted derivatives, and o f peanut agglutinin, specific for galactose only, showed that in soybe an agglutinin, Tyr108 is retained, and Pro134-Trp135 is replaced by Se r-Trp, whereas in peanut agglutinin, the former residue is replaced by Thr and the dipeptide by Ser-Glu-Tyr-Asn. Three mutants of ECorL were therefore constructed: L2, in which Pro134-Trp135 was replaced by Ser Glu-Tyr-Asn; Y108T, in which Tyr108 was replaced by Thr and the doubl e mutant L2; Y108T. They were expressed in Escherichia coli, as done f or recombinant ECorL [(1992) Eur. J. Biochem. 205, 575-581]. The mutan ts had the same hemagglutinating activity as native or rECorL. Their s pecificity for galactose, GalNAc and MebetaGalNDns was examined by inh ibition of hemagglutination and of the binding of the lectin to immobi lized asialofetuin; in addition, their association constants with Melp haGalNDns and MebetaGalNDns were measured by spectrofluorimetric titra tion. The results showed that Y108T had essentially similar specificit y as the native and recombinant lectins. The affinity of L2 and L2;Y10 8T for galactose was also the same as ECorL, but they had a lower affi nity for GalNAc and markedly diminished affinity for the dansyl sugars (up to 43 times, or 2 kcal, less). This appears to be largely due to steric hindrance by the two additional amino acids present in the cavi ty region in these mutants. Our findings also provide an explanation f or the inability of PNA to accommodate C-2-substituted galactose deriv atives at its primary subsite.