Mutational and structural analysis of the lectin activity in binding domain 2 of ricin B chain.
The study of the lectin binding sites of ricin B chain and of other homologous members of the small gene family that make up ricin-like molecules has revealed a number of key contact residues involved in sugar binding. In particular, on the basis of data generated by the X-ray crystallographic structure of ricin, comparisons of sequence homologies to other ricin-like molecules and substrate binding studies with these molecules, it has been proposed that His248 of Ricinus communis agglutinin (RCA) B chain may interfere with galactose binding in the second binding domain of that lectin. To test that hypothesis, single binding domain 2 (SBD2) of ricin B chain was expressed as a gene 3 fusion protein on the surface of fd phage to measure directly the effect of mutational changes on this binding site. Replacement of tyrosine with histidine at amino acid position 248 of SBD2 of ricin B chain was shown to reduce lectin activity. The sequences of RCA and ricin B chains were aligned and compared with the tertiary structure of ricin B chain to select various mutations that were introduced as controls in the study. One of these controls, Leu247 to Val247, displayed increased affinity for galactosides. The role of sequence changes is discussed in relation to the structural and functional divergence in these molecules.
ImmunoGen, Inc., Cambridge, MA 02139.
S M
SM
Lehar
J T
JT
Pedersen
R S
RS
Kamath
C
C
Swimmer
V S
VS
Goldmacher
J M
JM
Lambert
W A
WA
Blättler
B C
BC
Guild
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003090:Coliphages / Q000235:genetics; D017343:Genes, Plant; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D037121:Plant Lectins; D010947:Plants, Toxic; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012276:Ricin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012278:Ricinus / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D037121:Plant Lectins; D011993:Recombinant Fusion Proteins; D012276:Ricin