Pinpoint mapping of recognition residues on the cohesin surface by progressive homologue swapping.
The high affinity cohesin-dockerin interaction dictates the suprastructural assembly of the multienzyme cellulosome complex. The connection between affinity and species specificity was studied by exploring the recognition properties of two structurally related cohesin species of divergent specificity. The cohesins were examined by progressive rounds of swapping, in which corresponding homologous stretches were interchanged. The specificity of binding of the resultant chimeric cohesins was determined by enzyme-linked affinity assay and complementary protein microarray. In succeeding rounds, swapped segments were systematically contracted, according to the binding behavior of previously generated chimeras. In the fourth and final round we discerned three residues, reputedly involved in interspecies binding specificity. By replacing only these three residues, we were able to convert the specificity of the resultant mutated cohesin, which bound preferentially to the rival dockerin with approximately 20% capacity of the wild-type interaction. These residues represent but 3 of the 16 contact residues that participate in the cohesin-dockerin interaction. This approach allowed us to differentiate, in a structure-independent fashion, between residues critical for interspecies recognition and binding residues per se.
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
David
D
Nakar
Tal
T
Handelsman
Yuval
Y
Shoham
Henri-Pierre
HP
Fierobe
Jean-Pierre
JP
Belaich
Ely
E
Morag
Raphael
R
Lamed
Edward A
EA
Bayer
D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D003013:Clostridium / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005656:Fungal Proteins; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D040081:Protein Array Analysis; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D000097722:Cohesins
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004247:DNA; D005656:Fungal Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000097722:Cohesins