Pinpoint mapping of recognition residues on the cohesin surface by progressive homologue swapping

Dekel Nakar, T Handelsman, Yoram Shoham, HP Fierobe, JP Belaich, E Morag, R Lamed, Ed Bayer

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

Abstract

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 ∼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.

Original languageEnglish
Pages (from-to)42881-42888
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number41
DOIs
Publication statusPublished - 8 Oct 2004

Funding

We thank Meir Wilchek for critical reading of the manuscript.

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry
  • Cell Biology

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