Structural role of calcium for the organization of the cellulosome of Clostridium thermocellum.
The cellulosome of Clostridium thermocellum is a multipolypeptide complex of structural and catalytic subunits. Several of the catalytic subunits have at the carboxyl end a conserved duplicated region (CDR) which interacts with internally repeated elements (IREs) of scaffolding subunits such as CipA. This interaction requires calcium. The two parts of the CDR region here designated CDR1 and CDR2 (closest to the carboxyl end) each consist of about 20 amino acids residues. As shown in our previous paper [Choi, S.K., & Ljungdahl, L.G. (1996) Biochemistry 35, 4897-4905], treatment of the cellulosome with ethylenediaminetetraacetic acid (EDTA) under aerobic conditions disintegrates the cellulosome with formation of truncated catalytic subunits. The cleavage is at a specific asparagine residue located within CDR1 and occurs with complete loss of CDR2. Two branched peptides containing the amino acid sequences of CDR1 and CDR2 (designated bCDR1 and bCDR2) were synthesized, and specific antibodies were raised against them. These antibodies did not cross react with bCDR1 or bCDR2, respectively. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, it was observed that about 15 subunits of the cellulosome reacted with anti-bCDR1 and anti-bCDR2. In a similar experiment with EDTA-treated cellulosomes, these subunits reacted with anti-bCDR1 but not with anti-bCDR2, showing that they lost the bCDR2 epitope and were truncated. The peptide bCDR1 binds calcium, whereas bCDR2 does not. Furthermore, bCDR1 but not bCDR2 binds to CipA, presumably at IRE regions. This binding requires calcium. A model is proposed for the binding of the catalytic subunits to CipA which involves CDR1, an IRE, and calcium.
Choi
S K
SK
Department of Biochemistry and Molecular Biology, Center for Biological Resources Recovery, University of Georgia, Athens, 30602-7229, USA.
Ljungdahl
L G
LG
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D003013:Clostridium / Q000187:drug effects / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*
D001426:Bacterial Proteins; C080369:CipA protein, Clostridium; D008565:Membrane Proteins; D010455:Peptides; D002482:Cellulose; D004492:Edetic Acid; D002118:Calcium