A single surface tryptophan in the chitin-binding domain from Bacillus circulans chitinase A1 plays a pivotal role in binding chitin and can be modified to create an elutable affinity tag.
Site-directed mutagenesis was carried out to investigate the roles of a number of highly conserved residues of the chitin-binding domain (ChBD) of Bacillus circulans chitinase A1 (ChiA1) in the binding of chitin. Analysis of single alanine replacement mutants showed that mutation of an exposed tryptophan residue (Trp(687)) impaired the binding to chitin, while mutation of other highly conserved residues, most carrying aromatic or hydrophobic side chains, did not significantly affect the binding activity. Interestingly, replacement of Trp(687) with phenylalanine significantly reduced chitin-binding activity at lower salt concentrations (0-1 M NaCl) but allowed strong binding to chitin at 2 M NaCl. Since Trp(687) is conserved among the ChBDs belonging to the bacterial ChiA1 subfamily, the data presented suggest a general mechanism in which this exposed tryptophan, which is located in the cleft formed between two beta-sheets as revealed by the solution structure [J. Biol. Chem. 275 (2000) 13654], makes a major contribution to ligand binding presumably through hydrophobic interactions. Furthermore, modulation of the chitin-binding activity by the conserved amino acid replacement (W687F) and a shift in the ionic strength of buffer has led to the development of an elutable affinity tag for single column purification of recombinant proteins.
Ferrandon
Sébastien
S
New England Biolabs Inc., 32 Tozer Road, Beverly, MA 01915, USA.
Sterzenbach
Torsten
T
Mersha
Fana B
FB
Xu
Ming-Qun
MQ
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D002686:Chitin / Q000737:chemistry / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D010649:Phenylalanine / Q000737:chemistry; D010957:Plasmids; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis; D016415:Sequence Alignment; D014364:Tryptophan / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D002686:Chitin; D010649:Phenylalanine; D014364:Tryptophan; D002688:Chitinases; D000409:Alanine