Abstract
Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.
Highlights
Plant cell walls, composed mainly of cellulose, hemicellulose, and lignin, represent one of the most abundant sources of biomass on Earth [1], and microbial glycoside hydrolases play a
The crystal structures of the two proteins represent the only examples in Carbohydrate binding modules (CBMs) family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking
A single CBM may be able to bind to differently configured substrates, and this has been demonstrated for the Piromyces equi NCP1 CBM29-1 and CBM29-2, Caldanaerobius polysaccharolyticus Man5A CBM16-1 and CBM16-2, Clostridium thermocellum Lic26A-Cel5E CBM11 and CtCel9DCel44A CBM44 (5, 12, 14 –16)
Summary
Materials—The construction of the plasmids expressing the C. polysaccharolyticus (formerly Thermoanaerobacterium polysaccharolyticum and reclassified by Lee et al [22]) Man5A carbohydrate binding modules CBM16-1 and CBM16-2 were described by Bae et al [16]. Gene Expression and Protein Purification—The plasmids encoding either the wild-type or a mutant of CBM16-1 or CBM16-2 were transformed individually into BL21(DE3) CodonPlus RIL competent cells (Stratagene, La Jolla, CA) that were selected on LB plates supplemented with 100 g/ml ampicillin and 50 g/ml chloramphenicol and cultured at 37 °C. The data collected were fitted to a nonlinear regression using a single site binding model (MicroCal Origin software), and the thermodynamic parameters were calculated with both the Gibbs free energy equation ⌬G ϭ ⌬H – T⌬S and the relationship ⌬G ϭ –RT lnKa. Crystallization and X-ray Data Collection—Initial co-crystallization efforts of CBM16-1 variants in complex with either mannopentaose or cellopentaose focused around the crystallization conditions originally reported for wild-type CBM16-1, with the respective oligosaccharide [16]. Of the aromatic and polar residues, three differed in the two CBMs (Gln-21, Asn-97, and Gln-121 in CBM16-1 corresponded to Gly-21, Arg-97, and Glu-121 in CBM16-2)
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