Physical characterization and crystallization of the carbohydrate-recognition domain of a mannose-binding protein from rat.
A portion of rat mannose-binding protein A (MBP-A), a Ca(2+)-dependent animal lectin, has been overproduced in a bacterial expression system, biochemically characterized, and crystallized. A fragment corresponding to the COOH-terminal 115 residues of native MBP-A, produced by subtilisin digestion of the bacterially expressed protein, contains the carbohydrate-recognition domain (CRD). Gel filtration, chemical cross-linking, and crystallographic self-rotation function analyses indicate that the subtilisin fragment is a dimer, although the complete bacterially expressed fragment, containing the neck and CRD of MBP-A, is a trimer. Crystals of the minimal CRD, obtained only as a complex with a Man6GlcNAc2Asn glycopeptide, diffract to Bragg spacings of at least 1.7 A. Several trivalent lanthanide ions (Ln3+) can substitute for Ca2+, as assessed by their ability to support carbohydrate binding and to protect the CRD from proteolysis in a manner similar to that observed for Ca2+. These assays indicate that Ln2+ binds about 30 times more tightly than Ca2+ to the CRD, and that two Ca2+ or Ln3+ bind to each monomer, a result confirmed by determination of the Ho3+ positions in a Ho(3+)-containing crystal of the CRD. Crystals grown in the presence of Ln3+ belong to different space groups from those obtained with Ca2+ and are therefore not useable for traditional crystallographic phase determination methods, but are well-suited for high resolution structure determination by multiwavelength anomalous dispersion phasing.
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032.
W I
WI
Weis
G V
GV
Crichlow
H M
HM
Murthy
W A
WA
Hendrickson
K
K
Drickamer
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D002240:Carbohydrate Sequence; D002352:Carrier Proteins / Q000737:chemistry*; D002412:Cations; D002850:Chromatography, Gel; D003461:Crystallography; D004252:DNA Mutational Analysis; D007811:Lanthanum / Q000737:chemistry; D037102:Lectins / Q000737:chemistry*; D037241:Mannose-Binding Lectins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D002412:Cations; D037102:Lectins; D037241:Mannose-Binding Lectins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007811:Lanthanum; D002118:Calcium