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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.
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