Crystal Structure of the Cysteine-Rich Domain of Mannose Receptor Complexed with a Sulfated Carbohydrate Ligand

The macrophage and epithelial cell mannose receptor (MR) binds carbohydrates on foreign and host molecules. Two portions of MR recognize carbohydrates: tandemly arranged C-type lectin domains facilitate carbohydrate-dependent macrophage uptake of infectious organisms, and the NH2-terminal cysteine-rich domain (Cys-MR) binds to sulfated glycoproteins including pituitary hormones. To elucidate the mechanism of sulfated carbohydrate recognition, we determined crystal structures of Cys-MR alone and complexed with 4-sulfated-N-acetylgalactosamine at 1.7 and 2.2 Å resolution, respectively. Cys-MR folds into an approximately three-fold symmetric β-trefoil shape resembling fibroblast growth factor. The sulfate portions of 4-sulfated-N-acetylgalactosamine and an unidentified ligand found in the native crystals bind in a neutral pocket in the third lobe. We use the structures to rationalize the carbohydrate binding specificities of Cys-MR and compare the recognition properties of Cys-MR with other β-trefoil proteins

Crystallization and X-ray Structure Determination of Cytochrome c_2 from Rhodobacter sphaeroides in Three Crystal Forms

Cytochrome c_2 serves as the secondary electron donor that reduces the photo-oxidized bacteriochlorophyll dimer in photosynthetic bacteria. Cytochrome c_2 from Rhodobacter sphaeroides has been crystallized in three different forms. At high ionic strength, crystals of a hexagonal space group (P6_122) were obtained, while at low ionic strength, triclinic (P1) and tetragonal (P4_12_12) crystals were formed. The three-dimensional structures of the cytochrome in all three crystal forms have been determined by X-ray diffraction at resolutions of 2.20 Å (hexagonal), 1.95 Å, (triclinic) and 1.53 Å (tetragonal). The most significant difference observed was the binding of an imidazole molecule to the iron atom of the heme group in the hexagonal structure. This binding displaces the sulfur atom of Met 100, which forms the axial ligand in the triclinic and tetragonal structures

Crystal Structure of the Hemochromatosis Protein HFE and Characterization of Its Interaction with Transferrin Receptor

AbstractHFE is an MHC-related protein that is mutated in the iron-overload disease hereditary hemochromatosis. HFE binds to transferrin receptor (TfR) and reduces its affinity for iron-loaded transferrin, implicating HFE in iron metabolism. The 2.6 Å crystal structure of HFE reveals the locations of hemochromatosis mutations and a patch of histidines that could be involved in pH-dependent interactions. We also demonstrate that soluble TfR and HFE bind tightly at the basic pH of the cell surface, but not at the acidic pH of intracellular vesicles. TfR:HFE stoichiometry (2:1) differs from TfR:transferrin stoichiometry (2:2), implying a different mode of binding for HFE and transferrin to TfR, consistent with our demonstration that HFE, transferrin, and TfR form a ternary complex

Crystal Structure of Human ZAG, a Fat-Depleting Factor Related to MHC Molecules

Zn-α_2-glycoprotein (ZAG) is a soluble protein that is present in serum and other body fluids. ZAG stimulates lipid degradation in adipocytes and causes the extensive fat losses associated with some advanced cancers. The 2.8 angstrom crystal structure of ZAG resembles a class I major histocompatibility complex (MHC) heavy chain, but ZAG does not bind the class I light chain β_2-microglobulin. The ZAG structure includes a large groove analogous to class I MHC peptide binding grooves. Instead of a peptide, the ZAG groove contains a nonpeptidic compound that may be implicated in lipid catabolism under normal or pathological conditions

Crystallographic Analyses of Site-Directed Mutants of the Photosynthetic Reaction Center from Rhodobacter sphaeroides

Seven site-directed mutants of the bacterial photosynthetic reaction center (RC) from the 2.4.1 and WS 231 wild-type strains of Rhodobacter sphaeroides have been crystallized and their X-ray diffraction analyzed to resolutions between 3.0 and 4.0 Å. The mutations can be divided into four distinct categories: (1) mutations altering cofactor composition that affect electron transfer and quantum yield, His M202 → Leu (M202HL), His L173 → Leu (L173HL), and Leu M214 → His (M214LH); (2) a mutation in the proposed pathway of electron transfer altering electron-transfer kinetics, Tyr M210 → Phe (M210YF); (3) a mutation around the non-heme iron resulting in an iron-less reaction center, His M219 → Cys (M219HC); and (4) mutations around the secondary electron acceptor, a ubiquinone, affecting proton transfer and quinone turnover, Glu L212 → Gin (L212EQ) and Asp L213 → Asn (L213DN). Residues L173 and M202 are within bonding distance of the respective magnesiums of the two bacteriochlorophylls of the BChl special pair, while M214 is close to the bacteriopheophytin on the active A branch of the RC. The L173HL and M202HL crystal structures show that the respective bacteriochlorophylls are replaced with bacteriopheophytins (i.e., loss of magnesium) without significant structural perturbations to the surrounding main-chain or side-chain atoms. In the M214LH mutant, the bacteriopheophytin has been replaced by a bacteriochlorophyll, and the side chain of His M214 is within ligand distance of the magnesium. The M210YF, L212EQ, and L213DN mutants show no significant tertiary structure changes near the mutation sites. The M219HC diffraction data indicate that the overall tertiary structure of the reaction center is maintained in the absence of the non-heme iron

Crystallographic Analyses of Site-Directed Mutants of the Photosynthetic Reaction Center from Rhodobacter sphaeroides

Seven site-directed mutants of the bacterial photosynthetic reaction center (RC) from the 2.4.1 and WS 231 wild-type strains of Rhodobacter sphaeroides have been crystallized and their X-ray diffraction analyzed to resolutions between 3.0 and 4.0 Å. The mutations can be divided into four distinct categories: (1) mutations altering cofactor composition that affect electron transfer and quantum yield, His M202 → Leu (M202HL), His L173 → Leu (L173HL), and Leu M214 → His (M214LH); (2) a mutation in the proposed pathway of electron transfer altering electron-transfer kinetics, Tyr M210 → Phe (M210YF); (3) a mutation around the non-heme iron resulting in an iron-less reaction center, His M219 → Cys (M219HC); and (4) mutations around the secondary electron acceptor, a ubiquinone, affecting proton transfer and quinone turnover, Glu L212 → Gin (L212EQ) and Asp L213 → Asn (L213DN). Residues L173 and M202 are within bonding distance of the respective magnesiums of the two bacteriochlorophylls of the BChl special pair, while M214 is close to the bacteriopheophytin on the active A branch of the RC. The L173HL and M202HL crystal structures show that the respective bacteriochlorophylls are replaced with bacteriopheophytins (i.e., loss of magnesium) without significant structural perturbations to the surrounding main-chain or side-chain atoms. In the M214LH mutant, the bacteriopheophytin has been replaced by a bacteriochlorophyll, and the side chain of His M214 is within ligand distance of the magnesium. The M210YF, L212EQ, and L213DN mutants show no significant tertiary structure changes near the mutation sites. The M219HC diffraction data indicate that the overall tertiary structure of the reaction center is maintained in the absence of the non-heme iron