Bacterioferritin of Escherichia coli, also known as cytochrome b1, is a hollow, nearly spherical shell made up of 24 identical protein subunits and 12 haems. We have solved this structure in a tetragonal crystal form at 2.9 A resolution. We find that each haem is bound in a pocket formed by the interface between a pair of symmetry-related subunits. The quasi-twofold axis of the haem is closely aligned with the local twofold axis relating these subunits. The axial ligands of the haem are sulphurs of two equivalent methionyl residues (Met 52) from the symmetry-related subunits. A cluster of four water molecules is trapped in the gap between the upper edge of the haem and two extended protein loops which close off the haem from the outer aqueous environment. This is the first structure of a bis-methionine ligated haem-binding site and the first case of a twofold symmetric haem-binding site.
Although essential for most forms of life, too much iron is harmful. To cope with these antagonistic phenomena an iron-storage molecule, ferritin, has evolved. The structure of horse spleen apoferritin, which has recently been refined, consists of 24 symmetrically related subunits forming a near-spherical hollow shell. In ferritin the central cavity is occupied by an iron core of 'ferrihydrite', a geologically ephemeral mineral found in hot or cold springs and in mine workings, or produced in the laboratory by heating solutions of ferric salts. Ferritin itself forms most readily from apoferritin, in the presence of dioxygen, from FeII, not FeIII. Access to its interior is through small intersubunit channels, and the protein influences both the rate of FeII-oxidation and the form of oxide produced.
A complex of concanavalin A with methyl alpha‐D‐mannopyranoside has been crystallized in space group P212121 with a = 123.9 A, b = 129.1 A and c = 67.5 A. X‐ray diffraction intensities to 2.9 A resolution have been collected on a Xentronics/Nicolet area detector. The structure has been solved by molecular replacement where the starting model was based on refined coordinates of an I222 crystal of saccharide‐free concanavalin A. The structure of the saccharide complex was refined by restrained least‐squares methods to an R‐factor value of 0.19. In this crystal form, the asymmetric unit contains four protein subunits, to each of which a molecule of mannoside is bound in a shallow crevice near the surface of the protein. The methyl alpha‐D‐mannopyranoside molecule is bound in the C1 chair conformation 8.7 A from the calcium‐binding site and 12.8 A from the transition metal‐binding site. A network of seven hydrogen bonds connects oxygen atoms O‐3, O‐4, O‐5 and O‐6 of the mannoside to residues Asn14, Leu99, Tyr100, Asp208 and Arg228. O‐2 and O‐1 of the mannoside extend into the solvent. O‐2 is hydrogen‐bonded through a water molecule to an adjacent asymmetric unit. O‐1 is not involved in any hydrogen bond and there is no fixed position for its methyl substituent.
Crystallographic and computational methods have been used to study the binding of two monosaccharides (glucoside and mannoside) to concanavalin-A. The 2 structure of glucoside bound concanavalin-A is reported and compared with the 2 Ó Ó structure of the mannoside complex. The interaction energies of the substrate in each crystallographic subunit were calculated by molecular mechanics and found to be essentially the same for both sugars. Further energy minimisation of the active site region of the subunits did not alter this conclusion. Information from crystallographic B-factors was interpreted in terms of mobility of the sugars in the combining site. Molecular dynamics (MD) was employed to investigate mobility of the ligands at the binding sites. Switching between di †erent binding states was observed for mannoside over the ensemble in line with the crystallographic B-factors. A calculated average interaction energy was found to be more favourable for mannoside than glucoside, by 4.9 ^3.6 kcal mol~1 (comparable with the experimentally determined binding energy di †erence of 1.6 ^0.3 kcal mol~1). However, on consideration of all terms contributing to the binding enthalpy a di †erence is not found. This work demonstrates the difficulty in relating structure to thermodynamic properties, but suggests that dynamic models are needed to provide a more complete picture of ligandÈreceptor interactions.
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