STRUCTURE DETERMINATION AND REFINEMENT OF HOMOTETRAMERIC HEMOGLOBIN FROM URECHIS-CAUPO AT 2.5 ANGSTROM RESOLUTION

A 5 angstrom resolution multiple isomorphous replacement solution for hemoglobin isolated from Urechis caupo revealed a previously unobserved quaternary structure for tetrameric hemoglobin [Kolatkar, Meador, Stanfield & Hackert (1988). J. Biol. Chem. 263(7), 3462-3465]. We report here the structure of Urechis hemoglobin in the cyanomet state refined to 2.5 angstrom resolution by simulated annealing yielding R = 0.148 for reflections F > 3-sigma between 5.0 and 2.5 angstrom resolution. The starting model was fitted to a map originally derived from multiple-wavelength anomalous-dispersion phases to 3 angstrom resolution that was then subjected to cyclic twofold molecular averaging and solvent flattening. Structural analysis of the resultant model shows that the unique quaternary assemblage is possible due to several favorable interactions between subunits, including salt links, hydrophobic pockets and interactions mediated by bound water. The tetramer is stabilized by subunit-subunit interactions between the G/H turns and D helices within the crystallographic dimer, and the A/B turn regions and E helices between subunits related by a molecular twofold axis. Interestingly, each subunit has one cysteine residue (Cys21) located in the A/B turn. These twofold-related cysteinyl residues are near enough to one another to form a disulfide bridge but do not.