The X-ray structure of an oxygenated hemocyanin molecule, subunit II of Limulus polyphemus hemocyanin, was determined at 2.4 A resolution and refined to a crystallographic R-factor of 17.1%. The 73-kDa subunit crystallizes with the symmetry of the space group R32 with one subunit per asymmetric unit forming hexamers with 32 point group symmetry. Molecular oxygen is bound to a dinuclear copper center in the protein's second domain, symmetrically between and equidistant from the two copper atoms. The copper-copper distance in oxygenated Limulus hemocyanin is 3.6 +/- 0.2 A, which is surprisingly 1 A less than that seen previously in deoxygenated Limulus polyphemus subunit II hemocyanin (Hazes et al., Protein Sci. 2:597, 1993). Away from the oxygen binding sites, the tertiary and quaternary structures of oxygenated and deoxygenated Limulus subunit II hemocyanins are quite similar. A major difference in tertiary structures is seen, however, when the Limulus structures are compared with deoxygenated Panulirus interruptus hemocyanin (Volbeda, A., Hol, W.G.J.J. Mol. Biol. 209:249, 1989) where the position of domain 1 is rotated by 8 degrees with respect to domains 2 and 3. We postulate this rotation plays an important role in cooperativity and regulation of oxygen affinity in all arthropod hemocyanins.

The crystal structure of Limulus polyphemus subunit type I1 hemocyanin in the deoxygenated state has been determined to a resolution of 2.18 A . Phase information for this first structure of a cheliceratan hemocyanin was obtained by molecular replacement using the crustacean hemocyanin structure of Panulirus interruptus. The most striking observation in the Limulus structure is the unexpectedly large distance of 4.6 A between both copper ions in the oxygen-binding site. Each copper has approximate trigonal planar coordination by three histidine NE atoms. No bridging ligand between the copper ions could be detected. Other important new discoveries are (1) the presence of a cis-peptide bond between Glu 309 and Ser 310, with the carbonyl oxygen of the peptide plane hydrogen bonded to the N6 atom of the copper B ligand His 324; (2) localization of a chloride-binding site in the interface between the first and second domain; (3) localization of a putative calcium-binding site in the third domain. Furthermore, comparison of Limulus versus Panulirus hemocyanin revealed considerable tertiary and quaternary rigid body movements, although the overall folds are similar. Within the subunit, the first domain is rotated by about 7.5" with respect to the other two domains, whereas within the hexamer the major movement is a 3.1 O rotation of the trimers with respect to each other. The rigid body rotation of the first domain suggests a structural mechanism for the allosteric regulation by chloride ions and probably causes the cooperative transition of the hexamer between low and high oxygen affinity states. In this postulated mechanism, the fully conserved Phe 49 is the key residue that couples conformational changes of the dinuclear copper site into movements of the first domain.

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We determined the structures of Acanthamoeba profilin I and profifin I by x-ray crystallography at resolutions of 2.0 and 2.8 A, respectively. The polypeptide folds and the actin-binding surfaces of the amoeba profiins are very similar to those of bovine and human profifins. The (6) and/or by promoting the transfer of actin subunits from thymosin to the barbed end of actin filaments (7).Profilins bind to micelles and vesicles containing phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate (PIP2) (8) with Kd values in the range 1-100 ,uM (9, 10). PIP2 competes with actin for binding profilin (8), and biochemical evidence suggests that profilin may regulate the production of the second messengers inositol trisphosphate and diacylglycerol by inhibiting phospholipase C-'yl (10,11).Within the past year an NMR structure of Acanthamoeba profilin I (12), an NMR structure of human profilin (13) and the crystal structure ofthe f-actin-profflin complex (14) have become available. The amoeba and mammalian profilins have similar tertiary structures and the evidence suggests that they bind actin similarly. NMR spectroscopy also provided direct evidence that polyproline binds between the amino-and carboxyl-terminal a-helices of profilin (15,16).Acanthamoeba has three isoforms of profilin (17) All data were collected on a Siemens (Iselin, NJ) area detector using a Rigaku RU-200 operating in fine focus mode as the x-ray source and were reduced with either XENGEN (19) A. These data yielded very clean isomorphous and anomalous difference Patterson maps which were consistent with a single-site derivative. A second EMP data set was collected to 2.5 A. The initial native data were merged with data from an unsuccessful heavy-atom derivative (PtCl-). These additional data were particularly important to complete the Abbreviations: EMP, ethylmercuric phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate.