A three-dimensional crystal structure of the biotin-binding core of streptavidin has been determined at 3.1i-resolution. The structure was analyzed from diffraction data measured at three wavelengths from a single crystal of the selenobiotinyl complex with streptavidin. Streptavidin is a tetramer with subunits arrayed in D2 symmetry. Each protomer is an 8-stranded fl-barrel with simple up-down topology.Biotin molecules are bound at one end of each barrel. This study demonstrates the effectiveness of multiwavelength anomalous diffraction (MAD) procedures for macromolecular crystallography and provides a basis for detailed study of biotinavidin interactions.Streptavidin takes its name from the bacterial source of the protein, Streptomyces avidinii, and from hen egg-white avidin with which it shares an extraordinary ligand binding affinity (Kd -10-15M) for biotin (1). This similarity extends to many other properties (2), including a common tetrameric structure and a 33% identity in amino acid sequence between avidin and the homologous core of streptavidin (3, 4). Core streptavidin is proteolyzed naturally, but not always completely (3), at both ends of the 159-residue gene product to a 125-to 127-residue core (4) that matches quite precisely with the actual secreted avidin gene product (5). The biological functions of avidin and streptavidin are poorly understood, but they most probably involve antibiotic properties. Interest in the avidin family, however, transcends their natural biology. Their remarkable avidity for biotin motivates two types of study: (i) efforts to understand the chemical basis for the high affinity and (ii) attempts to optimize biotechnology applications that exploit this activity (6)(7)(8). We aim to examine these biophysical and biotechnological properties in refined crystallographic detail. Streptavidin has also been crystallized by others (ref. 9 and P. McLaughlin, personal communication).This structural study of streptavidin also has a second objective related to diffraction methodology. It seemed from the outset that selenobiotinyl streptavidin could be an apt subject for direct analysis from multiwavelength anomalous diffraction (MAD) data obtained with synchrotron radiation. Selenobiotin is a stable compound (10) sufficiently similar to biotin itself that the two molecules crystallize isomorphously (11). The high affinity (Kd 10-13 M) of avidin for desthiobiotin (2, 12) suggested to us that selenobiotin would also bind well. We expected anomalous diffraction ratios (13) from the four selenium atoms in the 54-kDa core streptavidin tetramer (up to 3%) that compared favorably with signals measured successfully from crambin (14) and myohemerythrin (15) and with those obtained in our lamprey hemoglobin test of MAD phasing (16,17).The theoretical basis for the MAD method and details of its implementation are presented elsewhere (13,17,18). Qualitatively, MAD experiments can be thought of as in situ multiple isomorphous replacements (MIR) generated by the variation in scattering fact...

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A novel x-ray diffraction technique, multiple-wavelength anomalous dispersion (MAD) phasing, has been applied to the de novo determination of an unknown protein structure, that of the "blue" copper protein isolated from cucumber seedlings. This method makes use of crystallographic phases determined from measurements made at several wavelengths and has recently been made technically feasible through the use of intense, polychromatic synchrotron radiation together with accurate data collection from multiwire electronic area detectors. In contrast with all of the conventional methods of solving protein structures, which require either multiple isomorphous derivatives or coordinates of a similar structure for molecular replacement, this technique allows direct solution of the classical "phase problem" in x-ray crystallography. MAD phase assignment should be particularly useful for determining structures of small to medium-sized metalloproteins for which isomorphous derivatives are difficult or impossible to make. The structure of this particular protein provides new insights into the spectroscopic and redox properties of blue copper proteins, an important class of metalloproteins widely distributed in nature.

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