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...

An example of two related enzymes that catalyse similar reactions but possess different active sites is provided by comparing the structure of Escherichia coli thioredoxin reductase with glutathione reductase. Both are dimeric enzymes that catalyse the reduction of disulphides by pyridine nucleotides through an enzyme disulphide and a flavin. Human glutathione reductase contains four structural domains within each molecule: the flavin-adenine dinucleotide (FAD)- and nicotinamide-adenine dinucleotide phosphate (NADPH)-binding domains, the 'central' domain and the C-terminal domain that provides the dimer interface and part of the active site. Although both enzymes share the same catalytic mechanism and similar tertiary structures, their active sites do not resemble each other. We have determined the crystal structure of E. coli thioredoxin reductase at 2 A resolution, and show that thioredoxin reductase lacks the domain that provides the dimer interface in glutathione reductase, and forms a completely different dimeric structure. The catalytically active disulphides are located in different domains on opposite sides of the flavin ring system. This suggests that these enzymes diverged from an ancestral nucleotide-binding protein and acquired their disulphide reductase activities independently.

A probability distribution function, cast in the representation of Hendrickson & Lattman [Acta Cryst. (1970), B26, 136-143], has been derived for the phase information from measurements of multiwavelength anomalous diffraction (MAD). This probability function readily permits one to determine figure-of-merit weights similar to those used in isomorphous replacement, and the coefficients that characterize this distribution function facilitate the combining of MAD phasing with results from other sources of phase information. This probability representation was derived in the course of a structural analysis of selenobiotinyl streptavidin from MAD data and applications have also been made in the structure determinations of interleukin-1 a and a drug complex with brominated DNA. IntroductionThe likelihood with which the phase angle of a structure factor F = IFI exp (iq~) attains a value between 0 and 2rr can be characterized by a phase probability distribution P(~o). Blow & Crick (1959) introduced a description of such phase probability assuming a Gaussian distribution for the errors e(~) from lack of closure in phase triangles in the method of isomorphous replacement. Thus P(~p)=Nexp[-e2(~p)/2E 2](1) where E is the expectation value for errors at the true phase angles. Somewhat later, Hendrickson & Lattman (1970) It is the phase probability coefficients A, B, C and D which uniquely characterize the distribution. The factor due to K is irrelevant to the form of the phase distribution and it can be combined into the normalization factor N to obtain unit integrated probability. A conspicuous advantage of using the functional form described by (3), apart from having a convenient representation of P(~0) by the four values A, B, C and D, is the possibility of combining phase information from various sources. If the distributions are mutually independent, by statistical arguments the distribution for combined information is just the product of the individual distributions. This combined distribution, by virtue of its exponential form, is then characterized by accumulated coefficients of Atotal equal to the sum of the individual A values, and similarly for the other coefficients. Multiwave!ength anomalous dispersionIn recent years a new method for phase determination, based primarily on synchrotron radiation, has been developed for application in macromolecular crystallography. This technique, known as multiwavelength anomalous-dispersion (MAD) phasing, makes use of the wavelength dependence of the anomalous-dispersion part of the atomic form factor f=fo+f,(•)+ if"(A) and has been cast into an algebraic form by Karle (1980). An algebraic formulation by Hendrickson (1985), which differs in the unknown quantities but is equivalent to Karle's approach (Karle, 1989), has been successfully applied in the structure determination of the protein streptavidin complexed with selenobiotin, an analog of its natural ligand biotin. The selenium atoms in this complex served as the source of anomalous scattering, and data from one ...