Crystal structure of core streptavidin determined from multiwavelength anomalous diffraction of synchrotron radiation.

Abstract

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...

Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca2+-Activated K+ Channel Expressed in a Human Cell Line

“…As shown in Figure 5, at a molar ratio greater than 0.5 mole StrAv tetramer per mole IbTx-LC-biotin, a new peak is observed corresponding to the elution time (18.1 minutes) of free, uncomplexed StrAv. This observation indicates that the StrAv tetramer binds a maximum of two molecules of IbTx-LC-biotin under the conditions of this experiment in comparison to the theoretical capacity of four molecules corresponding to the four known biotin sites on the tetrameric protein (Hendrickson et al, 1989;Green, 1990). This surprising result was verified by measuring the concentration of total biotin binding sites in the commercial StrAv preparation using a method based on fluorescence quenching of biotin-4-fluorescein (Kada et al, 1999).…”

“…This unusual behavior is attributed to the particular three-dimensional relationship of biotin binding sites in StrAv. The quaternary structure of StrAv tetramer consists of a dimer of dimers, with two adjacent biotin sites on neighboring StrAv monomers located within ∼19 Å of each other on each of two separate faces of the tetramer (Hendrickson et al, 1989;Green, 1990;Freitag et al, 1997). Biotin derivative molecules with long chain linkers have a propensity to interfere with pairwise binding of each other across the two separate binding faces of the tetramer.…”

Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin‐biotin system

“…We are interested in defining the high-affinity reaction coordinate of streptavidin, where the conformational change of a flexible binding loop is a prominent feature accompanying biotin association (Hendrickson et al, 1989;Weber et al, 1989). High-affinity protein-ligand interactions play an important role in biology and often are the goal of structure-based drug design projects.…”

“…The free energy of binding is the result of balancing the entropic costs/benefits of ordering of loops and release of bound water with the enthalpic benefits of burying nonpolar surface area and establishing bonding contacts. It is expected that loop folding may lead to energetic signatures similar to those associated with protein folding (Murphy et al, 1993;Spolar & Record, 1994).We are interested in defining the high-affinity reaction coordinate of streptavidin, where the conformational change of a flexible binding loop is a prominent feature accompanying biotin association (Hendrickson et al, 1989;Weber et al, 1989). High-affinity protein-ligand interactions play an important role in biology and often are the goal of structure-based drug design projects.…”

Crystallographic analysis of the pH‐dependent binding of iminobiotin by streptavidin

“…The substitution of the keto group on the ureido ring of biotin results in the guanidyl group in 2"iminobiotin. The ureido group of the ligands are polarized in the complexes of streptavidin with biotin and its analogues with unmodified ureido rings (Hendrickson et al, 1989;Weber et al, 1989; Athappilly et al, in prep.). This polarization, which results in a partially negative 0 2 ' with three lone pairs of electrons, is stabilized in the complexes by three tetrahedrally aligned hydrogen bonds accepted by 02'.…”