Three-Dimensional Structures of Avidin and the Avidin- Biotin Complex
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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5076-5080, June 1993 Biochemistry Three-dimensional structures of avidin and the avidin- biotin complex (deglycosylated avidin/divergent evolution/x-ray crystallography/streptavidin) ODED LIVNAH*, EDWARD A. BAYERt, MEIR WILCHEKt, AND JOEL L. SUSSMAN* Departments of *Structural Biology and tBiophysics, The Weizmann Institute of Science, Rehovot 76100 Israel Communicated by Christian B. Anfinsen, January 28, 1993 (receivedfor review November 14, 1992) ABSTRACT The crystal structures of a deglycosylated supplied by Belovo Chemicals (Bastogne, Belgium). It was form of the egg-white glycoprotein avidin and of its complex =10-fold less soluble than avidin, apparently due to removal with biotin have been determined to 2.6 and 3.0 A, respectively. ofthe carbohydrates. Crystals (space group P21212; a = 72.22 The structures reveal the amino acid residues critical for A, b = 80.42 A, c = 43.33 A, with two avidin monomers in the stabilization of the tetrameric assembly and for the exception- asymmetric unit) were grown at 19°C in hanging drops, con- ally tight binding ofbiotin. Each monomer is an eight-stranded sisting of 5 Al of protein solution (4 mg/ml) and 5 .lI from the antiparallel -barrel, remarkably similar to that of the genet- reservoir, which contained 23% polyethylene glycol 1000 and ically distinct bacterial analog streptavidin. As in streptavidin, 20 mM citrate buffer (pH 5.4). Crystallization conditions and binding of biotin involves a highly stabilized network of polar cell parameters were similar to those reported forglycosylated and hydrophobic interactions. There are, however, some dif- avidin (4). For larger crystals, macroseeding techniques were ferences. The presence of additional hydrophobic and hydro- applied (12). Crystals of the avidin-biotin complex were philic groups in the binding site of avidin (which are missing in obtained by soaking biotin into crystals of avidin. The cell streptavidin) may account for its higher affinity constant. Two constants ofthe avidin-biotin complex (a = 72.15 A, b = 80.37 amino acid substitutions are proposed to be responsible for its A, c = 43.54 A) differ only slightly from those of the native susceptibility to denaturation relative to streptavidin. Unex- protein. X-ray diffraction data were collected at room tem- pectedly, a residual N-acetylglucosamine moiety was detected perature on a Siemens/Xentronics multiwire areadetector and in the deglycosylated avidin monomer by difference Fourier processed by using the XDS data reduction program (13). synthesis. Structure Determination. Our starting model for the lite avidin-biotin complex was a partially refined structure of The biotin-binding proteins avidin (from egg white) and avidin (kindly provided by W. Hendrickson, Howard Hughes streptavidin (from the bacterium Streptomyces avidinii) oc- Medical Institute, Columbia University), which had been cupy a place of honor in many fields of biology. The reason determined by molecular replacement using streptavidin as a for interest in these proteins is 2-fold: (i) both proteins exhibit search model (7) and then refined to R = 0.257 at a resolution the highest known affinity (Ka 1015 M-1) in nature between range of6-3.2 A (A. Pahler and W. A. Hendrickson, personal a ligand and a protein (1), and (ii) largely as a consequence, communication). After initial rigid body refinement (14, 15), the avidin-biotin (and streptavidin-biotin) system has been the R factor for the avidin-biotin complex was 0.45 for 8.0-3.0 widely applied as a universal tool, particularly for diagnostic A resolution. Ourattempts to refine the structure using X-PLOR purposes (2). (15-17) failed to reduce the R factor below 0.28; the 2Fob, - Several groups have tried to crystallize egg-white avidin Fc,c electron density maps were discontinuous in some ofthe (3-5) with only limited success. During the course of our loop regions. When examining the fit ofthe avidin amino acid studies, which involved chemical and physical properties of sequence to the maps, it was clear that one ofthe strands (,B4; avidin, we succeeded in isolating an active deglycosylated Fig. 1) was fourresidues out ofregister; Lys-45 pointedtoward form of this protein (6), the structure of which we report a number of hydrophobic residues, and the electron density here.* Interestingly, the x-ray structure of the related, nat- near Ser-47 appeared too short for such a residue. To resolve urally nonglycosylated, bacterial protein streptavidin has this inconsistency in this region, we shifted the sequence four already been determined (7, 8). Comparison of these two residues relative to the electron density map (Fig. 2), resulting genetically remote structures permits us to decipher the in Lys-45 being replaced by Leu-49 and in Ser-47 being crucial elements for formation of such a strong binding site. replaced by Gly-51. Although these changes allowed us to fit It had been noted (9) that the primary structures of the two this part ofthe molecule better into the map, most ofthe loop proteins are similar (see Fig. 1) and that the conserved amino regions still lacked clear electron density, and the structure acid residues are mostly confined to six homologous seg- could not be refined to a significantly lowerR factor. We were ments (10, 11). The current study has revealed that the major able to overcome this problem by the use of noncrystallo- structural elements are also conserved and critical functional graphic symmetry averaging of the electron density maps groups are retained in the binding site. Nevertheless, there (refs. 18-20; unpublished results). We applied map averaging are some notable differences in their properties, many of and solvent flattening, starting from the initial model phases which can be explained in terms of the three-dimensional and then extendingthe phases from4 to 3 A resolution by using structures of avidin and streptavidin. the DEMON program suite (F. M. D. Vellieux, personal com- munication). The final averaged map was significantly im- MATERIALS AND METHODS proved, enabling us to trace the various regions of the mole- cule that could not be determined previously. Crystallization and Data Collection. "Lite avidin" (i.e., Following these two crucial steps that removed the initial avidin with most of the oligosaccharide chain removed) was model bias-i.e., the four-residue shift and the use of real- The publication costs of this article were defrayed in part by page charge tThe atomic coordinates and structure factors have been deposited payment. This article must therefore be hereby marked "advertisement" in the Protein Data Bank, Chemistry Department, Brookhaven in accordance with 18 U.S.C. §1734 solely to indicate this fact. National Laboratory, Upton NY 11973 (reference lAVI). 5076 Downloaded by guest on September 27, 2021 Biochemistry: Livnah et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5077 131 132 13 Table 1. Refinement results for avidin-biotin complex and avidin structures AV ARKCSLTGKWTDLG NA TIGAVNSRGEFfTGTlTUASMSNE-I .II II II Iilii Avidin-biotin Avidin StAVAAzAG1ITGTWYMQLG TFAVTAG-ADGALTGT GE ----AE 12 Program used X-PLOR X-PLOR 14 15 16 R factor 15.1% 18.8% 45 -0 Resolution range 8.0-3.0 A 8.0-2.6 A Av KESPLHGTENTIN--KRTQPTF GFTVNI ...- N-EiTIUV1TG C No. of observed reflections 4984* 7519* . 1.1-11 11 I Ii ..1 1 stAvSRYVLTGRYDSAPATDG;GTALGWTVA NNYRNAH A TMSG Y Completeness of data 98.5% 95.0%/c 52 Final model 2 avidin monomers 1892 atoms 1768 atoms 17 18 84 2 biotin molecules 32 atoms Av FIDRNGKEVLKiTMELLRS SVNDIGDDEKATRVGICIFTRLRTQRK 2 GlcNac 28 atoms 28 atoms StAvV- -GGEARIN TQLLTSGTTE-ANAMKSTLVGHMTFTKKPSAA Waters 74 molecules 103 molecules 97 Protein (Bir5) 11.3 A2 15.9 A2 FIG. 1. Alignment of primary structures of avidin (Av) and Biotin (BA50) 7.3 A2 streptavidin (StAv) based on the three-dimensional spatial position of GlcNac (Bir,) 20.3 A2 27.7 A2 the individual amino acids in the corresponding structures. N- and Waters (Bi.o) 23.9 A2 24.9 A2 C-terminal residues, shown in smaller typeface, are part of the rms deviations from ideality sequence but were not seen in the x-ray structures. Conserved Bond length 0.013 A 0.013 A residues are denoted by vertical lines. Similar residues (A, G; S, T; Bond angle 1.830 1.840 D, E; V, L, I, M; F, W, Y) are indicated by dots. The six homologous rms deviation of Cc between segments are enclosed in boxes. Positions ofthe respective 3-strands noncrystallographic are marked by arrows. Residues in the binding site, ofboth proteins, symmetry-related that interact directly with biotin are shown in white type. For each monomers 0.26 A 0.31 A protein, the position of the initial residue in each line is shown on the left of each row. The overall homology in the alignment of the two *AM Fobs > 0 were included in refinement. sequences is 30% identity (41% similarity); the respective values are 64% (74%) within the homologous segments and 7% (17%) outside of RESULTS these segments. The overall fold ofthe avidin monomer closely resembles that ofstreptavidin; it is constructed ofeight antiparallel (3-strands space noncrystallographic symmetry map averaging-the (Figs. 1 and 3), which form a classical (-barrel. The most avidin-biotin complex was fitted into the electron density striking differences in their tertiary fold lie in the size and map the program FRODO (21, 22). It was refined with by using conformation of the six extended loops that connect the X-PLOR (15-17) by using the simulated annealing slow-cooling protocol with tight noncrystallographic symmetry restraints strands. There are three regions of monomer-monomer interaction between monomers (see Table 1). Solvent molecules were the refinement No electron in avidin (Fig. 4). These interactions contribute to the rigidity gradually added during process.