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2.0-A Resolution (Protein Structure/Flavin Mononucleotide/Hydrogen Bonding/X-Ray Crystallography) K

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2.0-A Resolution (Protein Structure/Flavin Mononucleotide/Hydrogen Bonding/X-Ray Crystallography) K Proc. Nat. Acad. Sci. USA Vol. 70, No. 12, Part II, pp. 3857-3860, December 1973 The Binding of Riboflavin-5'-Phosphate in a Flavoprotein: Flavodoxin at 2.0-A Resolution (protein structure/flavin mononucleotide/hydrogen bonding/x-ray crystallography) K. D. WATENPAUGH, L. C. SIEKER, AND L. H. JENSEN Department of Biological Structure, University of Washington, Seattle, Wash. 98195 Communicated by Edmond H. Fischer, August 27, 1973 ABSTRACT The crystal structure of the oxidized form defined as of flavodoxin from Desulfovibrio vulgaris has been studied at 2.0-A resolution, and a detailed description of the region DF= 21 lFmean l-F(±)I /2jFmean!, around the flavin mononucleotide binding site is now available. The flavin is between a tyrosine group, roughly then DF for the 6669 Friedel pairs from the native crystal is parallel to it on one side, and a tryptophan, about 450 0.042 and for 999 centrosymmetrically related Friedel pairs from being parallel, on the other side. The two carbonyl of the Sm+3 derivative, it is 0.046. The data in the region groups and two nitrogen atoms of the flavin are hydrogen bonded to the peptide chain of the protein, while the two 2.5 < d < 2.6 A for both native and derivative crystals were methyl groups are exposed at the surface of the protein. replicates of previously collected sets. For the 514 duplicate The phosphate group of the flavin mononucleotide is reflections from the two native crystals, Die is 0.065, and for inside the protein and extensively hydrogen bonded to it. the 785 duplicate reflections from the Sm+3 derivative crys- The ribityl group is hydrogen bonded both to the protein it is 0.073. and to water on the surface of the protein. tals, The phases for the new data were determined by use of a Flavin-protein interactions have been studied extensively in single Sm+s derivative. The Sm+a site was the same as that recent years. Through kinetic, thermodynamic, various found in the 2.5A determination and the phase ambiguity spectral techniques, and other physico-chemical methods, of the single derivative was resolved by use of anomalous attempts have been made to understand flavin binding and scattering from Sm+a. The additional data were scaled and electron transport in flavoproteins. Any studies by these merged with the previously phased data to 2.5-A d spacing, techniques would be greatly aided by knowledge of the three- giving a complete data set from co to 2.0A d spacing, nearly dimensional structure of a flavoprotein and, in particular, doubling the number of terms used in calculating the electron the binding and environment of FMN (riboflavin-5'-phos- density map. The quality of the new map from which the in- phate) in it. The crystal structure of the oxidized form of terpretation of binding is derived is best illustrated by the flavodoxin from the sulfate-reducing bacterium Desulfovibrio electron density through the plane of the flavin group, Fig. 1. vulgaris (strain Hildenborough, NCIB 8303) has now been extended to 2.0-A resolution. The improvement of the elec- Description of the FMN binding and environment tron density map over the 2.5- resolution map enables us As reported previously (1), the structure of the flavodoxin to make a much more certain and detailed interpretation of can be described as consisting primarily of a central section the FMN environment than that already reported (1). of five strands of parallel pleated sheet with two sections of A similar investigation on the flavodoxin from Clostridium of a-helices on each side. The FMN is almost buried in one MP is underway by Martha Ludwig and coworkers at the side of the protein, as shown in the stereo view (Fig. 2). Only University of Michigan (2). those side groups involved directly in hydrogen bonding and the two aromatic groups near the flavin group are included Experimental in the figure to aid its clarity. The extensive system of hy- Crystallization and the method of data collection are the drogen bonds involving FMN apparently results in relatively same as previously described (1). Flavodoxin from D. vulgaris low thermal motion and little disorder, so that the electron crystallizes in space group P432,2 with unit cell dimensions density is particularly clear in this region of the map. It a = b = 51.6 A, c = 139.6 A, based on Xcus,, = 1.5418 A and should be noted, however, that much of the rest of the mole- unit cell volume of 372,000 A3. There is one molecule of the cule involves $-structure and a-helices and these are also protein of approximate molecular weight 16,000 per asym- stabilized by extensive hydrogen bonding. Thus the carbonyl metric unit. Ni-filtered CuKa radiation was used with a take- groups and many of the side groups are readily identified in off angle of 3.5°. A five-step (0.080 per step) co/20 scan across spite of the large solvent content of the crystals, about 60% the top of each peak was fit by least-squares with a Gaussian (see Fig. 1 of ref. 1). curve and used to obtain the integrated intensity. Over 13,000 The flavin group is buried in a pocket extending into the reflections (6669 Friedel pairs on the native crystal) were col- protein between segments of the polypeptide chain -and the lected on both the native crystal and a Sm+3 derivative in the two side groups of Trp 60 and Tyr 98. The orientation of these region 0.2 < sin O/X < 0.25 (2.0 A < d < 2.6 A). If the average aromatic groups with respect to the flavin group is shown in relative deviation from the mean for replicated reflections is Fig. 3. The tyrosine is nearly coplanar with the flavin (about 3857 Downloaded by guest on September 29, 2021 3858 Biochemistry: Watenpaugh et al. Proc. Nat. Acad. Sci. USA 70 (1973) FIG. 1. Electron density in the plane of the flavin at 2.0-A resolution. FIG. 2. Stereo view of the region around the FMN. Hydrogen bonds are indicated by single lines. Shaded atoms are water molecules. 150 between the normals to the planes) and could easily be rotated about the Cg-C. bond to be coplanar with it. The presence of the tyrosine near the chromophore is consistent with what has been observed in the related redox proteins ferredoxin (3) and rubredoxin (4). Although the tyrosine is sometimes replaced by phenylalanine or histidine in these proteins, depending on the bacterial source, the presence of an aromatic group would not appear to be completely coinci- dental. The tryptophan is not coplanar with the flavin (about 450 between the normals to the planes) and cannot be made IL- coplanar with it without a major change in the conformation of the protein. The pyrimidine ring of the flavin group is buried most deeply in the protein, allowing it to form strong hydrogen bonds as shown in Fig. 4. Only amino nitrogens and carbonyl groups of the main chain (no side groups) are involved in hydrogen bonding to the flavin. Fig. 4 shows N5 of the flavin N. hydrogen bonded to NH(62) of the main polypeptide chain, 04 to a water molecule that is hydrogen bonded to CO(62) and NH(100), NH3 to CO(100) and 02 to the two groups NH(95) and NH(102). It should be noted that with a small rotation residue 95 could bind to N1 rather than 02, and similarly residue 62 could bind to 04 rather than N5. FIG. 3. Orientation of Trp 60 and Tyr 98 relative to the The two methyl groups on the benzenoid portion of the flavin plane. Lower projection is rotated 900 relative to upper. flavin are on the surface of the protein and are apparently the Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Flavodoxin at 2.0-A Resolution 3859 FIG. 4. Proposed hydrogen bonding of the flavin to the protein. 10) r 15) FIG. 5. Proposed hydrogen bonding of the ribityl and phosphate portions of the FMN to the protein. Downloaded by guest on September 29, 2021 3860 Biochemistry: Watenpaugh et al. Proc. Nat. Acad. Sci. USA 70 (1973) TABLE 1. Partial sequence of three flavodoxins Comparison of D. vulgaris and Clostridium MP flavodoxins D. vulgaris P. elsdenii C. Past. The flavodoxins may be separated into two classes based on Res. no. (6) (7) (8) various physical and chemical properties (5). The pas- 1 Met Met Met teurianum type includes the flavodoxins isolated from the 2 Pro bacteria Peptostreptococcus elsdenii, Clostridium pasteurianum, 3 Lys Lys and Clostridium MP, while the rubrum type includes those 4 Ala Val Val from Rhodospirillum rubrum, Azotobacter vinelandii, and 5 Leu Glu Asn Desulfovibrio vulgaris. Either the entire chemical sequences or 6 Ile Ile Ile large segments of it are known for three different bacterial 7 Val Val Ile flavodoxins: D. vulgaris (6), P. elsdenii (7), and C. pasteuri- 8 Tyr Tyr Tyr anum (8). The three-dimensional structure of the C. MP 9 Gly Trp Trp 10 Ser Ser Ser flavodoxin is quite homologous with that of D. vulgaris, 11 Thr Gly Gly indicating that the over-all structure of the pasteurianum and 12 Thr Thr Thr rubrum type must be quite similar (2). In spite of this simi- 13 Gly Gly Gly larity, however, there are extensive differences in the chemical 14 Asn Asn Asn sequences. The only extended region of sequence homology 15 Thr Thr Thr between the two types is between residues 6 and 16, using the 16 Glu Glu Glu D.
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