Min-Sung Kim, Joon Shin, Weontae Lee, Heung-Soo Lee and Byung-Ha Ohsug - PDF document

Min-Sung Kim, Joon Shin, Weontae Lee, He
Min-Sung Kim, Joon Shin, Weontae Lee, Heung-Soo Lee and Byung-Ha Ohsugar-binding proteins with a novel folding architectureCrystal structures of RbsD leading to the identification of cytoplasmic published online May 8, 2003J. Biol. Chem.   10.1074/jbc.M304523200Access the most updated version of this article at doi:  Alerts:   When a correction for this article is posted•  When this article is cited•  to choose from all of JBC's e-mail alertsClick here by guest on January 4, 2021http://www.jbc.org/Downloaded from 0246810121416-1.4-1.2-1.0-0.8-0.6-0.4-0.20.0-6-4-20020406080100120140160Time (min)Molar Ratiokcal/mole of injectantcal/secRbsD+ D-ribose= 0.93 (0.01)mM= -5.6 (0.02) Kcal/mole051015202530-0.24-0.20-0.16-0.12-0.08-0.04-0.6-0.4-0.20.0020406080100Time (min)Molar Ratiokcal/mole of injectantFucU+ L-fucosecal/sec= 1.61 (0.08)mM= -6.1 (0.23) Kcal/mole01234567-0.09-0.08-0.07-0.3-0.2-0.1020406080100

Time (min)Molar Ratiokcal/mole of inject
Time (min)Molar Ratiokcal/mole of injectantcal/secRbsD+ D-ribose 5-phosphateabc by guest on January 4, 2021http://www.jbc.org/Downloaded from 5.0 4.03.0 2.01.06.0FucoseOnlyFucose + FucUH (ppm)Figure 5 by guest on January 4, 2021http://www.jbc.org/Downloaded from RBSD_BACSU: MKKHGAKILLGIVIAVPD----GVLKDLSLKPGLPAFQ : 52RBSD_LACLA: MKKEGAKIADDVPS----GVKKDLALTRGKPSFQ : 52RBSD_ECOLI: MKKGTSSVILGLVVCIPK----STTRDMALTQGVPSFM : 52RBSD_YERPE: MKKGVSAVILGIVIGIPA----TTTRDLALTRGVPGFL : 52RBSD_VIBVU: MKKSASYLVLGIPD----GVSRDLALTHGVPSFI : 52RBSD_STAEP: MKKTASSAILGIPN----DDKRDLAVTKSLPRFI : 52RBSD_THERM: MKKVGSKIVMGIPQ----GVKKDLVVDRGKPGLM : 52RBSD_MYCPU: MYNQDFQNETLIQTNQKIVVCIPV----GANIDLSLIANVPSFK : 54FUCU_ECOLI: MLKTISPLKVLMGDAHS---MGPQVRADGLLVSDLLQ : 55FUCU_SALTY: MLKTISPLKVLMGDAHS---LGPQVRADGLSVSDLLR : 55FUCU_HAEIN: MLKGIHPALKTLMGIVLAAHS---LHKNVRADGISIDILLE : 55FUCU_STRPN: MLKHIPKNLKTLMGIVLASAS---

CANKLRCDGVNIPELLD : 55FUCU_BRUME: MLKN
CANKLRCDGVNIPELLD : 55FUCU_BRUME: MLKNINPLAILMGLVIVAQA---AGVPVDFPGISATQVAE : 55FUCU_DANRE: MVILKGIPSLYVLMGLVLATSSVCKCGPVERADGVRIPELLK : 60FUCU_MUSMU: MVALKGIPKLFALMGIVLATSSICQCGPVERADGLDIPQLLE : 60FUCU_HOMSA: MVALKGVPALYALMGIVLAASSICQCGPMERADGLGIPQLLE : 60RBSD_BACSU: DTAALAEEMAEKVIAAAEIKASNQE-------NAKFLENFSE-------QERBSD_LACLA: EYLENILEKVYLAEEIKENNPEQ------LAILLTKSAD-------VERBSD_ECOLI: QVTNEMQEAAIIAEEIKHHNPQL------HETLLTHEQLQKHQGNTIERYTTH:106RBSD_YERPE: QVTQEMQENAYLAEEIVKNNPQL------HEALLVLTQLEQRQENQIA:106RBSD_VIBVU: ETMLSESQEGAIVATEFAEVSPEL------YQALVAEQCEEEKTGKVLS:106RBSD_STAEP: DETVLTEMEQKVYLAEEIKTANAQQ------LKAIKKLNDD-------VERBSD_THERM: ELLRELKERIILAKEMDEKSIQT------KQELLKLEKMN----GPVE:102RBSD_MYCPUIIENLHKALVLSNEIKEHNYD-------YLAYLISSKLP--------ISLSNNF : 99FUCU_ECOLI: AFELDSYAP-PLVMMAAVEGDTLDP---EVERRYRNASLQAP-C---PD:107FUCU_SALTY: AFELDSYAP-PLVMMAAVEGDTLDP---NVEARYRDASLEAP-C---PD:107FUCU_HAEIN: AFEFDAYDAPLLMMKAVEGDSLDP---NVET

RYLNAESAVGFT---PN:109FUCU_STRPN: SMPLDSY
RYLNAESAVGFT---PN:109FUCU_STRPN: SMPLDSYDSSIQFMNVVSGDDIP----KIWGTYRQMEGHGTDL---KT:108FUCU_BRUME: ALPLDDFDRPAAVMQAPNEMPAI------FKEFEAVEKAEGRK---IP:106FUCU_DANRE: AFPLDTYDES-AAVMDLVPSDLLKGLKVPIWDQYSELKQAGSD----GN:115FUCU_MUSMU: ALPLDTYESPAAVMDLVPSDKEKGLQTPIWKRYESLLEADCK----KT:115FUCU_HOMSA: ALPLDTYESPAAVMELVPSDKERGLQTPVWTEYESIRRAGCV----RA:115RBSD_BACSU: EEFLLTKD--AKARFTPGVLF------:131RBSD_LACLA: ETLLMNHE--VKARNTPIILQSGVAL------:132RBSD_ECOLI: EQFQQTAE--SQARCSPIILCAGVTF------:139RBSD_YERPE: EAFEQTKQ--SRARCSPIILGSGVTF------:139RBSD_VIBVU: EEFQRTES--SKARCTPFQAVF------:139RBSD_STAEP: SEMEMLKSPLNKGNITPIILESNTF------:134RBSD_THERM: KEFEMSKN--VKGRADIPVILVGGVIF------:135RBSD_MYCPU: EEFYLSSQNDVVLYLTPGKA-------:133FUCU_ECOLI: FAFERAQK--AFAIRAKILLKKGVTP------:140FUCU_SALTY: YAFERAQK--AFAICAKILLKKGVTP------:140FUCU_HAEIN: FDFTRAKQ--AYAVSIAKIIIKKGVTPIL----:144FUCU_STRPN: EDFERSKK--AYAATSLIILKKGVVVERENVQ :147FUCU_BRUME: FAFDRARG--AFARKRLFKKIRS-----:

140FUCU_DANRE: FAFERAKK--AFAATALLIIKKGVI
140FUCU_DANRE: FAFERAKK--AFAATALLIIKKGVIPPEEQC-:153FUCU_MUSMU: FEFERAKK--AFAAMALIILKKGTLDLGPS--:153FUCU_HOMSA: FEFERAKK--AFAATALLILRKGVLALNPLL-:154K102H20D28H98Y120N122 by guest on January 4, 2021http://www.jbc.org/Downloaded from H98H98K102K102H20D28D28N122N122H20gure 3acH98H98K102K102H20D28D28N122N122H20H98H20 by guest on January 4, 2021http://www.jbc.org/Downloaded from RbsD(wild-type)RbsD(H98A)OHOOHOH5432123 by guest on January 4, 2021http://www.jbc.org/Downloaded from 60Å20Å80Å90°CN11226343455Figure 1abcLys2Lys2 by guest on January 4, 2021http://www.jbc.org/Downloaded from Parameters Peak Edge Remote Wavelength (Å) CompletenesssymParameters symCompleteness, % Rmsd bond length (Å) Rmsd bond angle ( Most favored region Additionally allowed region Generously allowed region The numbers in parentheses are statistics from the highest resolution shell CompletenesssymΣ is average over symmetry equivale

nts. Figure of merit, defined as ) is t
nts. Figure of merit, defined as ) is the phase probability distribution. by guest on January 4, 2021http://www.jbc.org/Downloaded from B. subtilis (P36946), (Q9CF43), Yersinia pestisVibrio vulnificus (AAO07037.1), Staphylococcus (NP_326431); [FucU] (P11555), Haemophilus influenzaeStreptococcus pneumoniaeBrucella melitensis (Q8YHX8), zebra fish (CAD60840), mouse (AAH28662), human (XM_058320.2). Figure 5. Fucose only. O containing 10 mM phosphate (pH 7.0). The mixture was incubated for 2 hrs bewater peak was suppressed by the pulsed field gradient method as Figure 6. Isothermal titration calorimetric analysis. , Titration of , Titration of cU. The ITC measurements were L of 29.5 mM -ribose into 1.4 mL of 281 L of 10 mM M FucU solution, respectively. by guest on January 4, 2021http://www.jbc.org/Downloaded from -pyranoside form of the sugar is fitted into the map (1.95 Å, 1.1). The C4-OH group interacts with the C-term

inal carboxyl group via a water molecule
inal carboxyl group via a water molecule. The density of this water molecule is weak or unobserved in 2 out of 5 binding sites. These weak cleft. The bound ribose is displayed in CPK mode with the oxygen and carbon atoms in red and black, respectively. The bound water molecule is -ribose 5-phosphate. The phosphorylated sugar is in β-furanose form, which was determined at the beginning of the refinement. The final 2 map (2.05 Å, 1.1) is shown. His20 from one RbsD subunit is in cyan and rest of the residues from the adjacent subunit ed with the program Bobscript (26). Figure 4. Secondary structure assignment and sequence alignment. Sequences of RbsD homologues (top 8) and FucU homologues (bottom 8) are shown. The two groups are distinguished from each other by the substitutions at the positions corresponding to His98 and Lys102 in RbsD. The black boxes indicate the residues involved in the binding of ribose. The secondary structure

assignment, in the order from N to C ter
assignment, in the order from N to C terminus of the amino acid sequence, is shown at the top of the sequence. The red and blue letters indicate the amino acids that are 100% and greater than 70% position corresponding to His98 in RbsD, but a pair-wise alignment shows sD_MYCPU aligns with His98. The by guest on January 4, 2021http://www.jbc.org/Downloaded from Figure 1. Structure and ribose-binding site of RbsD. n in two different orientations. Ribose molecules bound to the intersubunit clefts are shown in ball-and-b, Ribbon diagram of RbsD monomer. The secondary structures are numbered in the order of appearance in the primary sequence. cage. A negatively charged ion (in cyan), which is putatively a Clin between two RbsD subunits related by the molecular two-fold axis that superimposes the pentameric rings. The symmetry-related pairs are shown in different colors. Water molecules are in red. Two histidine residues hydrogen-bond

ed to the bound water molecules are show
ed to the bound water molecules are shown. Figure 2, Ribose only. , Ribose + H98A mutant RbsD. Each sample contains 13.3 mM ribose and 1 mM sodium phosphate buffer (pH 7.4). The wild-type or mutant RbsD to a final concentration of 0.4 mM was mixed with ribose. Labeled are the resonance positions of the C-1 atom of the four different forms of C. In the experimental conditions, we could not observe the peak from the acyclic aldehyde form (0.13% ofhighly concentrated sample and prolonged acquisition time for its detection. Figure 3 by guest on January 4, 2021http://www.jbc.org/Downloaded from We thank Prof. C. Park (KAIST) for helpful discussions and appreciate S.-for help with the crystallization process. This study used the beamline 6B at the Pohang Accelerator Laboratory and was supported by Creative Research Initiatives and the NRL program (M1-0203-00-0020) (to W.L.) of the Korean Ministry of Science & Technology. M.-S.K. was su

pported by the Brain Korea 21 Project.
pported by the Brain Korea 21 Project. -ribose 5-phosphate complex), and 1OGF (the glycerol (e-mail) Tris(hydroxymethyl) aminomethane hydrochloride by guest on January 4, 2021http://www.jbc.org/Downloaded from 23. Bjorkman, A. J., and Mowbray, S. L. (1998) 24. Bjorkman, A. J., Binnie, R. A., M. A., and Mowbray, S. L. (1994) 30206-30211. 25. Gunn, F. J., Tate, C. G., Sansom, C. E., and Henderson, P. J. (1995) Mol. Microbiol.26. Esnouf, R. M. (1999) Acta Crystallogr. D 27. Hwang, T.-L., and Shaka, A. J. (1995) J. Magn. Reson. Series A112, by guest on January 4, 2021http://www.jbc.org/Downloaded from W., Stoneking, T., Nhan, M., Waterston, R., and Wilson, R. K. Nature11. Park, S. H., Pastuszak, I., Dr 12. Flowers, H. M. (1981) Adv. Carbohydr. Chem. Biochem.13. Chen, Y. M., Zhu, Y., and Lin, E. C. (1987) 14. Kim, M. S., Oh, H., Park, C., and Oh, B. H. (2001) Acta Crystallogr. 15. Otwinowski, Z., and Minor, W. (1997) 16. Terwill

iger, T. C., and Berendzen, J. (1999) Ac
iger, T. C., and Berendzen, J. (1999) Acta Crystallogr. D17. Terwilliger, T. C. (2000) Acta Crystallogr. D 18. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard. (1991) Crystallogr. A 110-119 19. Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. Acta Crystallogr. D 20. CCP4. (1994) Acta Crystallogr. D 21. Holm, L., and Sanders, C. (1993) Trends Biochem. Sci. 22. Andersson, C. E., and Mowbray, S. L. (2002) by guest on January 4, 2021http://www.jbc.org/Downloaded from 1. Willis, R. C., and Furlong, C. E. (1974) 2. Furlong, C. E. (1982) 3. Buckel, S. D. (1986) 4. Anderson, A., and Cooper, R. A. (1969) Biochem. Biophys. Acta5. Oh, H., Park, Y., and Park, C. (1999) 14011 6. Drew, K. N., Zajicek, J., Bondo, G., Bose, B., and Serianni, A. S. 7. Sigrell, J. A., Cameron, A. D., Jo

nes, T. A., and Mowbray, S. L. Structure
nes, T. A., and Mowbray, S. L. Structure8. Zhu, Y., and Lin, E. C. (1988) 9. Blattner, F. R., Plunkett, G., 3rd, Bloch, C. A., Perna, N. T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K., Mayhew, G. F., Gregor, J., Davis, N. W., Kirkpatrick, H. A., Goeden, M. A., Rose, D. J., Mau, B., and Shao, Y. (1997) 10. McClelland, M., Sanderson, K. E., Spieth, J., Clifton, S. W., Latreille, P., Courtney, L., Porwollik, S., Ali, J., Dante, M., Du, F., Hou, S., Layman, D., Leonard, S., Nguyen, C., Scott, K., Holmes, A., Grewal, N., Mulvaney, E., Ryan, E., Sun, H., Florea, L., Miller, by guest on January 4, 2021http://www.jbc.org/Downloaded from existence of the cytoplasmic sugar-binding proteins. While RbsD genes are found only in bacteria, FucU genes are also found in mouse and human genomes, which underscores the functional importance of the protein. The two genes must have been derived from the same ancestral gene a

nd evolved to have different sugar-bindi
nd evolved to have different sugar-binding affinities. It remains to be determined whether there are unidentified cytoplasmic sugar-binding proteins with different ligand-specificity that act on the transport of other neutral sugars. The two RbsD homologues described above are potential candidates. It is possible that remote homologues of RbsD may play roles in the influx of other sugars. The biochemical function of RbsD and FucU identified in this study is to bind -fucose, respectively. The sugar-binding activity of the proteins FucU are regulated as a part of the operon or the and FucU. The presented structures and the common biochemical function of the two proteins delineated in this study lay a footstep toward gaining a complete picture for the energy-driven by guest on January 4, 2021http://www.jbc.org/Downloaded from -anomers. We do not know how RbsD would exert its β-anomers of -ribose. The functional role may lie in faci

litating the influx of the sugar substra
litating the influx of the sugar substrate, the event upstream of In prokaryotic cells, fucose permease appears primarily responsible for the energy-driven uptake of -fucose. We do not know yet the anomeric specificity of FucU in -fucose due to unavailability of commercial C-13 labeled and difficulties in obtaining suitable crystals of the protein. It remains to be determined whether, like RbsD, FucU may bind specifically an anomeric form enzymes in the utilization the sugar. The membrane-bound fucose permease and ribose permease (RbsC) are unrelated proteins. The mutated ptsG is unrelated to RbsC, either, but the transporter(5). These observations suggest that RbsD and FucU do not interact directly with their respective permease component to exert function. Concluding remarks The existence of periplasmic ligand-binding proteins has long been known. The biochemical functions and action modes of these proteins are well characterize

d. The novel protein fold of RbsD and th
d. The novel protein fold of RbsD and the sugar- by guest on January 4, 2021http://www.jbc.org/Downloaded from Consequently, the binding of ribose to periplasmic RBP is substantially tight, M (24). What would be the biological role(s) of RbsD and FucU? We demonstrated that the biochemical function of RbsD and FucU is to bind specific forms of -ribose and fucose, respectively. The residues at the sugar-binding site in the both proteins are remarkably conserved. Therefore, the functional role(s) of the proteins must be related to this sugar-binding activity. We ruled out an effector function, because the ribose-bound structures of RbsD exhibit no of the uncomplexed RbsD and the oligom mutant cells lacking A 4-fold enhancement of the ribose-uptake by expressing RbsD in this mutant (5) is presumably owing to the action ofNoticeably, ribokinase specifically phosphorylates the -furanose form of ribose, the least populated ring form of

the sugar (7), while RbsD selectively -a
the sugar (7), while RbsD selectively -anomeric forms of equilibrium population of the sugar. Therefore, without directly competing -ribose. In playing the role, the low affinity of RbsD for ribose may be required for minimal interference with furanose form of d through the nonenzymatic by guest on January 4, 2021http://www.jbc.org/Downloaded from ribose (Fig. 6b). The analysis led to the value of 0.93 mM. The data -pyranose form binds to RbsD more strongly than the -furanose form of ribose. In the dapyranose form of -ribose contributes solely to the heat release in the titration, and used the equilibrium concentration of this form of the sugar in solution. Considering some contribution the slightly higher than 0.93 mM. FucU binds value of 1.61 mM (Fig. 6c). In analyzing the data, we assumed that FucU -forms of -fucose with the same affinity, because the anomeric [Figure 6] The binding affinity between RbsD and -ribose i

s quite low. The differential gain of fr
s quite low. The differential gain of free energy in the transfer of ribose from the solvent to the binding cleft of RbsD seems small due to ththe sugar molecule in water. The mribose is uniquely distinguished from that between periplasmic RBP and ribose. While the ribose-binding to RbsD does not induce a noticeable conformational change of the backbone or a side chain of the protein (data not shown), the binding of ribose to periplasmic RBP triggers a large domain movement that leads to the shielding of the bound ribose from the bulk solvent and the formation of new interactions between protein atoms (23). by guest on January 4, 2021http://www.jbc.org/Downloaded from FucU lacks an enzyme activity toward identity with human FucU (Fig. 4). We cloned and purified which shares 21% sequence identity with RbsD. The elution times of the two proteins from a size-exclusion column Superdex 200 were the same, eric quaternary structure simi

lar to that of RbsD. In FucU, the ribose
lar to that of RbsD. In FucU, the ribose-binding residues His20, Asp28, Tyr120, and Asn122 in RbsD are also 100% conserved, while His98 and Lys102 are erved as arginine and tyrosine, -ribose containing the 6-methyl group and having a different configuration at the C-2 position. Most likely, these substitutions provide FucU with specificity and affinity for binding fucose. We recorded the absence of FucU (Fig. 5). The after a 2 hr incubation of the mixture at room temperature, demonstrating that FucU lacks an enzyme activity of converting -fucose to a product. [Figure 5] Weak sugar-binding affinity of RbsD and FucU. We analyzed the interactions of RbsD and FucU with sugar molecules by ITC. The isothermal titration cated that the binding affinity of the dissociation constant by this method (Fig. 6a). However, it was possible to by guest on January 4, 2021http://www.jbc.org/Downloaded from sugar-binding affinity of the mut

ant is reduced. This is consistent with
ant is reduced. This is consistent with the deduced sugar-binding modes. Sugar-binding residues are virtually invariant. A sequence alignment reveals that Asp28, His98, Asn122, and His20 are invariant, while Lys102 and Tyr120 are 100% homologously conserved, among the 40 deposited sequences of RbsD homologues except for three entries. The conservation of the sugar-binding residues is significant in that Pro32 and Gly105 are the only two residues that are more than 97 % conserved among the RbsD homologues. One of the three exceptions is NP_326431 (annotated as gine (Fig. 4). The other two entries are NP_407075 and ZP_00060076, which contain a substitution of Lys102 with histidine and a substitution of Tyr120 with asparagine, respectively. Interestingly, two of the three genes are not a component of a canonical enzyme II, A & B (NP_326431) or araC-family transcriptional regulatory share sequence homology with RbsR. RbsDbinding specif

icity for sugars different from ribose.
icity for sugars different from ribose. The observation raises a possibility that the energy-driven tranribose and fucose may also require biochemical activity similar to that of RbsD in some organisms. [Figure 4] by guest on January 4, 2021http://www.jbc.org/Downloaded from -furanose form of -ribose was impossible. Especially, the electron density did group of the furanose form. We determined the binding mode of the furanose form indirectly by elucidating the 2.05 Å resolution structure of RbsD in complex with -ribose 5-phosphate, whose ring form is exclusively is the furanose form. As expected, the electron density for the five-membered ring was defined well enough to allow the unambiguous fitting of the sugar ring (Fig. 3c). The phosphorylated sugar that we used was the mixture of the -anomers. The electron density indicated -anomer of consistent with the NMR data. The reRbsD is not an anomerase. The atmissing (methyl

ene part) or diffused (phosphate group).
ene part) or diffused (phosphate group). As the phosphate form of -ribose would bind to the protein in the same mode observed for -ribose 5-phosphate. Notably, Asn122 does not interact with the bound ribose 5-phosphate. In order to confirm the correctness of the crystallographically deduced sugar-binding modes, we substituted His98 with alanine, whose imidazole ring provides a hydrogen bond to the both anomeric forms of the sugar (Fig. 3). The NMR spectrum of -anomers of (Fig. 2) than in the presence of the wild-type RbsD, indicating that the by guest on January 4, 2021http://www.jbc.org/Downloaded from determination of the structures of RbsD in complex with compounds. In order to avoid competition of glycerol for binding to the room temperature. With a crystal, less featured, ‘fat’ electron density was found at each of the ten intersubunit clefts (Fig. 3). The poorly defined electron density, despite the molecules in d

ifferent configurations. Given the NMR d
ifferent configurations. Given the NMR data demonstrating the binding affinity of RbsD for the both -furanose form -ribose, we first interpreted the density with the most populated pyranose form of -ribose. The modeled conformation mostly accounted for the electron density (Fig. 3a). The sugar-binding mode reveals that one provides a predominant contribution over the other. Asp28, His98, Lys102 (via a water molecule), Tyr120, and Asn122 of one subunit and only His20 ring of Tyr120 is packed against the hydrophobic part of the bound sugar, a typical pattern frequently observed in the protein-sugar interactions. The bound ribose molecule fits tightly into the intersubunit cleft and leaves almost no room (Fig. 3b). [Figure 3] . Interpretation of the ‘fat’ by guest on January 4, 2021http://www.jbc.org/Downloaded from -anomeric forms of the sugar. We considered the possibility of RbsD being an enzyme that could β-a

nomeric forms and the open -ribose. If R
nomeric forms and the open -ribose. If RbsD has this enzyme activity, the line broadening of the two NMR peaks would be due to chemical exchange -pyranose forms of open chain aldehyde. Such an enzyme activity could enhance the -furanose form of ribokinase. For example, when the -furanose form is depleted (a non-equilibrium situation), rapid conversion of -anomeric forms into the open chain form should increase the appearance of the -furanose form through the spontaneous conversion of the open chain form into the form. We cloned and overexpressed ribokinase and measured the enzyme activity of phosphorylating ribosRbsD according to the method reported in the literature (22). RbsD did not enhance the enzyme activity of ribokinathe protein does not have this hypothetical enzyme activity. We also the enzymatic assay of ribokinase, [Figure 2] . The exposure of the by guest on January 4, 2021http://www.jbc.org/Downloaded from

has been commonly observed that enzymes
has been commonly observed that enzymes subunits (for examples, 2-Cys peroxiredoxin, Rubisco, cyanase, muconolactone isomerase, and glutamine synthetase), indicating that the oligomer formation is vital for the enzyme activity. The interaction between RbsD and glycerol is weak when assessed by isothermal titration calorimetry . It was not possible to measure the dissociation constant method. The high concentration of glpresumably allowed the observation of the bound glycerol. Glycerol may be one of polyol compounds that are able to bind fortuitously to the binding pocket of RbsD that is designed to interact most strongly with (discussed below). The interaction of RbsD with glycerol is presumably physiologically irrelevant. RbsD lacks an enzyme activity toward riboseWe recorded C)ribose in the presence and absence of RbsD. The addition of RbsD did not give rise to new peaks, demonstrating that the protein lacks an enzyme activity t

o convert -ribose into other compound. I
o convert -ribose into other compound. Instead, it caused significant broadening of the resonances arising from the C-1 atoms of the β-furanose forms, but not those of the -pyranose forms (Fig. 2). These spectroscopic data indicate -anomeric forms of anomeric forms of the sugar. Sin-anomeric by guest on January 4, 2021http://www.jbc.org/Downloaded from non-globular shape of the quaternary structure of the protein. The subunit αlike loop on the other side (Fig. 1b). The N-terminal loop (residues 1-9) of one molecule is extended from the main body of the structure and tightly interacts with three adjacent molecules on the decameric assembly. A strong and round electron density was found surrounded by the amino group of Lys2 the pentameric rings (Fig. 1c). This cage, which is completely buried inside the protein, is most likely to arise from binding of a chloride ion. The same feature is found in the structure of RbsD, and Lys2 is

conserved in all but two RbsD homologue
conserved in all but two RbsD homologues in the this ion cage is important for the assembly and stability of the decameric structure. A search for analogous folding architectures in the PDB database using the program Dali (21) revealed that the structure of RbsD is distinct from any other known protein folds. The structure of a hypothetical trimeric protein YjgF (PDB code: est match, is obviously different from [Figure 1] We found that the clefts between the shown) when the crystals were immers by guest on January 4, 2021http://www.jbc.org/Downloaded from [Table 1] RESULTS AND DISCUSSION Description of the structure of RbsD RbsD throughout the text. The structure of RbsD was determined with multiple-wavelength anomalous dispersion phasing by using one selenomethionine derivative crystal of the protein (Table 1). The final model, refined against data to 2.3 Å resoglycerol molecules, 2 chloride ions, and 369 water

molecules. The electron densities for al
molecules. The electron densities for all the backbone atoms are well defined. The five RbsD molecules in the asymmetric unit of the fashion to form a pentameric ring struct is related by the crystallographic 2-fold axis (Fig. 1a). The inner and the outer diameter of the ring is ~20 and 80 Å, respectively, interactions between the two pentamericof the two, while the five molecules on We previously reported that RbsD is an octamer in solution on the basis of an analytical centrifugation analysis (14). However, the structure of RbsD, determined by the molecular replacement method, shows that it is also a decamer (data not shown). The in by guest on January 4, 2021http://www.jbc.org/Downloaded from fell within the limits of all the quality criteria of the program PROCHECK (20). refinement statistics of the RbsD NMR spectroscopy-fucose were collected with Bruker DRX500 spectromerter at relaxation time of 2.0 s, and spectral width o

f 10,000 Hz were used. For NMR spectra,
f 10,000 Hz were used. For NMR spectra, a total of 64 transients in 32k data points, relaxation time of employed in processing free induction decays with XWIN-NMR software (Bruker Instruments). The -pyranose form of of the water peak set to 4.6 ppm. . All measurements were carried out at 25 dialyzed against a buffer solution containing 20 mM sodium phosphate (pH 7.6) and 1 mM fucose were dissolved in the same buffer. The samples were degassed for 20 min and centrifuged to remove any residuals prior to the measurements. Dilution enthalpies were determined in separate experiments (titrant into buffer) and subtracted from the enthalpies of the binding between the by guest on January 4, 2021http://www.jbc.org/Downloaded from 8with unit cell dimensions of °. The asymmetric unit of the crystal contained a pentameric ring of selenomethionyl RbsD crystal at 100 K on the beamline 6B of the Pohang were briefly immersed in the sa

me precipitant solution containing 7% gl
me precipitant solution containing 7% glycerol. Ten selenium sites in the asymmetric unit of the crystal were located and used for phase determination at 2.8 Å with the program SOLVE (16) and phases were subsequently improved by density modification with the program RESOLVE (17). The electron density was of excellent quality showing nearly all features of protein side chains. The program O (18) was used for model building, and refinement was performed package (19). From the beginning of the chain-tracing, binding of glycerol to the protein was apparent. ribose-5-phosphatecrystals mounted on a capillary tubes using a Rigaku Raxis IVsystem on a rotating anode generator. For the structure determination of the complexes, small amount of complexes were solved by direct refinement of the structure of the by guest on January 4, 2021http://www.jbc.org/Downloaded from 7 and using a Q sepharose fast-flow column (Amersham Pharmacia B

iotech). The purified FucU was dialyzed
iotech). The purified FucU was dialyzed against 20 mM sodium phosphate buffer (pH 7.6) containing 1 mM Overexpression and purification of E. coli ribokinasegene was amplified by the PCR method from cell lysate. The PCR EX HTa vector (Invitrogen) and introduced BL21(DE3) strain. The protein was expressed as a fusion -tag at the N-terminus. The cells were grown in media containing 0.1 mg ml of ampicillin. The expression of the protein was induced by 1 mM IPTG at an optical density of 0.6 at 18 C for 7 h. Bacterial lysate was prepared by sonication in buffer A containing 20 mM Tris-HCl buffer (pH 7.4) and 0.1 M NaCl. The fusion protein bound to a Ni-NTA column (QIAGEN) was eluted with buffer A containing 200 mM imidazole after washing the column with buffer A containing 20 mM imidazole. The eluted fractions containing RbsK were dialyzed against buffer containing 30 mM Tris-HCl buffer (pH 8.0). determination. Subsequeturned out to

be easy to work with. The crystals belo
be easy to work with. The crystals belong to the space group C2 by guest on January 4, 2021http://www.jbc.org/Downloaded from n in Luria-Bertani media containing 0.1 mg ml ampicillin. The expression of the protein was induced by 1 mM since the both N- and C-termini are involved in the oligomerization of the B. subtilis RbsD was purified by ammonium sulfate precipitation (50-60% fractionation) and column chromatographic separation employing a Hitrap Q (Amersham Pharmacia Biotech) and a Hiload 26/60 Superdex 200 size-exclusion column (Amersham Pharmacia Biotech). The purified protein was in 20 mM Tris-HCl buffer (pH 7.4) containing 0.2 M NaCl and 2 mM dithiothreitol. The crystals of by hanging-drop vapor diffusion method against reservoir solution containing 20% polyethyleneglycol 4000, 19% isopropanol, and 100 mM sodium cacodylate (pH 6.5). Overexpression and purification of E. coli FucUn reaction (PCR) technique from c

ell lysate. The PCR products were ligat
ell lysate. The PCR products were ligat BL21(DE3) strain. The expression of recombinant FucU protein was induced by 1 mM IPTG at an sonication in a buffer solution containing 20 mM Tris-HCl (pH 7.4), 0.1 M NaCl, and 1 mM dithiothreitol. The protein was purified by ammonium by guest on January 4, 2021http://www.jbc.org/Downloaded from the degradation of the sugar (13). Or, containing oligo- and polysaccharides (11). The conservation of FucU in higher organisms including human highlights the functional significance of the protein. Both FucU and RbsD, containing no signal sequence, must be cytoplasmic proteins. We sought to elucidate the biochemical function of RbsD and FucU on the basis of the three-dimensional structcomplete picture of the energy-driven transport of ribose and fucose. We into a homodecameric assembly. mcal studies led to the conclusion -anomeric forms of -fucose, revealing the existence of cytoplasmic sugar-bindin

g proteins. The name is coined in remini
g proteins. The name is coined in reminiscent of the periplasmic sugar-binding proteins that are one component of the proteins involved in the active transport of various neutral sugars. A potential role of these proteins may lie in helping translocation of sugar substrates into cells. MATERIALS AND E. coli RbsDs were cloned from respective genomes and expressed in E. coli by guest on January 4, 2021http://www.jbc.org/Downloaded from exclusively in the furanose forms, the five-membered ring forms. The cyclic forms of the sugar interconvert spontaneously via the least populated acylic aldehyde. Surprisingly, ribokinase binds and phosphorylates the furanose form of -ribose, the least populated ring form (7). For an efficient supply of the -furanose form of enzymatic activity of increasing the interconversion between the different forms of the sugar may be required, or the spontaneous conversion of the sugar at physiological conditio

ns is fast enough so that such an enzyme
ns is fast enough so that such an enzyme activity is unnecessary. A sequence alignment shows that RbsD is found in a variety of bacteria and ose, Uclose paralogue of RbsD, with any compared pair of RbsD and FucU homologues sharing about 20% amino acid sequence identity with each other. Prokaryotic exists as a component of fucose regulon (8-10), -galactose) (11). The fucose regulon encodes seven components: kinase, fuculose 1-phosphate aldolase, 1,2-propanediol oxidoreductase, -fucose is the major component in various oligo- plant (12). The isomerization of by guest on January 4, 2021http://www.jbc.org/Downloaded from -ribose is the most abundant and important sugar that is a component of nucleic acids and many other biomolecules as well as an energy source. Escherichia coli high affinity ribose transport system consists of six operon (ACBK). Ribose-space (1). The sugar-bound form of RBP interacts with the membrane-bound perme

ase (encoded by membrane (2) coupled wit
ase (encoded by membrane (2) coupled with the action of the ATP-binding cassette component (encoded by ) (3). The transported biosynthetic or metabolic pathways (4). RbsR is the repressor that binds to operon. Among the six components, RbsD is the only protein whose function is unknown. The only clue about mutant operon but containing plasmid- and mutated glucose transporter on its genome, which -ribose at low affinity (5). -ribose in solution exists as multiple forms; aldehyde form (0.13%) (6). In contrast, -ribose in biomolecules is by guest on January 4, 2021http://www.jbc.org/Downloaded from SUMMARY RbsD is the only protein whose biochemical function is unknown among operon involved in the active transport of ribose. FucU, a paralogue of RbsD conserved from bacteria to human, is also the only protein whose function is unknown among the seven gene RbsD, which reveals a novel decameric toroidal assembly of the protein.

Nuclear magnetic resonance and other stu
Nuclear magnetic resonance and other studies on RbsD the protein binds specific forms of ribose, but it does not have an enzyme activity toward the sugar. Likewise, tivity toward this sugar. We conclude that RbsD and FucU are cytoplasmic sugar-binding proteins, a l role may lie in helping influx of the sugar substrates. by guest on January 4, 2021http://www.jbc.org/Downloaded from Crystal structures of RbsD leading tosugar-binding proteins with a novel folding architecture* , Weontae LeeCenter for Biomolecular Recognition and Division of Molecular and Life Department of Life Science, PTechnology, Pohang, Kyungbuk, 790-784, Korea Department of Biochemistry, College of Science, Yonsei University, Seoul, Pohang Accelerator Laboratory, Pohang, Kyungbuk, 790-784, Korea. Running Title: Cytoplasmic sugar-binding proteins Keywordssport/ribose/fucose/cytoplasmic sugar-binding protein/crystal structure by guest on January 4, 2021http://