The binding of β- and γ-cyclodextrins to glycogen phosphorylase b: Kinetic and crystallographic studies

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A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the inhibitor, and the new allosteric sites are currently under investigation as targets for inhibition of hepatic glycogenolysis under high glucose

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  The binding of    - and   -cyclodextrins to glycogenphosphorylase b: Kinetic and crystallographic studies NIKOS PINOTSIS, 1 DEMETRES D. LEONIDAS, 2 EVANGELIA D. CHRYSINA, 2 NIKOS G. OIKONOMAKOS, 2,3 AND  IRENE M. MAVRIDIS 1 1 Institute of Physical Chemistry, National Center for Scientific Research “Demokritos,” Athens, Greece 2 Institute of Organic and Pharmaceutical Chemistry, and  3 Institute of Biological Research and Biotechnology, TheNational Hellenic Research Foundation, Athens 11635, Greece(R ECEIVED  April 21, 2003; F INAL  R EVISION  May 29, 2003; A CCEPTED  May 30, 2003) Abstract A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the inhibitor,and the new allosteric sites are currently under investigation as targets for inhibition of hepatic glycogen-olysis under high glucose concentrations; in some cases specific inhibitors are under evaluation in humanclinical trials for therapeutic intervention in type 2 diabetes. In an attempt to investigate whether the storagesite can be exploited as target for modulating hepatic glucose production,   -,   -, and   -cyclodextrins wereidentified as moderate mixed-type competitive inhibitors of GPb (with respect to glycogen) with  K  i  valuesof 47.1, 14.1, and 7.4 mM, respectively. To elucidate the structural basis of inhibition, we determined thestructure of GPb complexed with   - and   -cyclodextrins at 1.94 Å and 2.3 Å resolution, respectively. Thestructures of the two complexes reveal that the inhibitors can be accommodated in the glycogen storage siteof T-state GPb with very little change of the tertiary structure and provide a basis for understanding theirpotency and subsite specificity. Structural comparisons of the two complexes with GPb in complex witheither maltopentaose (G5) or maltoheptaose (G7) show that  - and  -cyclodextrins bind in a mode analogousto the G5 and G7 binding with only some differences imposed by their cyclic conformations. It appears thatthe binding energy for stabilization of enzyme complexes derives from hydrogen bonding and van der Waalscontacts to protein residues. The binding of    -cyclodextrin and octakis (2,3,6-tri- O -methyl)-  -cyclodextrinwas also investigated, but none of them was bound in the crystal; moreover, the latter did not inhibit thephosphorylase reaction. Keywords:  Glycogen phosphorylase;  -cyclodextrin;  -cyclodextrin;  -cyclodextrin; oligosaccharide bind-ing; protein–carbohydrate interactions; X-ray crystallographyGlycogen phosphorylase (EC 2.4.1.1; GP) has been a mo-lecular target for structure-assisted design of inhibitors thatmight prevent unwanted glycogenolysis under high glucoseconditions. Several binding sites in GP, such as the cata-lytic, the allosteric, the inhibitor, and the new allosteric siteshave been explored as specific targets in kinetic, crystallo-graphic, and physiological studies (reviewed in McCormacket al. 2001; Somsak et al. 2001; Treadway et al. 2001;Oikonomakos 2002; Kurukulasuriya et al. 2003). However,the glycogen storage site has not been experimentally vali-dated as a target for modulating hepatic glucose productionin type 2 diabetes, and no medicinal chemistry studies havebeen reported on this target yet. This site was identifiedfrom crystallographic binding studies with maltopentaose(G5) and maltoheptaose (G7), that also bind to the catalyticsite and act as substrates (Johnson et al. 1988). In GPb, G5 Reprints requests to: Nikos G. Oikonomakos, Institute of Organic andPharmaceutical Chemistry, The National Hellenic Research Foundation, 48Vas. Constantinou Avenue, Athens 11635, Greece; e-mail: ngo@eie.gr;fax: 30-210-7273758(831).  Abbreviations:  GP, glycogen phosphorylase; GPb, muscle glycogenphosphorylase b; GPa, muscle glycogen phosphorylase a; glucose,   - D -glucose; Glc-1-P,   - D -glucose 1-phosphate;   -,  -,  -CD,   -,  -,  -cyclo-dextrin; TM  CD, octakis (2,3,6-tri- O -methyl)-  -cyclodextrin; G5, malto-pentaose; G7, maltoheptaose; RMSD, root-mean-square deviation.Article and publication are at http://www.proteinscience.org/cgi/doi/ 10.1110/ps.03149503. Protein Science  (2003), 12:1914–1924. Published by Cold Spring Harbor Laboratory Press. Copyright © 2003 The Protein Society 1914  can bind to the glycogen storage site by filling five majorand two minor subsites. The precise role of the glycogenstorage site is not fully understood, but it might serve as aregion through which the mammalian enzyme is attached toglycogen particles in vivo, to produce an effective highconcentration of end groups and act as additional regulatorysite whereby occupation by glycogen is a prerequisite forcatalysis. Also, model building studies (McLaughlin et al.1984) have shown that the minor site might be consistentwith the position of oligosaccharide at an   -1,6 branch po-sition, providing an explanation for the increased affinity of GP for branched polysaccharides like glycogen over linearglucans like maltodextrins (Hu and Gold 1975). Inhibitors,specific for this site, would be therefore of most interest.Acarbose, a pseudo-maltotetraose with the cyclitol unit of   -1,4-linked to a 4-amino-4,6-dideoxyglucose residue, is apotent inhibitor of many   -glucosidases,   -amylases, andsucrase-isomaltase, and is considered to be an analog of aglucosyl cation like transition state (Truscheit et al. 1981).Binding studies in the crystal of GPa showed that it boundat the major site of glycogen storage site in an analogousway to maltopentaose inhibitor. Kinetic studies have shownthat acarbose is a reasonable inhibitor of GPa ( K  i    26mM) in competition experiments with 30 mM G5 (Gold-smith et al. 1987). Apart from acarbose, specific inhibitorsof the enzyme that bind at the glycogen storage site have notbeen previously described.Given the structural analogy of cyclic oligosaccharides tolinear ones, we investigated the effect of    -,   -, and   -cy-clodextrins (CDs) on the catalytic and structural propertiesof GPb. The cyclodextrins are torus-like macrorings built upfrom   - D -glucopyranose units connected with an   -1,4 gly-cosidic linkage (Fig. 1). The   -CD molecule comprises 6glucopyranose units, while   - and   -CD comprise 7 and 8units, respectively. As a consequence of the  4 C 1  conforma-tion of the glucopyranose units, primary and secondary hy-droxyl groups are situated on either side of two edges of thering. The ring is a truncated cone whose cavity is lined bythe hydrogen atoms and the glycosidic oxygen bridges, re-spectively. The secondary O2 hydroxyl group of one glu-copyranose unit forms a hydrogen bond with the O3 hy-droxyl group of the preceding glucopyranose. As a result, acomplete belt of hydrogen bonds is formed in the secondaryside that is stronger in   -CD (Makedonopoulou and Mavri-dis 2000), making it the most rigid and the less water-soluble of all three CDs. In contrast, this hydrogen bond beltis not complete in   -CD (one of the glucose moieties isdistorted) and in   -CD (more distorted conformation), re-sulting in a higher flexibility and aqueous solubility com-pared to that of    -CD (Szejtli 1998).In this work we present a detailed description of the bind-ing of    -,   -,   -CDs and TM  CD to GPb through kineticand X-ray binding studies. The kinetic results show that   -,  -, and   -CDs are moderate inhibitors of the enzyme, with  -CD being the best ( K  i    7.4 mM), while TM  CD did notinhibit GPb. To elucidate the key interactions responsiblefor inhibition we performed crystallographic binding studieswith the above compounds and analyzed the structures of those that showed binding, namely GPb-  -CD and GPb-  -CD complexes at 1.94 Å and 2.3 Å resolution, respectively.The detailed interactions of    - and   -CDs with the proteinprovide a structural explanation for the kinetic propertiesand show that the cyclic oligosaccharides bind at the gly-cogen storage site of GPb in a mode analogous to the G5and G7 binding, with some changes imposed by differencesin their conformations. To improve the precision of ourunderstanding of the molecular basis of linear oligosaccha-ride recognition, we have also determined the crystal struc-tures of the GPb-G5 and GPb-G7 complexes at 2.2 Å reso-lution. Results and Discussion The kinetic parameters ( K  i s) of    -,   -, and   -CDs are sum-marized in Table 1. The kinetic experiments with GPb, inthe direction of glycogen synthesis, showed that all three Figure 1.  Chemical structure of    -CD and numbering scheme used in aglucose unit (IUPAC-IUB 1983). Binding of    - and   -cyclodextrins to phosphorylasewww.proteinscience.org  1915  cyclodextrins exhibited mixed-type inhibition with respectto glycogen substrate at constant concentrations of AMP (1mM) and Glc-1-P (4 mM). Lineweaver-Burk plots (1/  v  ver-sus 1/[glycogen]) yielded straight lines of which the slopesand the vertical axis intercept increased with increasing con-centrations of inhibitor.   -CD ( K  i    7.4 mM) was found tobe a better inhibitor than   -CD ( K  i    14.1 mM) and   -CD( K  i    47.1 mM). TM  CD had no effect on the enzymicactivity of GPb when added in concentrations varied from10–50 mM, in the presence of 1 mM AMP, 4 mM Glc-1-P,and 0.035% (w/v) glycogen.Crystallographic data collection, processing, and refine-ment statistics for the structure determinations of GPb com-plexed with G5, G7,   -CD, and   -CD, respectively, aresummarized in Table 2. The overall architecture of the na-tive T-state GPb showing the location of the catalytic, theinhibitor, the allosteric, the new allosteric, and the glycogenstorage site is presented in Figure 2. For both the G5 and G7complexes, electron density maps indicated that five glu-cose residues (S3 to S7) bound at the major site of theglycogen storage site, and there was no binding at the minorsite. For the GPb–  -CD complex, all glucose residues (S3 toS10) were visible in the electron density map, while for theGPb–  -CD complex the electron density maps suggestedpartial occupancy of    -CD of the glycogen storage site. Nobinding was observed at the catalytic site. The electron den-sity maps for GPb crystals soaked with   -CD and TM  CDindicated no binding at the glycogen storage site. We de-scribe briefly the G5 and G7 interactions at the glycogenstorage site and in more detail the   -CD and   -CD interac-tions at this site. The binding of maltopentaose and maltoheptaoseto glycogen phosphorylase b A major and a minor glycogen storage site have been de-fined from the crystal structures of the GPb–G5 complex at Table 1.  Kinetic parameters for maltoheptaose and cyclodextrins Oligosaccharide K i  (mM)Maltoheptaose 1.0 a  -Cyclodextrin 47.1 ± 5.1 b  -Cyclodextrin 14.1 ± 3.7 b  -Cyclodextrin 7.4 ±1.3 ca Data taken from Kasvinsky et al. (1978). b Calculated from double-reciprocal plots with varied glycogen concentra-tions (0.02%, 0.025%, 0.035%, and 0.05% w/v), constant concentrations of Glc-1-P (4.0 mM) and AMP (1 mM) and 9.4 mM oligosaccharide. c As above, except that three different concentrations of    -cyclodextrin(4.7, 9.4, and 14.0 mM) were used. Best fit lines (not shown) were com-puter generated according to the equation for linear mixed-type inhibition(Segel 1975) by fitting all of the data at once and using the nonlinearregression program  GraFit   (Leatherbarrow 1992). The kinetic parametersdetermined in this way for GPb inhibition by   -cyclodextrin were as fol-lows: V    147.4 ± 13.7 IU/mg,  K  s    0.023 ± 0.006% (w/v),  K  i    7.4 ±1.3 mM. Table 2.  Diffraction data and refinement statistics GPb complex G5 G7   -CD   -CDResolution (Å) 30.0–2.2 30.0–2.20 30.0–1.94 30.0–2.3Outermost shell (Å) 2.24–2.20 2.24–2.20 1.96–1.94 2.38–2.30No. of observations 442,007 456,706 714,419 360,103No. of unique reflections 49,992 49,982 68,504 43,859R symma 0.079 0.096 0.076 0.110(outermost shell) (0.426) (0.467) (0.499) (0.483)Completeness (%) 95.9 95.7 93.4 96.5(outermost shell) (%) (85.0) (84.2) (85.0) (84.9) 〈  I   /   (  I  ) 〉 b (outermost shell) 12.7 (2.8) 13.8 (2.2) 13.3 (2.1) 11.0 (2.0)Wilson B factor (Å 2 ) 28.5 32.0 17.9 28.1  R crystc 0.182 0.184 0.193 0.189  R freed 0.215 0.217 0.218 0.232No. of protein atoms 6575 6571 6597 6571No. of ligand atoms 56 56 22 88No. of water molecules 300 304 324 272RMSD in bond lengths (Å) 0.006 0.006 0.006 0.006RMSD in angles (°) 1.2 1.2 1.3 1.2Average B factor (Å 2 )Protein atoms 36.2 36.3 28.9 35.3Water molecules 41.5 41.2 37.3 38.3Ligand atoms 59.8 61.0 64.3 67.3 a  R symm    ∑ h ∑ i |  I  ( h ) −  I  i ( h ) |∑  h ∑ i  I  i ( h ) where  I  i ( h ) and  I  ( h ) are the  i th and the mean measurements of the intensityof reflection  h . b  (  I  ) is the standard deviation of   I  . c  R cryst    ∑ h | F  o  −  F  c |  /  ∑ h F  o , where  F  o  and  F  c  are the observed and calculated structure factors amplitudes of reflection  h , respectively. d  R free  is equal to  R cryst  for a randomly chosen 5% of the reflections that were not included in the refinement. Pinotsis et al. 1916  Protein Science, vol. 12  2.5 Å resolution (Johnson et al. 1988). The major site isdefined by the binding of five glucose units in subsitesS3-S4-S5-S6-S7, with the reducing end of the oligosacha-ride bound in subsite S3. This site is made up of helices  12and   13 (residues 396 to 418 and 420 to 429, respectively)and the loop connecting the two antiparallel strands,   15(residues 430 to 432) and   16 (residues 437 to 411; Fig. 2).The minor site, defined by the binding of glucosyl residuesS8 and S9, lies above the nonreducing end of the major site.It involves GP residues from the top of helix   12, the loopconnecting the antiparallel   -sheets   8 (residues 198 to209) and  9 (residues 212 to 223) and Val354 from helix  9(residues 344 to 355). In the complex structures of GPa–G7–caffeine–glucose and GPa–G5–phosphate, seven gluco-syl moieties (S1 to S7) were found at the major site and four(S8 to S11) at the minor site (Goldsmith et al. 1982, 1989;Goldsmith and Fletterick 1983). In contrast, in the complexGPa–G5–caffeine–glucose, only three glucosyl residues (S4to S6) and one glucose (S10) were found in the minor site(Goldsmith et al. 1989). In the GPb–heptulose–2-P–G7–AMP complex (Johnson et al. 1990), the minor oligosac-charide site is not occupied.One molecule of either G5 or G7 bound at the GPb stor-age site occupying the major site, while no binding wasobserved at the minor site. The mode of binding and theinteractions that G5 and G7 exhibit with GPb are almostidentical with those for the GPa–G5–phosphate, GPa–G5–caffeine–glucose, and GPa–G7–caffeine–glucose complexes Figure 2.  A schematic diagram of the muscle GPb dimeric molecule viewed down the twofold for residues 13 to 838. One subunitis colored gray and the other subunit light gray. The positions are shown for the catalytic, the allosteric, the inhibitor, the new allostericinhibitor site, and the glycogen storage site. The catalytic site, which includes the essential cofactor pyridoxal 5  -phosphate (notshown), is buried at the center of the subunit accessible to the bulk solvent through a 15 Å long channel. Glucose (shown inball-and-stick representation), a competitive inhibitor of the enzyme that promotes the less active T state through stabilization of theclosed position of the 280s loop, binds at this site. The allosteric site, which binds the activator AMP, is situated at the subunit–subunitinterface some 30 Å from the catalytic site. The inhibitor site, which binds purine compounds, nucleosides, or nucleotides at highconcentrations, and flavopiridol (shown bound; Oikonomakos et al. 2000) is located on the surface of the enzyme some 12 Å from thecatalytic site and, in the T state, obstructs the entrance to the catalytic site tunnel. The new allosteric inhibitor site, located inside thecentral cavity, formed on association of the two subunits, binds N-Benzoyl-N  -  - D -glucopyranosyl urea molecule (shown; Oikono-makos et al. 2002). The glycogen storage site (with   -CD bound) is on the surface of the molecule some 30 Å from the catalytic site,40 Å from the allosteric site, and 50 Å from the new allosteric site. Binding of    - and   -cyclodextrins to phosphorylasewww.proteinscience.org  1917  (Goldsmith et al. 1982, 1989; Goldsmith and Fletterick 1983) derived from medium-resolution X-ray crystallo-graphic analyses, and that for the GPb–heptulose–2-P–G7–AMP complex (Johnson et al. 1990) determined at a reso-lution of 2.86 Å resolution. The resolution of the presentstructures allows us to describe the binding site for G5 andG7 in some detail. Both structures indicated five well-or-dered structural waters that form a network of hydrogenbonds that link oligosaccharide to protein residues; thesewaters were not observed in the previous medium-resolu-tion structures.The bound G5 has a regular helical structure stabilized bythe O2–O3 hydrogen bonds formed between successivesugars with a break between glucose residues S4 and S5(Fig. 3A). The temperature factors are lower (50–62 Å 2 ) atglucose residues S4, S5, and S6, and increase (65–80 Å 2 )toward the two ends of the oligosaccharide chain. The O3hydroxyl group of glucose residue S4 forms a hydrogenbond with Ser429 from helix   13, and O2 hydroxyl is in-volved in a water-mediated interaction with Val431 O,Arg426 O, and Gln433 N through another water molecule.The O2 hydroxyl group of glucose residue S5 is hydrogen Figure 3.  (Continued on next page) Pinotsis et al. 1918  Protein Science, vol. 12
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