Tissue-specific Structure/Function Differentiation of the Liver Isoform of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase*

Yong-Hwan LeeDagger §, Yang Li, Kosaku Uyeda||, and Charles A. Hasemann**

From the Dagger  Structural Biology Core, Molecular Biology, University of Missouri, Columbia, Missouri 65211, the || Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390,  Research Service, Veterans Affairs Medical Center, Dallas, Texas 75216, and ** Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan 48105

Received for publication, September 5, 2002, and in revised form, October 11, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The crystal structures of the human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in three different liganding states were determined and compared with those of the rat testis isozyme. A set of amino acid sequence heterogeneity from the two distinct genes encoding the two different tissue isozymes leads to both global and local conformational differences that may cause the differences in catalytic properties of the two isozymes. The sequence differences in a beta -hairpin loop in the kinase domain causes a translational shift of several hydrophobic interactions in the dimeric contact region, and its propagation to the domains interface results in a 5° twist of the entire bisphosphatase domain relative to the kinase domain. The bisphosphatase domain twist allows the dimeric interactions between the bisphosphatase domains, which are negligible in the testis enzyme, and as a result, the conformational stability of the domain is increased. Sequence polymorphisms also confer small but significant structural dissimilarities in the substrate-binding loops, allowing the differentiated catalytic properties between the two different tissue-type isozymes. Whereas the polymorphic sequence at the bisphosphatase-active pocket suggests a more suitable substrate binding, a similar extent of sequence differences at the kinase-active pocket confers a different mechanism of substrates bindings to the kinase-active pocket. It includes the ATP-sensitive unwinding of the switch helix alpha 5, which is a characteristic ATP-dependent conformational change in the testis form. The sequence-dependent structural difference disallows the liver kinase to follow the ATP-switch mechanism. Altogether these suggest that the liver isoform has structural features more appropriate for an elevated bisphosphatase activity, compared with that of the testis form. The structural predisposition for bisphosphatase activity in the liver isozyme is consistent with the liver-unique glucose metabolic pathway, gluconeogenesis.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Due to the two independent catalytic domains in a single polypeptide chain, the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6-Fru(P)-2-kinase/Fru-2,6-P2ase),1 catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate (Fru-2,6-P2), a most potent allosteric regulator with dual functions as follows: an activator of phosphofructokinase and, simultaneously, an inhibitor of fructose-1,6-bisphosphatase, the key rate-limiting enzymes of glycolysis and gluconeogenesis, respectively (1-3). The functional duality allows Fru-2,6-P2 to play a critical role in control of the rates of both glycolysis and gluconeogenesis. An elevated cellular concentration of Fru-2,6-P2 increases glycolytic flux, whereas a lowered concentration of Fru-2,6-P2 results in a net increase in gluconeogenic flux. Because the liver is the major organ for vertebrate glucose homeostasis, regulation of the activity in the liver isoform of 6-Fru(P)-2-kinase/Fru-2,6-P2ase and the resulting Fru-2,6-P2 regulates glucose homeostasis in an organism (1-5). Reflecting the diversity of tissues and their functions, at least six different tissue-specific isoforms from five distinct genes express varying ratios of kinase to bisphosphatase activity. A single isoform generally predominates in each distinct tissue (1, 4).

The determination of crystal structures of the rat liver Fru-2,6-P2ase domain (6) and the rat testis 6-Fru(P)-2-kinase/Fru-2,6-P2ase (7) has led a great progress in our understanding of the catalytic mechanisms of this enzyme system (8, 9). However, one of the most fundamental questions about this enzyme system has remained unanswered. How are the structures of the tissue-specific isozymes of 6-Fru(P)-2-kinase/Fru-2,6-P2ase related to their differential functions in tissues that have specific physiological roles and, accordingly, different optimums of glucose metabolism? Reflecting such differences in roles, the two isozymes have been shown to have different kinetic properties, summarized in Table I (adapted from Ref. 10). Despite a reasonably high amino acid sequence identity (74%), the liver isoform kinase shows a 4-fold higher affinity for Fru-6-P but half the Vmax compared with the testis form, whereas the bisphosphatase has at least 20-fold increased affinity for Fru-2,6-P2 and a 2-fold increase in Vmax (10). These kinetic differences suggest that glucose metabolism is uniquely related to the physiological roles of the given tissues and not just a simple housekeeping function for energy production (10, 12, 13). This is more evident by the fact that only the liver isozyme of the two isozymes is phosphorylated by the cAMP-dependent protein kinase upon the signal of low levels of glucose and/or glucagon. The dephosphorylation is achieved by xylulose 5-phosphate (Xyl-5-P)-dependent protein phosphatase 2A upon the detection of insulin and/or high glucose levels (14-18). Phosphorylation of the liver 6-Fru(P)-2-kinase/Fru-2,6-P2ase induces an as yet unknown conformational change by which the predominant activity switches from the kinase to the bisphosphatase that leads to a decrease in the cellular concentration of Fru-2,6-P2 (15, 16) and hence stimulating gluconeogenesis.

                              
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Table I
Comparison of the kinetic parameters of the isotype bifunctional enzymes
This table is adopted from the original (10), in which the experimental conditions were kept identical to compare the kinetic properties of the two different tissue-specific isozymes.

Therefore, investigation of the structural differences in tissue isoforms will provide a new insight into the structure/function relationship, which is differentiated according to the physiological roles of the tissues expressing those isozymes. To better understand this unique enzyme system and to provide a better target for drug design, the crystal structure of the human liver isoform, which has kinetic properties nearly identical to the rat liver form of better than 98% (as suggested by sequence identity) (11), was determined and compared with the rat testis form, and the results are reported here.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Preparation and Crystallization of the Human Liver Bifunctional Enzyme-- The W0 mutant form of human liver 6-Fru(P)-2-kinase/Fru-2,6-P2ase (all Trp residues and Pro2 mutated to Phe and Arg, respectively) was employed for this study because it has better expression in Escherichia coli with the kinetic properties indifferent from those of the wild type.2 The same mutant form was used for the studies of the testis form (7). The protein was expressed in E. coli BL21(DE3) and purified using DEAE and Mono Q anion-exchange chromatography. Crystals were prepared by hanging drop vapor diffusion at 12 mg ml-1 protein (in 20 mM Tris-HCl, pH 7.25, 10 mM NaPi, 15% glycerol, 0.5 mM EDTA, 10 mM dithiothreitol, 6 mM ATPgamma S·Mg or ADP, and 5-10 mM Fru-6-P) mixed 1:1 with a mother liquor of 50 mM Tris-HCl, pH 7.5, 20-25% ethylene glycol, and 8% polyethylene glycol 4000. Crystals in a size of 0.2 × 0.2 × 0.05 mm were grown in 4-5 days.

Data Collection and Processing-- Crystals were submerged into liquid propane for flash freezing, and the low temperature (120 K) was maintained by an X-stream (MSC, The Woodlands, TX) cryogenic cooling device. Diffraction data were collected on an R-Axis IV image plate detector mounted on a Rigaku FR590 rotating anode generator, which was operated at 50 mA, 100 kV. The x-ray beam was focused using an Osmic confocal multilayer optic system. The data were integrated using DENZO and merged and scaled with SCALEPACK (20). The reduced data were formatted for the CCP4 suite (21), and 10% of the data were marked for free R-factor measurements in subsequent structure refinements. As shown in Table II, complete data sets with various resolution ranges depending on the liganding states were collected. All crystals belong to a C2 space group regardless of the bound ligands. The data statistics are summarized in Table II.

                              
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Table II
Statistics of reflection data and structure refinements
Numbers in parentheses are values in the highest resolution shell. Rsym = Sigma h(Sigma j|Ih,j - <Ih>|/Sigma Ih,j), where h = set of Miller indices and j = set of observations of reflection h. R.m.s.d. values are deviation from ideal values.

Structure Determination and Refinement-- The structure of the ATPgamma S complex was determined first by the molecular replacement method using X-PLOR (22). A single polyalanine form of the testis structure (Protein Data Bank accession code 1bif) was used as a search model. An X-PLOR rotation and subsequent translation search was performed using a data subset between 15.0 and 4.0 Å. A self-rotation search as well as the measurement of solvent fraction suggested a non-crystallographic dimer of the liver enzyme in 1 asymmetric unit. Assignment of the side chains and non-crystallographic dimerization was made using O (23). The initial model went through iterated cycles of manual model rebuilding and X-PLOR refinement, followed by two cycles of "refmac" refinement (21). During this procedure, a single model was the subject of all manual model corrections to prevent the accumulation of structural errors developed during the refinement cycles. When Rcrys/Rfree, reached 0.254/0.289, one molecule of ATPgamma S per protein molecule was built into the model, referring to an  Fo - Fc  omit map. As shown in Table II, the final model has a Rcrys/Rfree factor of 0.217/0.257 using a total of 7,545 scatterers, including solvent molecules, against 42,142 reflections. Of the 864 residues in the dimer, 89.3% are within the "most favorable region" of main chain dihedral angles distributions and none in the "disallowed region," when judged using "procheck" (21). The final structure was deposited in the Protein Data Bank under the accession code of 1K6M.

The subsequent structures of the liver isozyme complexed with either ADP or Pi were determined by using the structure of the ATPgamma S complex as the starting model and by following the structural refinements procedures similar to that used for the ATPgamma S complex. But, to guarantee a freedom to structural refinements, the indices of reflections in the free data were kept as those of the ATPgamma S complex. The B-factor refinement of the Pi complex was stopped after one cycle of group B-factor refinement, and the solvent molecules were not added to the structure due to a limited resolution. The statistics of reflection data and structure refinements are summarized in Table II.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Similarity of Overall Folding in the Liver and Testis Isoforms-- The crystal structures of the liver isozyme reveal a head-to-head homodimer of 432 amino acid residues per monomer (residues 39-470) (Fig. 1a and 2). Depending on the liganding conditions, each subunit contains an ATPgamma S, ADP, or Pi molecule bound to the kinase domain, and two inorganic phosphates, regardless of the liganding conditions, bound to the bisphosphatase domain (Table II). No electron density is interpretable for the N-terminal regulatory fragment of residues 1-38, in which Ser-32, the cAMP-dependent phosphorylation site (24-27), and a number of amino acid sequence differences are located (Fig. 2). The structure of this fragment thus remained undetermined in both the liver and testis isozymes.


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Fig. 1.   Stereo views of dimeric arrangement of the liver form and a comparison of the structures of the two isoforms. a, dimeric arrangement of the functional liver isozyme subunits about the crystallographic 2-fold axis of rotation is shown as a Calpha trace. Each monomer is colored differently, and the residue numbers are labeled every 25 residues. b, the monomeric structures of each isozyme were superimposed by a least square fit of the testis kinase domain onto that of the liver form using O (23). The liver kinase and bisphosphatase domains are colored red and blue, respectively, and those of the testis form are both in gray. Note that the liver Fru-2,6-P2ase domain is rotated ~5° relative to the same domain of the testis form. The secondary structure motifs involved in dimeric contacts are labeled (beta 1-5, beta 1-6, and alpha 7). The positions of amino sequence differences related to different dimeric interactions and the bisphosphatase twist are shown as the dark dots. All the structural figures were produced and rendered using Molscript (33) and Raster3D (19).


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Fig. 2.   Comparison of the amino acid sequences of the liver and testis isozymes. Identity of the sequences was compared along with their secondary structures. The identical sequences are represented by - and deletions or insertions by a period.

As predicted from a reasonably high sequence identity (74%) of tissue isozymes (Fig. 2), the liver bifunctional enzyme has a structure very similar to that of the testis isoform, consistent with similarities in their catalytic mechanisms (7, 9-11, 28). As shown in Fig. 1b, where the ribbon diagrams of the two isozyme kinase domains are superimposed, the overall folding of the liver form is very similar to that of the testis form. The similarity in folding patterns is maintained throughout both the kinase and phosphatase domains. The main structural characteristics are conserved in the liver form, i.e. the major dimeric interactions through the kinase domains, the constellation of catalytic residues in the active sites of each domain, and the manner of intramonomer domain-domain interactions.

All this together allowed us to assign the secondary structural motifs of the liver form according to those of the testis form (7) with no ambiguity, and for convenience of comparison, the amino acid sequences are compared with the assigned secondary structures in Fig. 2. Unless specifically mentioned, for the entire text of this report, all the amino acid sequence and numbers are according to the liver isozyme. For example, T224Q is Thr-224 in human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and is Gln in the same position of the testis isozyme.

Bisphosphatase Twist and Differentiated Dimeric Interface-- Although the number is small, the amino acid sequence differences between the two tissue isozymes seem to cause a variety of conformational differences. That includes a twist of the bisphosphatase domain relative to the kinase domain, resulting in an altered dimeric interface. Despite the fact that the amino acid residues involved in this interface are relatively well conserved, the bisphosphatase domain of the liver form is twisted by 5° around the dimeric axis from its position in the testis form (Fig. 1b). We considered three possibilities of this twist as follows: an indirect effect of the mobile N terminus, a difference in crystal contacts between the two isoforms, and an intrinsic difference coming from the amino acid sequence. A possibility of an effect of phosphorylation of Ser-32 at the mobile N terminus is ignorable since the enzyme was expressed in bacterium and purified in the absence of any protein phosphatase inhibitor such as fluoride. To test its relevance to liganding states and crystal contacts, the three structures of the testis isoform all in different crystal contacts and liganding states (ATPgamma S, AMP-PNP, and Pi) (7, 9, 28) and the three structures of the liver form in three different liganding states (ATPgamma S, ADP, and Pi) were cross-compared by superimposing onto each other. The positional differences of Calpha s were calculated and presented as Table III. The liver form ADP complex and the testis form AMPPNP complex were excluded from this table, because they were so similar to the liver form ATPgamma S complex and the testis form ATPgamma S complex, respectively, with a difference range of <0.5 Å. It is evident that the overall difference (~2 Å) between the two tissue isozymes is mainly caused by the bisphosphatase domain (~3 Å). This difference is not significantly affected by the liganding states, which cause less than 1 Å in magnitude. Legitimacy of the positional difference was tested by rotating the testis bisphosphatase domain by 5°, and the result, which is 2.8 Å, was found to be close to the value in the table. Because no different space group with reasonable resolution is available for the liver form, it is impossible to directly infer the effect of crystal contacts on the liver form. However, comparisons between the structures of the testis form, which have three different space groups (P1 for the Pi complex, C2 for the AMPPNP complex, and P3121 for the ATPgamma S complex), allowed an indirect observation. When these three structures were compared, the positional differences caused by the different crystal contacts are neither domain-specific nor significant (<0.8 Å). This observation becomes more convincing by the fact that the major source of crystal contacts in both isoforms is provided by the conserved dimeric kinase interactions. All this together suggests that the rotated bisphosphatase is a featuring conformation of the liver form.

                              
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Table III
Average positional differences of Calpha among the isotype bifunctional enzymes and their domains (Å)
The positional differences of the Calpha atoms of the two isozymes each in two different liganding conditions (ATP and Pi) are compared after superimposing the structures onto each other. The numbers in parentheses are representing the kinase and bisphosphatase domains, respectively. The space groups of the crystals are also in parentheses. The structures of testis·Pi and Testis·ATPgamma S were acquired under the PDB codes, 1bif and 3bif, respectively.

Moreover, it was found that the amino acid sequence differences in the kinase domain can cause a twist of the liver form bisphosphatase domain. As in the testis form, the kinase/bisphosphatase interface of the liver enzyme includes a structure in which one beta  hairpin between beta 1-5 and beta 1-6 (residues 215-230 in the liver form and 213-228 in the testis form) in the kinase domain forms a set of hydrophobic interactions with the following helix alpha 7 in the kinase domain, which in turn makes tight interactions with the bisphosphatase helix alpha 13 (7). A major portion of this junction also serves as the dimeric interface through intermonomeric kinase domain interactions (Fig. 3a). Sequence heterogeneity introduced at the three residues, T224Q, R225S, and Q232A, of this hairpin may have caused the bisphosphatase domain twist and, also, affected the dimeric interactions. As shown in Fig. 3a, the hairpin loop in the liver type forms a new intermolecular salt bridge with Glu-208 of the dimeric partner molecule, because of a newly introduced positive charge in Arg-225, and this tends to pull the loop upward. This upward movement is strengthened by T224Q, which forms a new hydrogen bond with the carbonyl O of Asp-221 in the same subunit, losing the intersubunit hydrogen bonding with Arg-230. Q232A also helps this structural change by introducing a new hydrogen bond to Ser-215. As a result, the hairpin structure is rotated by ~3° and shifted by 0.4-0.6 Å with respect to that of the testis form. To adjust for this structural change and to maintain tight hydrophobic interactions with the hairpin, the helix alpha 7 has made a translational shift (Fig. 3b), which is helped by a new hydrogen bond between Q232A and Ser-215 (Fig. 3a). The shift of alpha 7 induces a similar type of movement in alpha 13. In this way, a small shift of the beta -hairpin is propagated to the rest of the structural components of the junction between the kinase and bisphosphatase domains, eventually causing a 5° swing of the bisphosphatase domain (Fig. 1b).


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Fig. 3.   Stereo views of junction of the kinase and bisphosphatase domains. a, differences in the beta 1-5 and -6 hairpin viewed from the dimeric interface. The three residues, T224Q, R225S, and Q232A, which cause the bisphosphatase twist are shown. The two liver kinase domains are represented by different colors (magenta and blue), and those of rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase are shown in light gray. The dimeric interactions in the liver form are shown with the dark dotted lines, and the related interactions in the testis form are colored light gray. The 2-fold rotation axis is represented by a black-diamond . Note that the dimeric interaction pattern in the liver form causes an upward and rotational shift of the beta 1-5/beta 1-6 hairpin loop. b, the side chains of the residues involved in the kinase/bisphosphatase domain interface are shown demonstrating the positional differences between the liver and testis isozymes. The liver kinase and bisphosphatase domains are colored red and blue, respectively, while light gray is used for the testis form. Glu-208 from the magenta loop is from the dimeric partner kinase subunit. Note that the positional differences of the residues and the secondary structure motifs involved in this interdomain junction gradually increase from the top to the bottom.

Intramolecular rotation of the bisphosphatase domain appears to enhance the overall stability of the bisphosphatase domains in the liver form by introducing new dimeric interactions between the Fru-2,6-P2ase domains. As summarized in Table IV, the conformation distinguishes the liver isozyme from the testis form, causes a 4-fold increase in the buried surface area between the two bisphosphatase domains, and decreases the distances in the dimeric kinase/bisphosphatase interaction as well. Interestingly, neither the increased contact areas nor the decreased distances between the two bisphosphatase domains are caused by amino acid sequence differences within the bisphosphatase domain. The polymorphic sequences in the kinase domain that induce the twist between the kinase and bisphosphatase domains are the sole cause of increases in the dimeric interactions between the bisphosphatase domains.

                              
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Table IV
Comparison of the dimeric parameters
The buried surface areas were calculated using "areaimol" (30).

When deviations from mean B-factors of the testis ATPgamma S complex and the liver ATPgamma S complex are plotted, significant differences in the stability of the 6-Fru(P)-2-kinase (residues 39-249) and Fru-2,6-P2ase (residues 250-470) domains of the two isozymes are revealed (Fig. 4). A very similar pattern was also shown in other structures (data not shown for clear comparisons). This comparison dramatically shows that the Fru-2,6-P2ase domain of the liver isozyme is the more stable half of the monomer liver isozyme. Conversely, the 6-Fru(P)-2-kinase domain of the testis isozyme is the more stable domain. This distinct domain stability is not an artifact of different crystal contacts between the two isoforms. As shown on the bottom of the plot, most of the crystal contacts are shared, and the several unique contacts do not explain the unique stability of the tissue-specific isoforms. This difference was apparent throughout the structure refinement process, where the liver bisphosphatase domain always had an overall more interpretable density map than the kinase domain, opposite the situation seen in the testis structures. The liver bisphosphatase domain is more stable than its own kinase domain and the testis bisphosphatase domain. On the contrary, the testis kinase domain is more stable than its bisphosphatase domain and the liver kinase domain. However, it is unclear whether the increase in the buried surface area by the twisted bisphosphatase causes the liver bisphosphatase domain to be more stable.


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Fig. 4.   Distribution of the temperature (B-) factors in the two isozymes. The Calpha atom B-factors of the structures of the two isoforms in the same liganding state are compared with each other. The B-factors are expressed as standard deviations from the average values of each structure: dark line, the liver form; gray, the testis form. The functional roles of the protein motifs are labeled above the plots. The motifs used as crystal contacts are underlined as follows: solid line, the liver form; dotted line, the testis form.

Differentiated Substrate-binding Loops in the Kinase Domain-- Outside of the N-terminal regulatory motif (residues 1-38), polymorphic amino acid sequences between the liver and testis isozymes are most frequent in the substrate binding loops of the kinase domain: the Fru-6-P loop (residues 80-120) and the ATP loop (residues 160-200) (Fig. 2). In the testis isozyme, it was shown that the last turn of helix alpha 5 (residues 162-175) undergoes unwinding upon binding of ATP to recruit Lys-172 to the bridge oxygen between the beta - and gamma -phosphates of the bound ATP (9, 29). This ATP-driven conformational change has been suggested to be transmitted to the Fru-6-P loop via the loop coupling composed of the salt bridges between Asp-177, Arg-78, and Arg-193 and the hydrogen bonds between Arg-181 and the carbonyl oxygens of Tyr-85 and Lys-86 (9, 29). Thus, the ATP binding was suggested to be a prerequisite for the subsequent binding of Fru-6-P to the kinase domain in the testis kinase reaction, following the ordered binding of substrates.

Like the testis form and as shown in Fig. 5a, the structure of the liver form also has a "switch-on" conformation induced by the bound ATP. Neither of the two additional structures of the liver enzyme complexed with ADP or Pi shows the "switch-off" conformation, which is shown by the testis enzyme upon bindings of ADP or Pi (Fig. 5b). Furthermore, substitution of cysteine for Arg-183 in the liver isozyme (C183R) breaks the outer lobe coupling of the ATP and Fru-6-P loops (Fig. 5c). It is believed that the sequence differences in this region stabilize the "switch-on" conformation of the liver enzyme. Mutation in G164E appears to greatly enhance the hydrophobic interactions between I165V and Val-222 by introducing more flexibility into the first turn of the switch helix (alpha 5), and as a result, the distance between Ile-165 and Val-222 is shortened to 3.8 Å from 4.9 Å. In addition, a different sequence at E168A introduces a new hydrogen bond to the main chain N of Gln-172. Furthermore, the residue differences of V188A, L189T, and L193M add new sources of interactions to a local hydrophobic pocket made of Tyr-180, Leu-175, Phe-192, and Ile-170 (Fig. 5a), which has been shown to be important in the binding of ATP and Fru-6-P (9).


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Fig. 5.   Sequence-related differences in the substrate loops of the kinase domain. a, ATP-induced switch motion in the liver isozyme. The alpha 5 helix of the liver form (blue) is compared with that of the testis form (gray). The dotted lines demonstrate the additional sequence-related interactions unique in the liver isoform stabilizing the ATP switch: dark dots, hydrogen bonds; green dots, hydrophobic interactions. b, conformation of the alpha 5 helix upon bindings of ADP or Pi. Note that Lys-174 of testis form (gray) swings out from the bound Pi, and the same result is shown in the ADP complex (data not shown). c, lost coupling between the substrate loops in the liver kinase domain. The sequence difference at C183R causes a loss of hydrogen bonds in the liver form, which serve to couple the two substrate loops in the testis form. The ATP and Fru-6-P loops of the liver enzyme are colored magenta and blue, respectively, whereas the testis loops are in gray. The hydrogen bonds in the testis isozyme are shown with dark dotted lines.

These sequence-related structural differences altogether appear to stabilize the switch-on conformation of the liver isoform by locking down the switch-on conformation and to isolate the Fru-6-P loop from the conformational changes in the ATP loop. To have the "off" (ATP-free) conformation suggested in the testis enzyme, the new hydrophobic interactions introduced by V188A, L189T, and L193M have to be broken. Thus, it appears that the helix (alpha 5) of the liver form would experience a much higher energy barrier than that of the testis isoform when it returns to the ATP-free conformation. Because of the additional hydrogen bond through the side chain of E168A and the strengthened local hydrophobic interactions, it is likely that the ATP-induced switch-on conformation of the liver form is more stable than that of the testis form. The two substrate loops are mutually independent from the conformations of the other, resulting in an enhanced affinity for Fru-6-P. This observation allows the conclusion that the substrate binding to the liver kinase follows a random fashion, as originally suggested by the previous kinetic studies of the liver form (30), and that stabilized ATP-induced switch-on conformation together with the decoupled substrate loops may contribute to a 4-fold higher affinity of the liver kinase for Fru-6-P (1, 10).

Differentiation in the Bisphosphatase Domain-- As mentioned earlier, a prominent feature of the liver isozyme is the increased stability of the bisphosphatase domain induced by the bisphosphatase twist and the concomitant increase in dimeric interactions between the bisphosphatase domains (Table IV). In addition, polymorphic amino acid residues apparently contribute to an enhanced stability of loops involved in substrate binding to the bisphosphatase domain. The sequence change at R266K introduces a new salt bridge between Arg-266 and Glu-262. In addition, a change at S273P stabilizes the loop (residues 260-280), which surrounds the 2-P position of the bisphosphatase-active site, by forming a new hydrogen bond with the main chain N of Leu-263 (Fig. 6).


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Fig. 6.   The substrate loops in the liver bisphosphatase domain. The sequence difference-related new interactions that stabilize the loops surrounding the 2-P motif and the 6-P motif of Fru-2,6-P2 are shown using dark dotted lines. Different colors of the ribbon diagram represent different subunits: magenta, the first subunit of the liver form; green, the other subunit in dimeric contact; and gray, rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Note that the sequence difference at E347L causes a change in the arrangement of a local hydrophobic patch and that the interaction between Glu-335 and Lys-363 is introduced by the bisphosphatase twist in the liver isoform.

The amino acid difference at residue E347L pushes Glu-347 away from the hydrophobic pocket consisting of E347L, Leu-351, and Ile-455 and includes Tyr-359 instead (Fig. 6). The rearranged hydrophobic pocket shifts the loop 358-361 such that a salt bridge between Arg-360 and Asp-369 becomes more stable. The distance of this salt bridge is shortened from 3.5 to 2.7 Å, and as a result, the B-factors of Arg-360 and Asp-369 decrease by ~27 and 7 Å2, respectively, from those of the testis form. In addition, a new salt bridge between Glu-335 and Lys-363 is formed by the enhanced dimeric bisphosphatase interactions, and as a result, the B-factors are decreased by 15 and 19 Å2 from those of the testis form. This conformational change makes the loop (residues 335-363) more stable, which serves as a lid of the active site of the bisphosphatase domain (6, 7) and harbors a number of residues interacting with the 6-phosphate moiety of substrate and product (31, 32). This loop has served as a popular target of the earlier structure/function studies and has been shown to be critical in the proper binding of both Fru-6-P and Fru-2,6-P2. The site-directed mutants, Arg-352 right-arrow Ala, Lys-356 right-arrow Ala, and Arg-360 right-arrow Ala, showed more than 100-fold decreases in binding of both Fru-6-P and Fru-2,6-P2. Thus, in addition to the domain stabilization by the twist, it is likely that stabilization of both the 2-P and the 6-P loop structures by the amino acid sequence of the liver isozyme plays an important role in the 20-fold increase in its affinity for Fru-2,6-P2 compared with that of the testis form, reflecting the differences in their kinetic and allosteric properties, consistent with their different physiological roles (1-3, 11-13).

Physiological Significance-- Sequence differences between the human liver and rat testis isozymes result in both local and global differences in the structures of these isozymes. The local conformational differences in the liver form kinase domain result in a loose coupling (or decoupling) of the Fru-6-P- and ATP-binding loops and a stabilized switch-on conformation even without ATP. These structural differences may allow the Fru-6-P binding to be independent of ATP binding unlike the testis form, in which ATP-binding is prerequisite for Fru-6-P binding. This structure-related functional difference may be related to the physiological roles of the tissues that harbor these isozymes. To regulate the blood glucose levels, the liver appears to harbor an isozyme that is more sensitive to the changes in Fru-6-P concentration, which is a more accurate indicator of glucose concentration than that of ATP. Meanwhile, the testis isozyme, which functions to maintain a steady level of energy production, appears to adopt a structure more sensitive to the changes in ATP concentration, a more accurate indicator of the cellular energy level than Fru-6-P. Stabilization of the whole bisphosphatase domain by the "domain twist" and its Fru-2,6-P2-binding loops by the local sequence differences in the liver isozyme appears to enhance its affinity for Fru-2,6-P2. Considering that the Fru-2,6-P2 concentration is a critical factor in determining the flux through the glucose pathways in liver, it is reasonable that the liver bisphosphatase domain has a keen sensitivity to the concentration of Fru-2,6-P2 to actively engage in such a mission. However, by having lower affinity for Fru-2,6-P2, the testis isozyme is likely to have the bisphosphatase domain functioning not to determine direction of the pathways but to regulate only the rate of glycolysis.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK52089 (to C. A. H.) and DK16194 (to K. U.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1K6M) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

§ To whom correspondence should be addressed: Structural Biology Core, Molecular Biology, 125 Chemistry Bldg., University of Missouri, Columbia, MO 65211. Tel.: 573-884-5767; Fax: 573-882-2754; E-mail: leeyon@missouri.edu.

Published, JBC Papers in Press, October 11, 2002, DOI 10.1074/jbc.M209105200

2 K. Uyeda, unpublished data.

    ABBREVIATIONS

The abbreviations used are: 6-Fru(P)-2-kinase/Fru-2, 6-P2ase, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Fru-2, 6-P2, fructose 2,6-bisphosphate; Fru-2, 6-P2ase, fructose-2,6-bisphosphatase; Fru-6-P, fructose 6-phosphate; PFK, phosphofructokinase; FBPase, fructose-1,6-bisphosphatase; PP2A, protein phosphatase 2A; Xyl-5-P, xylulose 5-phosphate; ATPgamma S, adenosine 5'-O-(thiotriphosphate); AMP-PNP, adenosine 5'-(beta ,gamma -imino)triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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