R-State AMP Complex Reveals Initial Steps of the Quaternary Transition of Fructose-1,6-bisphosphatase*

AMP transforms fructose-1,6-bisphosphatase from its active R-state to its inactive T-state; however, the mechanism of that transformation is poorly understood. The mutation of Ala to leucine destabilizes the T-state of fructose-1,6-bisphosphatase. The mutant enzyme retains wild-type levels of activity, but the concentration of AMP that causes 50% inhibition increases 50-fold. In the absence of AMP, the Leu enzyme adopts an R-state conformation nearly identical to that of the wild-type enzyme. The mutant enzyme, however, grows in two crystal forms in the presence of saturating AMP. In one form, the AMP-bound tetramer is in a T-like conformation, whereas in the other form, the AMP-bound tetramer is in a R-like conformation. The latter reveals conformational changes in two helices due to the binding of AMP. Helix H1 moves toward the center of the tetramer and displaces Ile from a hydrophobic pocket. The displacement of Ile exposes a hydrophobic surface critical to interactions that stabilize the T-state. Helix H2 moves away from the center of the tetramer, breaking hydrogen bonds with a buried loop (residues 187– 195) in an adjacent subunit. The same hydrogen bonds reform but only after the quaternary transition to the T-state. Proposed here is a model that accounts for the quaternary transition and cooperativity in the inhibition of catalysis by AMP.

FBPase is a homotetramer (subunit M r of 37,000 (11)) and exists in at least two distinct quaternary conformations called R and T (12)(13). AMP induces the transition from the active R-state to the inactive (or less active) T-state. Substrates or products in combination with metal cations stabilize the Rstate conformation. A proposed mechanism for allosteric regulation of catalysis involves three conformational states of loop 52-72 called engaged, disengaged, and disordered (14). AMP alone or with F26P 2 stabilizes a disengaged loop (15,16), whereas metals with products stabilize an engaged loop (10, 16 -18). In active forms of the enzyme, loop 52-72 probably cycles between its engaged and disordered conformations (14,17). Fluorescence from a tryptophan reporter group at position 57 is consistent with the conformational states for loop 52-72 observed in crystal structures (19,20). Thus far, the engaged conformation of loop 52-72 has appeared only in R-state crystal structures and the disengaged conformer has appeared only in T-state structures; however, disordered conformations of the dynamic loop have appeared in both the R-and T-states (16,17,21,22).
The precise sequence of events that attend the R-to T-state transition in FBPase has been elusive. Crystal structures of the R-and T-states are the endpoints of the allosteric transition and leave much to speculation regarding intermediate conformational states of FBPase. The immediate consequences of AMP binding to the R-state are unknown. Does the dynamic loop become disengaged in response to the binding of AMP or in response to the allosteric transition to the T-state? How does the binding of AMP destabilize the R-state and stabilize the T-state? A recent study (23) revealed the first intermediate state of porcine FBPase, a T-like conformation due to the binding of an allosteric effector to the center of the tetramer. The results of that study indicated the potential for trapping intermediate conformational states of FBPase by crystallization.
The mutation of Ala 54 to leucine disrupts key packing interactions of the disengaged loop conformation. The resulting Leu 54 enzyme has wild-type catalytic properties and retains cooperativity in AMP inhibition but exhibits a 50-fold increase in the IC 50 for AMP. In the absence of AMP, Leu 54 FBPase is in its canonical R-state. However, two crystal forms grow in the presence of saturating AMP. In one crystal form, the enzyme is in its T-state but with a disordered dynamic loop. In the other crystal form, the tetramer is in an R-like quaternary state with an engaged dynamic loop. The latter crystal form reveals the immediate consequences of AMP association in the absence of an allosteric transition. The observed conformational changes suggest the mechanism by which AMP leverages the allosteric transition in FBPase.

EXPERIMENTAL PROCEDURES
Materials-F16P 2 , F26P 2 , NADP ϩ , and AMP were purchased from Sigma. DNA-modifying and restriction enzymes and T4 polynucleotide kinase and ligase were from Promega. Glucose-6-phophate dehydrogenase and phosphoglucose isomerase came from Roche Applied Science. Other chemicals were of reagent grade or equivalent. Escherichia coli strains BMH 71-18 mutS and XL1-Blue came from Clontech and Stratagene, respectively. The FBPase-deficient E. coli strain DF 657 came from the Genetic Stock Center at Yale University.
Mutagenesis of Wild-type FBPase-The mutation of Ala 54 to leucine was accomplished by specific base changes in a double-stranded plasmid containing the gene coding for FBPase using the Transformer TM site-directed mutagenesis kit (Clontech). The mutagenic primer for Ala 54 3 Leu was 5Ј-GGCGGGCATCCTGCACCTC-3Ј (the codon with the point mutation is underlined in boldface). The selection primer for mutagenesis, 5Ј-CAGCCTCGCCTCGAGAACGCCA-3Ј (digestion site underlined in boldface), changed an original NruI site on the plasmid into a XhoI site. The mutation and the integrity of the construct were confirmed by sequencing the promoter region and the entire open reading frame. The Iowa State University sequencing facility provided DNA sequences using the fluorescent dye-dideoxy terminator method.
Expression and Purification of Wild-type and Leu 54 FBPases-Cellfree extracts of wild-type and Leu 54 FBPases were subjected to heat treatment (63°C for 7 min) followed by centrifugation. The supernatant solution was loaded onto a Cibracon Blue-Sepharose column previously equilibrated with 20 mM Tris-HCl, pH 7.5. The column was washed first with 20 mM Tris-HCl, pH 7.5. Enzyme was eluted with a solution of 500 mM NaCl and 20 mM Tris-HCl of the same pH. After pressure concentration (Amicon PM-30 membrane) and dialysis against 10 mM Tris-HCl, pH 8.0, the protein sample was loaded onto a DEAE-Sepharose column equilibrated with 10 mM Tris-HCl, pH 8.0. Purified enzyme was eluted with a NaCl gradient (0 -0.5 M) in 10 mM Tris-HCl, pH 8.0, and then dialyzed extensively against 50 mM Hepes, pH 7.4, for kinetic investigations and for crystallization experiments. Purity and protein concentrations of FBPase preparations were confirmed by SDS-polyacrylamide gel electrophoresis (24) and the Bradford assay (25), respectively.
Data Collection-Data were collected at Iowa State University from single crystals on a Rigaku R-AXIS IVϩϩ rotating anode/image plate system using CuK ␣ radiation from an Osmic confocal optics system and a temperature of 110 K. Data were reduced with the program package CrystalClear provided with the instrument.
Structure Determination, Model Building, and Refinement-Crystals of Leu 54 FBPase are isomorphous to either the AMP/Zn 2ϩ -product complex (16) or the Zn 2ϩ -product complex (10). Phase angles used in the generation of initial electron density maps were based on model 1EYJ or 1CNQ of the Protein Data Bank from which water molecules, metal cations, small molecule ligands, and residues 52-72 had been omitted. Residues 52-72 were built into the electron density of omit maps using the program XTALVIEW (28). Ligands were added to account for omit electron density at the active site and/or the AMP site. The resulting models underwent refinement using CNS (29) with force constants and parameters of stereochemistry from Engh and Huber (30). A cycle of refinement consisted of slow cooling from 1000 to 300 K in steps of 25 K followed by 120 cycles of conjugate gradient minimization and concluded by the refinement of individual thermal parameters. Thermal parameter refinement employed restraints of 1.5 Å 2 on nearest neighbor and next-to-nearest neighbor main chain atoms, 2.0 Å 2 on nearest neighbor side chain atoms, and 2.5 Å 2 on next-to-nearest neighbor side chain atoms.
In subsequent cycles of refinement, water molecules were fit to difference electron density of 2.5 or better and were added until no significant decrease was evident in the R free value. Water molecules in the final models make suitable donor-acceptor distances to each other and the protein and have thermal parameters under 60 Å 2 . Stereochemistry of the models was examined by the use of PROCHECK (31). 54 3 Ala Mutation-The C␤ atom of Ala 54 is at the center of a cluster of hydrophobic side chains, which forms only when the dynamic loop is in its T-statedisengaged conformation. A mutation at position 54 to a large side chain should disrupt packing interactions and thereby destabilize the disengaged conformation of the dynamic loop. In contrast, ample room is available for large side chains at position 54 in the R-state-engaged conformation of the loop. The Ala 54 3 Leu mutation then should shift the equilibrium population of AMP complexes of FBPase toward the R-state.

Rationale for the Leu
Expression and Purification of Wild-type and Leu 54 FB-Pases-Expression and isolation procedures described above provide wild-type and Leu 54 FBPases in at least 95% purity, as judged by SDS-polyacrylamide gel electrophoresis (data not shown). Gels indicated no proteolysis of the purified enzymes.
Kinetics Experiments-Kinetics parameters for Leu 54 and wild-type FBPases are in Table I. The determination of k cat and K m for F16P 2 (listed as K m F16P2 in Table I)   Activity ratio, pH 7.5:9.5 3.5 Ϯ 0.5 pH 7.5-9.5 for wild-type and Leu 54 FBPases (each above 3) are indicative of tetrameric enzymes with intact (nonproteolyzed) polypeptide chains. The Hill coefficient for Mg 2ϩ was determined at a saturating concentration of F16P 2 (20 M) and concentrations of free Mg 2ϩ ranging from 0.1 to 5.0 mM. Data were fit to Equation 1, where v is the velocity, V m is the maximum velocity at saturating concentrations of F16P 2 and Mg 2ϩ , A is the concentration of Mg 2ϩ , n is the Hill coefficient for Mg 2ϩ , and A 0.5 is the concentration of Mg 2ϩ that gives v/V m of 50%. The Hill coefficient for AMP was determined at saturating F16P 2 (20 M), Mg 2ϩ concentrations of 0.8 and 0.15 mM for wild-type and Leu 54 FBPases, and AMP concentrations ranging from 0 to 500 M. Data were fit to Equation 2, where v is the velocity, V 0 is the velocity at an AMP concentration of zero, I is the concentration of AMP, n is the Hill coefficient for AMP, and I 0.5 is the concentration of AMP that gives v/V 0 of 50%.
where i runs over multiple observations of the same intensity and j runs over all of the crystallographically unique intensities. The kinetic mechanism of AMP inhibition with respect to Mg 2ϩ was determined from assays that employed saturating (20 M) F16P 2 , five different Mg 2ϩ concentrations ranging from 0.8 to 3.0 mM for wild-type enzyme or 0.2 to 0.6 mM for Leu 54 FBPase, and five different AMP concentrations ranging from 0 to 150 M. A model for linear competitive inhibition (Equation 3) provided the best result (goodness-of-fit of Ͻ4%), where v is the velocity, V m is the velocity at an inhibitor concentration of zero saturating concentrations of F16P 2 and Mg 2ϩ , A is the concentration of Mg 2ϩ , I is the concentration of AMP, K a is the Michaelis constant for Mg 2ϩ , and K i AMP is the dissociation constant for AMP from the enzyme-inhibitor complex. Equation 3 constrains the Hill coefficients for Mg 2ϩ and AMP to 2, consistent with independent determinations of these quantities.
The kinetic mechanism of F26P 2 inhibition with respect to F16P 2 was determined from assays that employed saturating Mg 2ϩ (5 mM for wild-type enzyme and 2 mM for Leu 54 FBPase), five different concentrations (1-6 M) of F16P 2 , and five different concentrations (0 -1.0 M) of F26P 2 . A model for linear competitive inhibition provided the best fit to the data (goodness-of-fit of Ͻ3%), where V m is the velocity at an inhibitor concentration of zero and saturating concentrations of F16P 2 and Mg 2ϩ , B is the concentration of F16P 2 , I is the concentration of F26P 2 , K b is the Michaelis constant for F16P 2 , and K i F26P2 is the dissociation constant for F26P 2 from the inhibitor-enzyme complex.
Product Complex of Leu 54 FBPase (Protein Data Bank code 1YXI)-Crystals belong to the space group I222 (a ϭ 52.8, b ϭ 82.8, and c ϭ 165.5 Å) and are isomorphous to those of wildtype FBPase in its R-state, containing one subunit of the tetramer in the asymmetric unit of the crystal (10, 16 -18). Electron density for residues 1-6 is weak or absent. The model begins at residue 7 and continues to the last residue of the sequence. Thermal parameters vary from 10 to 70 Å 2 . The model has stereochemistry (as determined by PROCHECK (31)) comparable to that of structures of equivalent resolution. Statistics for data collection and refinement are in Table II.
The product complex of Leu 54 FBPase is identical to that of the wild-type enzyme but for clear electron density showing the leucyl side chain at position 54. One molecule each of Fru-6-P and P i bind to the active site with three atoms of Mg 2ϩ . The dynamic loop (residues 52-72) is in its engaged conformation. Superposition of the Leu 54 subunit onto the wild-type subunit reveals no deviation in the relative positions of C␣ atoms in excess of 0.43 Å. Superposition of the Leu 54 tetramer onto canonical wild-type R-and T-states clearly indicates an R-state complex. We refer the reader to other descriptions of R-state product complexes (10, 16 -18) for more detailed descriptions of comparable structures.
AMP-Product R-like Complex of Leu 54 FBPase (Protein Data Bank Code 1YYZ)-Crystals belong to the space group I222 (a ϭ 53.8, b ϭ 82.6, and c ϭ 166.6 Å). They contain one subunit in the asymmetric unit and are isomorphous to those of wildtype FBPase in its R-state (10, 16 -18). Electron density for residues 1-9 is weak or absent. The model begins at residue 10 and continues to the last residue of the sequence. Thermal parameters vary from 10 to 75 Å 2 . The model has stereochemistry comparable to that of structures of equivalent resolution (31).
The subunit of the AMP-product complex of Leu 54 FBPase has one molecule each of Fru-6-P and P i with three atoms of Mg 2ϩ at the active site. In addition, strong electron density is present in the allosteric inhibitor pocket, which represents a bound molecule of AMP (Fig. 1). The dynamic loop (residues 52-72) adopts the engaged conformation. Superposition of the Leu 54 tetramer onto canonical wild-type R-and T-states reveals a change in quaternary state (Table III). Subunit pair C1-C2 has rotated 3°relative to C3-C4 (Fig. 2). The subunit rotation lies between that of the canonical R-state (0°rotation) and T-state (15°rotation) and differs from the intermediate quaternary state (9°rotation) stabilized by the allosteric inhibitor OC252 (Fig. 2). Hereafter, we will use the label I R to represent the R-like state of the AMP-product complex of Leu 54 FBPase and I T to represent the T-like state of the OC252 complex.
The I R structure reveals the effect of AMP binding in the absence of a complete quaternary transition. The superposition of the I R subunit onto the subunits of the R-state tetramer removes coordinate displacements due to the partial (3°) rotation of the subunit pairs in the I R -state, revealing conformational changes at the tertiary level. In such a comparison, conformational changes are evident only in the N-terminal element and helices H1 and H2. The 6-amino group of AMP draws backbone carbonyl 17 (helix H1) toward itself while pushing away the side chain of Val 17 in avoiding unacceptable Protein Data Bank identifiers 1EYK and 1CNQ represent the canonical T-and R-states, respectively. I T is the T-like state of the OC252 complex of FBPase (Protein Data Bank code 1Q9D). I R is the R-like state of the AMP/product complex of Leu 54 FBPase reported here. The determined angle of rotation is sensitive to the subset of C␣ atoms used in the calculation of the rotation matrix. The use of all of the C␣ atoms, including those of the dynamic loop, results in an angle of 18°for the Rto T-state transition. In contrast, the use of C␣ atoms that deviate by less than 1 Å in the initial superposition gives an angle of 13°. Superpositions and root mean square deviations here are based on C␣ atoms from the following residues: 33-49, 75-265, and 272-330. Values in boldface come from superpositions of tetramers. Other values come from superpositions of C1-C2 dimers. The values from C1-C2 dimers provide an estimate of coordinate uncertainty due to random and systematic errors.  Table III are the basis for superposition of subunit pair C1-C2. This drawing was prepared with MOLSCIRPT (38). nonbonded contacts (Fig. 3A). The interactions between AMP and Val 17 translate helix H1 by 0.5 Å toward the center of the tetramer and move the C-terminal end of helix H1 ϳ1.0 Å toward the bound AMP molecule. The movement compresses the N-terminal end of helix H1 into residues 193-195 of subunit C1 and displaces the side chain of Ile 10 from a hydrophobic cluster of residues (Fig. 3, B and C). Helix H2 moves along its axis 0.5 Å away from the center of the tetramer. Helix movements sever hydrogen bonds between Thr 14 and Asn 35 and between Thr 39 and Glu 192 , the latter being a linkage between subunits C1 and C4 (Table IV). Moreover, the hydrogen bond between Thr 46 and backbone carbonyl 189 (another C1-C4 contact) may be weakened. Lys 42 remains in its intersubunit salt link with Glu 192 with little change to its other hydrogen-bonding interactions. The loss or weakening of hydrogen bonds involving Thr 39 , Thr 46 , and Glu 192 observed in the I R structure is not evident in a direct comparison of the canonical R-and T-states (Table IV).
The superposition of the I R -state subunit onto the subunits of the T-state tetramer of wild-type FBPase reveals tertiary conformational change due to the 12°subunit-pair rotation. This includes the movement of the dynamic loop from its engaged to  3. Tertiary conformational changes between R-and I R -states. Dotted lines represent selected donor-acceptor interactions of 3.2 Å or less. Solid green lines represent potential nonbonded contacts of 2.5 Å or less. Panels A-C, superposition of the I R -state subunit (red) onto each subunit of the R-state tetramer (black) reveals tertiary conformational changes induced by the binding of AMP and the resulting 3°subunit-pair rotation. Hydrogen bonds (dotted red lines) involving the 6-amino group of AMP and a nonbonded contact (green line) with the side chain of Val 17 induce helix movement and shears hydrogen bonds between Thr 14 and Asn 35 (panel A) as well as Glu 192 and Thr 39 (panels A-C). In addition, the movement of helix H1 displaces Ile 10 from its R-state hydrophobic contacts (panels B and C). This drawing was prepared with MOLSCIRPT (38). Inset, shown are regions of the tetramer (purple) and viewing directions (boldface arrows) corresponding to panels A-C. The viewing direction for panel C is 45°inclined to the plane of the tetramer. disengaged conformer, a displacement of Ͼ30 Å for some C␣ carbons of that loop. For the most part, other conformational changes involve modest displacements in atoms not exceeding 0.5 Å. These changes involve almost every atom in FBPase in a correlated set of collective movements. Immediately evident is the additional translation of helix H2, outward from the center of the tetramer and along its axis, and the occurrence of unacceptable contacts between loops 190 (Fig. 4). The unacceptable contacts are relaxed in the T-state subunit by movements in loops 190 in subunits C1 and C4 away from a molecular axis of 2-fold symmetry.
AMP-Product T-state Complex of Leu 54 FBPase (Protein Data Bank Code 1YZ0)-Crystals belong to the space group P2 1 2 1 2  (a ϭ 59.7, b ϭ 166.1, and c ϭ 78.9) and are isomorphous to those of AMP complexes of FBPase (16,17). The subunit pair C1-C2 is in the asymmetric unit of this crystal form. Regions of weak or absent electron density include residues 1-8 and 55-72. The model begins at residue 9 and continues to the last residue of the sequence, but segment 55-72 is unreliable as evidenced by thermal parameters as high as 113 Å 2 . The model has stereochemistry generally comparable to that of structures derived from data of comparable resolution (31). Statistics for data collection and refinement are in Table II. The enzyme in this crystal form is in the T-state (quaternary transition angle of 15°); however, unlike loop-disengaged AMP complexes of the wild-type enzyme, the dynamic loop in T-state Leu 54 FBPase is disordered. Moreover, hydrogen bonds normally well established in the T-state of the wild-type enzyme seem to be marginally weaker in T-state Leu 54 FBPase. The active site retains Fru-6-P and Mg 2ϩ bound with low occupancy to site 1.

DISCUSSION
Conformational changes between the R-and I R -states of Leu 54 FBPase are consistent with two models for quaternary change: 1) a concerted model in which AMP drives the quaternary transition by acting on a set of interconnected levers and 2) a sequential model in which AMP raises the energy level of the R-state while simultaneously lowering that of the nascent T-state (Fig. 5). The point of departure for each model is an AMP-induced translation of helices H1 and H2 in opposite directions, helix H1 toward and helix H2 away from the center of the tetramer. The two models differ in regard to the consequences of helix H2 movement. For the concerted model, helix H2 retains its interactions with loops 190, whereas in the sequential model, interactions between helix H2 and loops 190 are broken.
In the concerted model, two sets of coupling interactions distribute forces throughout the entire tetramer due to the binding of one AMP molecule. One set of interactions involves Thr 39 , Lys 42 , and Thr 46 of helices H2 with Glu 192 and backbone carbonyls 189 and 190 of loops 190 (Table IV). These interactions link subunits C1-C4 and C2-C3. A second set of interactions defines the C1-C2 and symmetry-related C3-C4 subunit interfaces (13). The binding of one molecule of AMP, for instance subunit C1, results in the aforementioned movements of helices H1 and H2. The movement in helix H2 of subunit C1 exerts an outward force on loops 190 of subunits C1 and C4. The C1-C2 and C3-C4 subunit interactions, however, constrain loops 190 to a fixed distance from the center of the tetramer. Loops 190 can only follow the outward movement of helix H2 by the rigid body rotations of subunit pairs C1-C2 and C3-C4 (Fig.  5). The two sets of coupling interactions ensure that all loops 190 and their associated subunits undergo rigid-body motions and that all helices H2 undergo an outward movement in response to the binding of one or more molecules of AMP.
The concerted model suffers from two significant shortcomings. First, the coupling interactions between helix H2 and loops 190 have weakened in the I R state. Only the interactions involving Lys 42 appear unaffected by movements in helices H2, and we suggest below that even this critical salt link may rupture during the quaternary transition. The weakened linkages between helices H2 and loops 190, however, may be the consequence of having four AMP molecules bound to an R-state tetramer. Two bound molecules of AMP convert R-state hybrid tetramers of FBPase into their T-states (32). Hence, the I Rstate of the Leu 54 FBPase may represent an "over-torqued" tetramer, the existence of which is possible only because the mutation at position 54 eliminates the T-state as a lowenergy alternative. The second shortcoming of the concerted model is not so easy to dismiss. A concerted mechanism for FBPase requires cooperativity in the binding of AMP molecules to any pair of sites. A hybrid tetramer of FBPase that constrains AMP binding to subunits C1 and C2, however, exhibits noncooperative inhibition, even though it undergoes a quaternary transition (32).
In considering the sequential model for the quaternary transition, we note first that the subunit rotation observed in the R- FIG. 4. Tertiary conformational changes between T-and I Rstates. Superposition of the I R -state subunit (red) onto each subunit of the T-state tetramer (black) reveals tertiary conformational changes induced by the 12°subunit-pair rotation. An additional translation of helix H2 along its axis is evident (panels A-C). The disengaged loop (residues 52-72) covers the hydrophobic surface exposed by the displacement of Ile 10 (panel C). Close contacts between loops 190 of subunits C1 and C4 and Glu 192 and Lys 42 (solid green lines) are relaxed by conformational changes. The conformational change in Glu 192 re-establishes its hydrogen bond with Thr 39 (panels A-C). Regions of FBPase depicted and viewing directions are as indicated in Fig. 3. This drawing was prepared with MOLSCIRPT (38).
to T-state transition cannot happen as a rigid-body motion. In the R-state, loop 190 from subunit C1 is in contact with the symmetry-related loop from subunit C4. Progress toward the T-state results in unacceptable contacts between loops 190 from subunits C1 and C4. Loop 190 must undergo conformational change, but multiple hydrogen bonds fix its conformation in both the R-and T-states (Table IV). The movement of helix H2 releases a conformational restraint on loop 190 in a neighboring subunit by the disruption or weakening of hydrogen bonds involving Thr 39 and Thr 46 . In this environment of fewer restraints, loop 190 is more likely to relax unfavorable contacts that occur during the quaternary transition. Moreover, the movement in helix H2 favorably positions Thr 39 and Thr 46 for the formation of strong hydrogen bonds in the T-state. In fact, the hydrogen bond involving Glu 192 and Thr 39 ruptured in the R-to I R -state transition reforms in the T-state. The mechanism is fully reversible. The loss of AMP from the T-state presumably causes helix H2 to move back toward the center of the enzyme, breaking or weakening hydrogen bonds between subunits C1 and C4 and repositioning Thr 39 and Thr 46 in favor of R-state interactions.
The sequential model can accommodate both cooperative and noncooperative mechanisms of AMP inhibition. A mixture of hybrid mutants of FBPase that force AMP binding to opposite halves of the tetramer exhibits cooperative inhibition (32). Hence, subunit coupling must exist between top and bottom halves of the tetramer. Coupling interactions necessarily involve subunits C1 and C4, because subunits C1 and C3 have no direct interactions in the R-state. The binding of AMP to subunit C1 may not only disrupt hydrogen bonds between helix H2 of subunit C1 and loop 190 of subunit C4, but it may also weaken symmetry-related hydrogen bonds between helix H2 of subunit C4 and loop 190 of subunit C1. Hence, a second molecule of AMP would divert less of its binding energy to the movement of helix H2 in subunit C4 and, as a consequence, bind with higher affinity. All of the reported mutations of Lys 42 , Arg 49 , Glu 192 , Ile 190 , and Gly 191 and some of the mutations of Lys 50 abolish AMP cooperativity (22,(33)(34)(35). These residues are part of helix H2 or loop 190 and are near to or part of the coupling interactions between subunits C1 and C4.
A second coupling pathway between AMP binding sites may involve Arg 22 . The mutation of Arg 22 to methionine eliminates cooperativity in AMP inhibition (36). Arg 22 is near the AMP pocket, has high thermal parameters in the R-state, and does not participate in intersubunit hydrogen bonds in either the Ror T-state structures. Replacing subunit C1 of R-state Leu 54 FBPase with an I R subunit generates a model that approximates FBPase with one bound molecule of AMP (Fig. 6). In that model, Arg 22 of subunit C1, due to conformational changes induced by AMP, makes a strong hydrogen bond with backbone carbonyl 108 of subunit C4. The formation of the symmetryrelated hydrogen bond involving Arg 22 of subunit C4 should FIG. 5. Models of concerted and sequential conformational change. The subunits of the FBPase tetramer are simplified to helix H2 (rectangle) and loop 190 (oval). The viewing direction is down a molecular axis of 2-fold symmetry with subunit C1 and C2 above the plane (boldface lines) and subunit C3 and C4 below the plane. In the concerted model (left), AMP molecules bind successively in any order to the subunits of tetramer until the combined torque (represented by open arrows) on each subunit pair exceeds the energy barrier that separates the R-and T-states. The binding of at least two AMP molecules is necessary for the concerted conformational change. In the sequential model (right), the first molecule of AMP can bind to any subunit with equal affinity, causing the outward movement of helix H2 of only that subunit (filled arrow). If binding occurs at subunit C1, interactions between subunit C1 and C4 are weakened. The second molecule of AMP binds to subunit C4, because less binding energy is spent in the movement of helix H2 in that subunit. The R-to T-state transition can occur in response to the binding of at least two molecules of AMP. For the sequential mechanism, transition to the T-state restores hydrogen bonds lost by the AMP-induced movements of helices H2 in the R-state.
induce conformational changes in subunit C4 that favor the binding of AMP.
The concerted and sequential models both assume an energy barrier between R-and T-states. In the sequential model, hydrogen bonds involving Thr 39 and Thr 46 contribute significantly to the barrier but not so in the concerted model because these interactions are retained. At least one other interaction may contribute significantly to the energy barrier between Rand T-states. Unacceptable contacts between the side chains of Glu 192 (subunit C4) and Lys 42 (subunit C1) are likely during the quaternary transition (Fig. 4B). A conformational change in the side chain of Glu 192 eliminates bad contacts with Lys 42 and re-establishes its hydrogen bond with Thr 39 . The conformational change in Glu 192 may require a transitory loss or weakening of its salt link with Lys 42 . The presumed loss of this salt link could favor the dissociation of the tetramer into subunit pairs (C1-C2 from C3-C4) and thereby represent the first step in FBPase subunit exchange kinetics (32, 37).
The models above have yet to consider conformational change in the dynamic loop (residues 52-72). In all of the reported structures of FBPase, the dynamic loop is either engaged or disordered in R-like states and either disordered or disengaged in T-like states. The AMP-induced movement of helix H1, which probably occurs in concert with that of helix H2, displaces Ile 10 from a hydrophobic surface. That surface interacts with side chains of the dynamic loop in its disengaged T-state conformation (Fig. 4C). In the R-state, Ile 10 effectively blocks the disengaged conformer of the dynamic loop. The formation of the disengaged conformer appears as a significant thermodynamic driving force in the quaternary transition to the T-state. A modest change in the conditions of crystallization (substitution of glycerol for 2-methy-2,4-pentanediol) transforms the AMP-product complex of Leu 54 FBPase from a loop-disordered T-state to a loop-engaged R-state. Direct interactions between cryoprotectant and enzyme are unlikely, because no bound cryoprotectant molecules appear in either crystal structure. The AMP-product complex of Leu 54 FBPase probably has substantial populations of I R -and T-states in solution, allowing the growth of different crystal forms under nearly identical conditions.
The potential significance of C1-C4 interactions in FBPase has been suggested by others (13,22), but crystal structures of the R-and T-states did not reveal the transitory loss of hydrogen bonds across the C1-C4 subunit interface. As a consequence, the basis for a sequential mechanism of quaternary change remained hidden. The sequential model presented here reconciles properties of AMP inhibition in wild-type, mutant, and hybrid FBPases with known conformational changes in the tetramer. The present study also suggests that AMP-ligation of the R-state does not displace the dynamic loop from its engaged conformation. Instead, the dynamic loop leaves the engaged conformation in the T-state for reasons now poorly understood.
FIG. 6. Stereoview of the proposed role of Arg 22 in cooperativity of AMP inhibition. The view is down a molecular 2-fold axis toward the center of the tetramer. The superposition of the I R -state subunit (boldface lines) onto subunit C1 of the R-state (fine lines) represents possible relaxation events due to the binding of AMP to subunit C1 (top). AMP-induced conformational change in helix H1 would allow the formation of a hydrogen bond between Arg 22 (subunit C1) and backbone carbonyl 108 (subunit C4) and stack the side chains of Arg 22 and Phe 89 (subunit C4). A second superposition of an I R -state subunit (boldface lines) onto subunit C4 of the R-state (fine lines) represents the relaxation of subunit C4 to the altered conformation of AMP-bound subunit C1 (bottom). The proposed interaction involving Arg 22 (subunit C1) carries over to the symmetry-related Arg 22 (subunit C4). As a consequence, the AMP pocket of subunit C4 may adopt a conformation that approximates the AMP-bound conformation of subunit C1, even in the absence of AMP.