Crystallographic structure of allosterically inhibited phosphofructokinase at 7 Å resolution

.J. Mol. Hiol. (1986) 191. 713TN

Crystallographic Structure of Allosterically Inhibited Phosphofructokinase at 7 A Resolution P. R. Evans, G. W. Farrants? and M. C. Lawrence1 M RC Laboratory for Molecular Biology University Postgraduate Medical School. Hills Road, Cambridge CB2 2&H, England (Received 24 February

1986, and in revised form

11 June 1986)

of the allosterically inhibited form of phosphofructokinase from Bacil1u.s has been determined by X-ray crystallography to 7 A resolution by molecular replacement using the known structure of the active state as a starting model. Comparing the inhibited state with the active state, the tetramer is twisted about its long axis such that one pair of subunits in the tetramer rotates relative to the other pair by about 8” around one of the molecular dyad axes. This rotation partly closes the binding site for the cooperative substrate fructose-g-phosphate, explaining its weaker binding to this conformational state. Within the subunit, one domain rotates relative to the other by 4.5”. which further closes the fructose-g-phosphate site, without closing the cleft between the domains of the same subunit: this motion causes little change to the catalytic site. This T-state model is consistent with the simple allosteric kinetic scheme in which the active and the inhibited conformations differ in t,heir affinities for fructose-g-phosphate, but not in t’heir catalytic rates. It does not explain the heterotropic allosteric effects.

The structure

stearothermophilus

1. Introduction

from Bacillus stearothermoto 2.4 A resolution from crystals grown in the presence of the co-operative substrate fructose-6-phosphate, and some details are known about the ligand binding sites (Evans & Hudson, 1979: Evans et al., 1981). The enzyme is a tet,ramer with identical subunits arranged about a set of three mutualby perpendicular &fold axes (called p, q and r in this paper). Each subunit has a two-domain structure. Three binding sites have been identified on each subunit. The active site, in which fructose-6-phosphate and ATT’ bind, lies in a cleft between the two domains of the subunit. The fructose-6-phosphate binding site also involves residues from a neighbouring subunit related by the molecular r axis. The allosteric activator ADP and the inhibitor phosphoenolpyruvate bind in a third site between another pair of subunits related by the molecular p axis. The kinetics of the B. stearothermophilus enzyme reaction (H. Hengartner & R. Mulvey, unpublished results) are similar to those of the highly homologous (Hellinga & Evans, 1985) I?. coli enzyme (Blangy et al., 1968) and may be described in terms of the two-state model of Monod et ~2. (1965). This paper describes the observed structure of the inactive T-state of the enzyme, crystallized in the presence of the non-physiological allosteric inhibitor 2-phosphoglycolate and in the absence of substrates.

the

Phosphofructokinase (EC 2.7.1 .ll) is an allosteric enzyme that catalyses the main control step in 1979). Phosphofructokinase gIycoIysis (Uyeda. of fructosecatalyses t,he phosphorylation B-phosphate by ATP in the presence of Mg2+ to form fructose-l .6-bisphosphate. The enzyme shows homotropic co-operative kinetics with respect to the fructose-6-phosphate, but not with substrate respect, to the other substrate ATP, heterotropic activation by ADP and heterotropic allosteric allosteric inhibition by phosphoenolpyruvate. We have proposed that the allosteric transition between the active (R) and inactive (T) conformations consists of a rearrangement, of essentially rigid subunits into a new quaternary structure (Evans et 01.. 1981). This was based on the conclusions of Blangy pt al. (1968). that the enzyme behaves as a K-system. i.e. the two states differ in their affinit.y for the substrate fructose&phosphate, but not in their catalytic rate once the substrate has bound. The crystal structure of the active conformation of t Present address: Department of Pathology, The Cancer Research Institute, The Norwegian Radium Hospital, Montebello, 0310 Oslo 3, Norway. $ Present address: Electron Microscope Unit. Universit’v of Cape Town. Rondebosch, 7700 South Africa. ” oozd-2836186/200713-OH503.00;0

enzyme

obtained

philu.s has been solved

713

0 1986 Academic Press Im.

(London)

Ltd

714

P. R. Evans

2. Materials and Methods (a) Crystal growth and data collection Phosphofructokinase from B. stearothermophilus was purified by the method of Hudson et al. (1978). Crystals of the enzyme were grown from a solution containing 5 to 10 mg protein/ml, approx. 1.5 to 2 M-pOtaSSiUm tartrate at pH 7.8, 10 mM 2-phosphoglycolate, 1 mw-dithiothreitol and 0.5 mM-EDTA. The crystals were grown in small glass vials containing between 10 and 100 ,ul. The crystals diffract fairly weakly to about 2.9 A and cannot be transferred out of their mother liquor without becoming disordered. X-ray photographs show that the crystals belong to space group P2,2,2,, with cell dimensions of 131.5 Ax 114.1 A x 96.1 A, and with one tetramer per asymmetric unit. Intensity data for the native protein and a few putative heavy-atom derivatives were collected to 7 A resolution on a diffractometer. Native data were collected from a total of 12 crystals. The final data set was constructed from those of the best 7 and contained 2551 independent reflections. The overall merging R-factor (~lli-Il/~n~ was 0.10. Data collection to higher resolution on a rotation camera was frustrated by the poor quality of the crystals. A partial data set to 2.9 A resolution from 16 crystals showed major discrepancies in the degree of order of the different crystals, which prevented their merging to a consistent set of intensities. Various attempts were made to make heavy-atom derivatives by soaking crystals in a solution identical to t,he mother liquor of crystallization with the addition of heavy-atom compounds. In many cases the crystals diffracted poorly after this soaking. The best of these derivatives, and the only one that was useful in the subsequent analysis, was soaked with O-25 m&i-methyl mercury nitrate. Crystals were also grown from phosphofructokinase that had reacted with one equivalent of p-hydroxymercuribenzoate per subunit, as estimated by back titration with 5,5’-dithio bis(2nitrobenzoic acid), but these crystals, although isomorphous with the native protein, were very sensitive to radiation.

et al. T&ate. These methods allow change of tertiary quaternary structure. These steps are described in detail below.

as well as

(c) Location of the internal symmetry elements The T-state crystals contain a tetramer in the asymmetric unit, and the anticipated 222 symmetry should be apparent in the diffraction pattern. The Patterson function at 7 A resolution showed one strong peak of integrated size 18% of the origin peak on the 2:= l/2 Harker section. This feature can be interpreted as arising from a molecular dyad axis (later identified as q) lying parallel to the crystallographic y axis (see for example Eagles et al., 1969). From the position of the peak we calculated that the axis q passes through J: =0.12a, z= 0.47~ in the x-z plane. The peak was elongated in the x direction indicating that the q axis is tilted slightly away from the crystallographic y axis towards the crystallographic z axis. For a molecule displaying 222 symmetry there exist three rotations of 180” which superimpose the Patterson functions of the initial and rotated molecules, and these rotations should appear as distinct peaks in the selfrotation function. The self-rotation function was calculated from the 10 to 7 A data (1612 reflections) using the fast rotation function program of Crowther (1972): Fig. 1 shows the stereographic projection of the 180” rot&ion section down the crystallographic y axis. The peak at. the centre of the section together with the 4 major peaks at 90” intervals around the circumference correspond to rotations about the crystallographic screw The molecular dyad axis q, parallel to the dyad axes. crystallographic y axis, leads to a peak superimposed on t,he peak at the centre of the projection. The peaks corresponding to the remaining 2 molecular dyad axes p and r can then be identified with the minor peaks

(b) Strategy for structure determination The structure of the T-state enzyme was obtained by using the known structure of the R-state as a starting model, and then systematically altering the atomic coordinates of the model until best agreement was achieved between structure factors calculated from the model and the native T-state X-ray data. This strategy should succeed as long as the structural differences between the two conformations are not too large. The key steps were as follows. (1) Initial estimates of the position and orientation of the T-state molecular symmetry axes in the unit cell were obtained from the native Patterson and a self-rotation function, together with cross-rotation, translation and packing function searches using the unmodified R-state tetramer as the search object. The essential correctness of these estimates was confirmed by analysis of the methyl mercury isomorphous replacement data. (2) A rigid-body refinement procedure was used to determine the displacement of the subunits (treated as rigid entities) in the T-state tetramer relative to those of the R-state. (3) Restrained refinement methods were used to calculate the changes in the relative atomic co-ordinates within each subunit on the transition from the R- to the

Figure 1. Stereographic projection of the 180” rotation section of the native self-rotation function. The large peaks in the centre and at the sides are the crystallographic screw dyad axes, and the smaller peaks around the edge are the molecular p and r dyad axes. The molecular q dyad axis is superimposed on the crystallographic screw dyad axis in the centre of the section.

The Structure

of Inhibited

I

k $

I

x,:1 1’

0.45

& 0.30 -

0.15

1‘\~\\

Q______-

0.00 0.0

I 0.1

--

__--

,’ ,’,’ I 03

1 02

I 0.4

!

Y/b

Figure 2. Translational

R-factor searches along the y axis. The 4 lines correspond to orientations defined by Eulerian angles (in degrees): (a) p&$’ = (-20, 0, 0); (b) (-20, 0. -4); (c) (-20, 0, +4); (d) (-19.8, -2.8. +4.7) in descending order. The broken line shows a packing search, counting the number of a-carbon atoms closer than 6 A to an a-carbon atom in a neighbouring molecule.

displaced by about 20” from the crystallographic screw dyad peaks on the circumference of the projection. The p and r molecular axes are thus rotated by about 20” with respect to the crystallographic x and z axes, and the 222 molecular symmetry is confirmed. The streaking of the peaks again suggests a slight tilt of the q axis with respect to the crystallographic system. (d) Location of the model in the new crystal form The orientation of the molecule in the T-state cell was determined by a cross-rotation function, comparing the observed intensities with intensities calculated from the R-state tetramer placed in a large (208A x 208 A x 208 A) Pl cell, using reflections from 10 to 7 A resolution. The expected 4 peaks for superimposing 2 tetramers are reduced to 2 because of the molecular dyad parallel to a crystallographic screw dyad. These were found as the 2 largest peaks in the cross-rotation function map in positions consistent with the orientation of the molecular axes from the self-rotation function. The cross-rotation function was also calculated using the R-state subunit as a search object in a 90 a x 100 .& x 90 A Pl cell. In this case the same 2 peaks were present, but 4 larger peaks were found in unrelated parts of the map. The position of the centre of the tetramer along the x and z directions of the cell is given by the native Patterson. The position along y was determined by an R-factor search? using a program written by E. J. Dodson (Derewenda et al., 1981). The R-state tetramer was used as a model search object, in several orientations close to that determined by the rotation function analyses. The R-factor between observed and calculated structure factors was then computed as a function of the search object position. Figure 2 shows some of these searches at orientations defined by Eulerian angles p, 8, 4 (as defined bv eqn (7) of Sussmann et al. (1977)) close to (-20”, 0”, 0”): this shows a clear minimum at about y= 0.14 for orientations around (-20”, - 2”. +4”). The possible positions along y were also checked to see whether the molecules overlapped in the cell: the broken line in Fig. 2 shows the number of a-carbon atoms in different molecules closer than 6 A to each other. The minimum in the R-fartor is clearly in an acceptable region of crystal parking.

Phosphofructokinase

715

(e) Isomorphous replaceme?lt Confirmation of the tetramer and subunit positions can from an analysis of the isomorphous replacement data. A difference Fourier map was computed using methyl and native T-state phosphofructokinase mercury structure factors in the 15 to 7 A resolution range (2214 reflections). Phases for this map were calculated from a model R-state tetramer placed in the orientation given by the rotation function analyses and in the position given by the translational search. We would expect to see peaks at positions equivalent to the binding sites for mercury in the R-state structure, at the 3 cysteine residues 73, 119 and 283. The map showed strong peaks near the predicted positions of Cys73 and Cys283 in all 4 subunits, but not clearly on any of the Cysll9 sites. The peaks near the predicted mercury positions were the strongest 8 peaks in the map, at 4 to 7 standard deviations, but below this level there were many “noise” peaks (about 13 between 3 and 4 standard deviations). The maps calculated with phases from the refined models gave slightly higher peak heights than with the phases from the initial model, but with no better discrimination from the other peaks. Further refinement of this derivative showed that, despite these peaks in the difference map, it, was not sufficiently isomorphous to give useful phases. (f) Rigid subunit rejinement On the initial assumption that the R- and T-state molecules differ only in the relative positions of the subunits within the tetramer, a rigid-body refinement procedure was used to refine simultaneously the position and orientation of the molecular axes in the unit cell and the relative positions and orientations of the subunits within the tetramer, maintaining the 222 symmetry of the molecule. The refinement used a model tetramer constructed from 4 R-state subunits, the tetramer structure being described by 12 parameters (see Table 1): 6 defining the position and orientation of the molecular axes in the T-state cell (set initially to the values from the Patterson function, rotation function and translational searches) and 6 defining the position and orientation of the subunits relative to the molecular axes (set initially to give a tetramer identical to the R-state). The usual leastsquares residual c (E”,- Fc)* was minimized with respect to these 12 parameters plus a scale factor between FOand FC, using 2435 reflections in the resolution range 40 to 7 A (refinement 1 in Table 1). The final R-factor between observed and calculated amplitudes was 0.44.

(g) Rejinement of changes within the subunits The internal differences between the R- and T-state subunits were first investigated by a modified form of the Jack-Levitt restrained refinement (Jack & Levitt, 1978). In this procedure, the X-ray residual is minimized simultaneously with the potential energy of the model. The first stage (DERIV) calculates a diagonal matrix and gradient vector from a (FO-F,) difference Fourier map, which together define individual atom shifts. However, at 7 A resolution, shifts of individual atomic positions cannot be resolved from the difference map. To overcome this difficulty and maintain correlated movement of neighbouring atoms, a moving average filter was applied to the set of estimated shifts produced by DERIV, then these averaged shifts were passed to the energy refinement. The moving average filter is constructed so as to produce the same shift Li: for all atoms in a given

P. R. Evans et al.

716

Table 1 Rigid-body A. Pwametws

for T-state models

relating molecular ares to crystal axes, R rind t

~(4

4.7”

B. Paranbel-s

parameters

em -2.8”

4@)

- 19.8”

L

1.54

6

14.1

**:5

(A)

relating R-state wmdel subunit to rejked vmdel. Q and d (in the molecular fmvtrj K-fact,or 4 4 4 e(Q) 4(Q) P(Q)

(A)

(deg.1 Rejinement 1.2

-3.6

-0.5

1.3 0.3 2.6

-3.5 -3.: -4.1

-0.6 - 0.1 -0.4

0.0

0.2

-0.2

-0.1 0.0 0.2

0.2 0.3 0.4

-0.4 -0.2 -0.7

1. Rigid subunitt 2. Smoothed atom, 222 symmetry relaxed Whole subunit Domain 1 Domain 2 3. Smoothed atom, 222 symmetry enforced Whole subunit Domain 1 Domain 2

1.0 0.2 2.5

-3.5 -3.1 -4.0

-0.6 -0.2 -0.6

-0.2 -0.1 0.2

0.1 0.3 0.3

-0.4 -0.1 -0.7

4. CORELY Whole subunit Domain 1 Domain 2

1.8 0.0 4.1

-3.8 -3.5 -4.7

- 1.o 04 - 1.2

-0.1 0.1 0.6

0.0 0.3 0.3

-0-3 0.1 -0.8

044 W29

0.34

0.36

Rigid-body superimposition of a-carbon atoms of T-state models from various refinements on the H-state model. The co-ordinates in the crystal frame are given by R[C,(Qx+d)]+ t. where II and Q are rotation matrices characterized by Eulerinn angles p&# (eqn (7) of Sussmsnn el al. (1977), rotation of 4 about t, followed by a rotation of 6, about x. followed by a rotation of p about y), d and t are translation vectors. and f; is the jth rot~ation matrix for the molecular point group symmetry (222). Domain 1 is residues 1 to 138 and 249 to 304. domain 2 is 139 to 248 and 305 to 319. t Refined parameters: all others are from superposition of a-carbons of T-state model on R-state model.

residue i. this shift being computed as: i? = 1 G(d;; cqAjjlc G(dj: a). i j where Aj is the shift vector estimated by DERIV for atom j. C$ is the distance between atom j and the a-carbon atom of residue i. and G(dj; a) is a Gaussian weight function of mean zero and nominal standard deviation o = 7 A. The summation extends over all backbone atoms in residues i- 12 to i+ 12. This technique should effectively retain any concerted motions of clusters of residues as well as maintaining the side-chain/backbone structure within each residue. The initial model was taken from the rigid subunit retinement. Eight cycles of refinement were first done with t.he 4 subunits treated independently so as to avoid assuming a position for the molecular axes (refinement 2 in Table 1). The position and orientation of the molecular axes were then determined by least-squares from the relined co-ordinates of the 4 subunits. These differed insignificantly from those from the rigid subunit refinement. To compare the internal changes within the 4 subunits, the co-ordinates of equivalent atoms in the 4 subunits were averaged about the 222 axes to obtain 4 averaged subunits with exact 222 symmetry. Correlation coefficients were computed:

(1) where sj is the position vector of the ith atom in the jth subunit (with no 222 symmetry enforced), ai is that of t,he ith atom in the jth averaged subunit (222 symmetry enforced), and xj is the ith atom in the jth subunit of the

reference rigid-body refinement model. These coefficients measure the deviation of the resultant independently refined T-state subunits from 222 symmetry: ‘j = 1 for all j implies that 222 symmetry has been maintained. where rj = 0 for all j implies that shifts for equivalent atoms in the 4 subunits show no correlation. The correlations were found to be T, = 0.86, rz = 0.87. rj = 0.88 and r, = 0.89. implying that the 4 subunits had deformed in similar ways. The co-ordinates of related atoms in the 4 subunits were averaged about the 222 axes to obtain 4 “averaged subunits” with precise 222 symmetry. The R-factor for independent subunits was 0.29, but rose to 0.35 for a tetramer assembled from the average subunit. The refinement was then repeated maintaining the symmetry throughout the calculation molecular (refinement 3 in Table 1). The (1p,-F,) difference map used to get the gradients for DERIV was averaged about the molecular axes (Rricogne, 1974, 1976), using an envelope calculated from the co-ordinates. The X-ray and energy refinement was then done on one subunit only. The final shifts were applied in a symmetric fashion to all 4 subunits and the tetramer reassembled. The cycle was repeated 7 times. Two refinements were done, the first using the model from the rigid-body refinement as a starting model, the second the averaged subunit from the independent subunit refinement. In both cases, the final R-factfor between the observed and calculated structure factors was 0.34. Finally, the structure was also refined using the program CORELS (Sussmann et al., 1977), dividing each subunit successively into 2, 6 and 8 pieces. The molecular 222 symmetry was maintained by averaging the rigidbody parameters over the 4 subunits between each cycle (Leslie. 1984). The subunit was divided first into its 2

The Structure of Inhibited

Phosphofructokinase

domains, and then subdivided into smaller pieces by inspection of the model and of difference maps (refinement 4 in Table 1): the 6 groups (cycles 7 to 11) corresponded to the 3 layers of a-helix/P-sheet/a-helix in each domain. while for the 8 groups (cycles 12 to 18) each domain was divided across the B-sheet. The final R-factor was 0.36.

glycolate, presumably because hydrolysis of phosphoenolpyruvate releases Pi, which is probably an allosteric activator. The 2-phosphoglycolate crystals are rapidly smashed by addition of the homotropic co-operative ligand fructose6-phosphate. In contrast, the crystals previously studied (Evans & Hudson, 1979; Evans et al., 1981) were crystallized in the presence of fructose-6-phosphate and phosphate, and they can bind other substrates and activators and change space group when transferred away from activating ligands into tartrate solution. These crystals correspond to the active R-state.

(h) Molecular averaging Molecular averaging (Bricogne, 1974, 1976) was used to improve the quality of the phases obtained from the restrained refinements. An initial F, map was calculated from the R-state model placed in the correct position in the T-state cell, using reflections in the 40 to 7 A resolution range. The averaging was done about the 222 axes and 9 cycles of averaging performed. The final R-factor between observed F values and structure amplitudes calculated from the averaged maps was 0.195. Figure 3 shows part of the averaged F, map calculated with phases from the 9th cycle of averaging. Superimposed on this are the a-carbon atoms of the initial R-state model (open bonds) and the model from the CORELS refinement 4 (filled bonds). This T-state model clearly fits the map much better than the R-state model (particularly around residues 222 and 240) despite the fart that the latter provided the starting phases for the averaging.

3. Results and Discussion (a) Crystal forms We identify this crystal form, grown in the 2-phosphopresence of the allosteric inhibitor glycolate with the inactive T-state of the molecule. 2-Phosphoglycolate is a weaker inhibitor than phosphoenolpyruvate, but its kinetic effects are similar (It. Mulvey, unpublished results) and it is more stable to hydrolysis. Very similar crystals may be grown with phosphoenolpyruvate, but they are much less stable under storage or X-ray 2-phosphoexposure than those grown with

717

(b) Assessment of the results Several models of the T-state conformation have been obtained by different rigid-body and smoothed refinement methods. We need to assess whether these models represent the true difference from the R-state model used as a starting point in this molecular replacement exercise. At this low resolution, the recognition of known structural features, which were not present in the starting model, is much less useful as a criterion of success than at higher resolution. However, the phases calculated from these models bring up peaks in difference maps for the methyl mercury derivative in positions corresponding to mercury sit,es in the R-state structure, which suggests that the position of the tetramer in the cell is correct. The changes to the structure of the subunits themselves seems plausible because the refinements by different methods (2, 3 and 4 in Table 1) give broadly similar results, and because the independent subunits (refinement 2) deformed in equivalent ways. Most importantly the map calculated by averaging by t)he non-crystallographic symmetry is more consistent with the various T-state models than R

Figure 3. Part of the averaged F, map calculated with phases after 9 cycles of 4-fold averaging, showing the better fit of the T-model from CORELS refinement 4 (a-carbon atoms drawn with filled bonds) than the initial R-state model (acarbon atoms drawn with open bonds), and the reduced size of the fructose-6-phosphate site (F6P) in the T-state. The initial R-stat)e model provided the starting phases for the averaging (without the substrates).

P. R. Evans et al.

718

Table 2 Differences between phases from models and averaging Mean phase difference for acentric reflections (deg.)

Model (a) Initial R-state model (b) Model after refinement (c) Model after refinement

1 4

43.4 39.8 39.4

Proportion of centric reflections with same phase oG31 0.83 0.83

Differences between phases from molecular averaging and calculated phases from various models (15 to 7 A resolution): (a) initial model of R-state structure in T-state cell (starting point for averaging); (b) model after rigid subunit refinement 1; (c) model after CORELS refinement 4.

with the initial R-state model (Fig. 3). This map was generated using only the starting R-state model and fourfold averaging, and no information about the models altered by the various refinement techniques. The phases calculated from the T-state model are also closer to the averaged phases than those calculated from the starting model (Table 2), i.e. both the fourfold averaging and the refinement away from the starting R-state model shift the phases in the same direction. This is also shown by the vector correlation coefficient between Average- Initial and Fcorels- Initial (defined in a similar way to eqn (1)) being 0.65. On the other hand, when difference maps (F,-- F,) were examined to see if there was any indication of regions of the model that fitted the data badly, they had quite large features (maximum peak height 0.15 to 0.2 of the highest peak in the 2F,-F, map) distributed around the subunit, in similar places for all models, but these features were not obviously interpretable, nor were they at the edges of the subunit in the regions of largest difference between models. This suggests that the models are not entirely correct, but again the resolution is too low to know how to improve them.

(c) Structural changes Because of the uncertainties about the details of the models, it is only valid to make conclusions on the broad features of the various models. Table 1 summarizes the relationship between the R-state model and the T-state model from the various refinements. The transformations which best superimpose the a-carbon atoms are given as Eulerian angles p, 0, I#J and a translation vector. This shows that the models obtained by the different refinement methods are very similar: in refinements 2, 3 and 4 where the tertiary structure within the subunit was allowed to change, the best superimposition of the whole subunit differs only a little from the result of the rigid subunit refinement 1. and the two domains of the subunit show a relative shift that is consistent between refinements 2, 3 and 4. The differences between the a-carbon atoms of the R-state and the T-state model (from the CORELS refinement 4) are shown in Figures 4 and 5: the models from the other refinements (including the rigid subunit refinement 1) look similar despite their differences in tertiary structure. Axes are drawn in the Figures represent,ing the various screw rotations relating the whole

Figure 4. The m-carbon atoms of 2 subunits of the R-state structure CORELS refinement 4) (continuous lines), looking down the molecular r ATP, and the activator ADP are shown in their positions in the R-state. states are shown for the whole subunit (dotted axis), the large domain 1 small domain 2 (continuous axis). The open ribbons mark the walls of the closer together in the T-state. The broken axis is the rotation axis of the The other 2 subunits of the tetramer lie behind those shown.

(broken lines) and the T-state structure (from axis. The substrates fructose-g-phosphate and The best axes of rotation between R and T (bottom right and top left, open axis), and the fructose-6-phosphate binding site, which move small domain 2 relative to the large domain 1.

The Structure of Inhibited

Phosphofructokinase

719

4

Figure 5. The a-carbon atoms of 2 subunits of the R-state structure (broken lines) and the T-state structure (from CORELS refinement 4) (continuous lines), looking down the molecular p axis. The large domain rotation axis (open) is almost perpendicular to the paper, and is difficult to see. The other 2 subunits lie behind those shown. subunit

or the individual

domains in the R- and

as the axis of rotation with the minimum translation (McLachlan, all 1979): these transformations include a small (0.1 to 0.3 A) translation along the rotation axis. The changes between the R- and T-states can be described as quaternary and tertiary structure changes. The quaternary structure change is a rotation of the subunit about 4” about an axis at 20” to the molecular p axis (shown dotted in Figs 4 and 5). The tertiary structure changes can most simply be described as a twist of one domain relative to the other by 4.5” about an axis along the long dimension of the subunit (shown as a broken line in Figs 4 and 5). Putting these two motions together, the two domains of the subunit rotate about different axes: the large domain 1 rotates

1968). This simple model requires the homotropic co-operative substrate fructose-6-phosphate to bind with low affinity to the T-state (for the Escherichia co& enzyme (Blangy et al., 1968) the dissociation constant K,=25mM compared to K, = 0.0125 MM). The T-state structure presented here explains the low affinity for fructose6-phosphate by the closure of its binding site; indeed it is not clear that fructose-g-phosphate could bind at all: if that were the case, it would require some modification to the kinetic scheme. The K-system model also assumes that the catalytic rate is the same for both conformational states, which is consistent with our structure. The region around the catalytic site between fructose6-phosphate and ATP is changed lit.tle, because the twisting of the subunit is about an axis that passes close to this catalytic site (the broken axis in Figs 4

about

and 5). The present

T-states,

expressing

an axis

the rigid-body

parallel

to the

transformation

molecular

p axis,

while the small domain 2 rotates about an axis at 43” to the p axis. Because these axes pass close to the p axis, the subunit interface between the p axisrelated subunits changes little in the R-T state transition (Fig. 5). The T axis-related contact (Fig. 4) is changed more, although the change in contact

area is not large, since the rotation

model

does not at this

explain the action of the heterotropic

stage

effecters ADP

and phosphoenolpyruvate. The subunit interface across the p axis, which includes the effector site, is

changed least in the R to T state transition, and there is no visible change in the effector site itself. The overall change of st*ructure between R and T

axis for

the whole subunit passes through the subunit contact region. However, the two P-sheet strands close to the r axis are brought closer together in the T-state, presumably expelling some water molecules that

are present

in the R-state.

This larger

contact

area in the T-state may account for its greater stability compared to the R-state in the absence of activating ligands. The change between the R and T states may be summarized as a twisting of the tetramer around the q axis, as shown in Figure 6. The most significant result of these changes is to close the fructose-6-phosphate binding cleft in the T-state structure, by the movement of the regions shown as ribbons in Figure 4. This change explains the low affinity of this T-state conformation for fructose-6-phosF)hate.

4. Conclusions The data from steady-state kinetics fit well to a scheme for a two-state K-system (Blangy et al.,

Figure 6. A schematic tetramer showing the overall directions of shifts between the R and T states. The motion is a relative twist of the 2 ends of the subunits about the q axis.

P. R. Evans et al.

720

states is best considered as a twisting of the tetramer about its long q axis (Fig. 6). This motion partly closes the fructose-6-phosphate site without making large changes in the subunit contact, although the r axis contact area is probably larger in the T-state. The details of the changes in these contacts must await a high resolution structure. We thank H. W. Hellinga for helpful discussions. Figures 4 and 5 were produced by a computer program written by Lesk & Hardman (1982). G.W.F. was supported by an MRC research studentship, and M.C.L. by a South African MRC post-doctoral fellowship.

References Blangy. D.; But. H. & Monod, J. (1968). J. Mol. Biol. 31. 13-35. Bricogne; G. (1974). Acta Crystallogr. sect. A, 30, 395-405. Bricogne, G. (1976). Acta Crystallogr. sect. A, 32, 832-847. Crowther, R. A. (1972). In The Molecular Replacement Method (Rossmann, M. G.. ed.). pp. 1733178. Gordon and Breach, New York. Derewenda, Z. S., Dodson, E. J.. Dodson, G. G. & Edited

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by R. Hubef