Urry D.W. (Ed.) What Sustains Life? : Consilient Mechanisms for Protein-Based Machines and Materials

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8.4 ATP

Synthase:

The Twofold Rotary Protein Motor of Oxidative Phosphorylation

405

-ATP

6-empt

(E)

FIGURE 8.30. Fi-motor of ATP synthase, horizontal

cross section of space-filling representation, showing

three noncatalytic a-ATP subunits; the P-empty, p-

P-ADP

(D)

ATP,

and P-ADP catalytic subunits with sites circled

• and the y-rotor. (Adapted from Urry.^°^)

like structure as a generally hydrophobic

double-stranded a-helical coiled coil and the

lower third continues on as a single a-helix.

The y-subunit passes through a central aqueous

cavity like a skewer, but with hydrophobic

bearings. One hydrophobic bearing occurs as it

passes double stranded into the orange-like

structure, and most strikingly the primary

hydrophobic bearing occurs as the single-

stranded section of the ysubunit terminates

without quite passing to the outside. The 8-

subunit is a small polypeptide that binds to the

stem of the y-subunit where it enters the (ap)3

structure. The (aP)3 structure functions as both

a dynamic motor housing and a site of catalysis

for the Fi-motor.

8.4.4.2.2

The Rotational Directions

of the y-rotor

The y-subunit is driven to rotate in a clockwise

direction by the Fo-motor and, when the Fi-

motor is functioning as an ATPase, it rotates in

a counterclockwise direction due to the energy

derived from the hydrolysis of ATP to form

ADP and

Pi.

This occurs in each of three active

sites,

one in each of the three P-subunits,

arranged with near-threefold symmetry, as

shown in Figure 8.30. The lack of exact three-

fold symmetry comes from the inherent asym-

metry of the y-rotor at the core of the (aP)3

structure and from different occupancy states

of the three P-subunits.

406

8. Consilient Mechanisms for Protein-based Machines of Biology

FIGURE 8.31. P-(Empty) face of the y-rotor of ATP

synthase identified as the very hydrophobic face

by the calculation shown of Table 8.2 to give

2AGHA(empty face) ~ -20kcal/mole. Shown at right

are the a-ATP and p-ADP ligands, at left the a-ATP

and p-ATP ligands, and the a-ATP ligand behind.

The neutral residues are light gray, the aromatic

residues are black, the other hydrophobic residues

are gray, and the charged residues are white.

(Adapted from Urry.^^^)

8.4.4.2.3

Early Crystal Structure Data

The early crystal structure data, utilized in

Figure 8.30, proved to be particularly interest-

ing as it occurred with noncatalytic ATP bound

in each the three a-subunits, but most interest-

ingly because the three |3-subunits were solved

with ATP bound at one unit, with ADP bound

at the second unit, and with an empty third (3-

subunit. This structure provides many opport-

unities with which to begin analyses of

mechanism. Among other things, this structure

permits definition of three faces of the y-rotor,

as discussed immediately below.

8A.4.3 The Three Faces of the y-rotor

Our perspective of the hydrophobic consilient

mechanism as it would apply to ATP synthase

requires that there be an asymmetric hydro-

phobic rotor and that a variable apolar-polar

repulsion occur between the different occu-

pancy states of the catalytic p-subunits and the

rotor. If the hydrophobic consilient mechanism

is relevant, then the structure utilized in Figure

8.30 should occur with a distinctly and hydro-

phobically asymmetric y-rotor.

8.4.4.3.1

The Hydrophobic Face Defined by

p-Empty Subunit

In the static crystal structure, on the basis of the

hydrophobic consilient mechanism one expects

the side of the y-rotor adjacent to the p-empty

subunit to be the most hydrophobic. By the

apolar-polar repulsive free energy of hydra-

tion, AGap, the polar nucleotide ligands should

repulse the most hydrophobic side of the

y-rotor.

The ligands themselves allow correct identi-

fication of the orientation. By using the labeled

cross section of Figure 8.30 as a guide, posi-

tioning the a-ATP and p-ADP at right, the a-

ATP and P-ATP at the left, and an a-ATP

behind allows direct observation of the P-

empty face of the y-rotor, as shown in Figure

8.31.

The hydrophobicity scale in Table 5.3 lists the

contribution of each amino acid residue to the

Gibbs free energy of hydrophobic association,

AGHA- Table 5.3 also provides the information

required to calculate numbers for the relative

hydrophobicities of the faces of the y-rotor. The

resulting numbers are tabulated and summed in

Table 8.2, where the IlAGnACP-empty face) ~

-20kcal/mole. This is indeed a very hydropho-

bic value.

8.4.4.3.2

The Polar Face Defined by the

p-ADP Subunit

Identification of the face defined by the p-ADP

subunit comes from placing the a-ATP and P-

ATP ligands at right, the a-ATP ligand and P-

empty site at left, and the third a-ATP ligand

behind, as shown in Figure 8.32. With K4, D5,

K260,

E261, and E264 contributing to this face,

it is clearly a polar face. On tabulating the con-

tribution of the visible amino acid residues for

this face and summing, as shown in Table 8.2,

2:AGHA(P - ADP face) « +9kcal/mole. The cal-

culation confirms a quite polar face.

8.4.4.3.3

The Neutral Face Defined by the

p-ATP Subunit

Identification of the face defined by the P-ATP

subunit comes from placing the a-ATP ligand

and p-empty site at right, the a-ATP and p-

ADP ligands at left, and the third a-ATP ligand

behind, as shown in Figure 8.33. Interestingly,

as shown in Table 8.2, tabulation of the amino

TABLE

8.2. Values for ZAGHA(y-rotor faces).^

p-empty

Res.

No.

Thr2/3

Leu

Lys

Thr7

Leu 10/2

He 263/3

Leu 262/2

Glu 261/2

Thr259

He 258

Val 257/2

Gin 255

Arg 254

Arg 252/2

Asn 251

Phe 250

Leu 248

Thr247

Sum

face

AGHA

-0.20

-4.05

+2.94

-0.60

-2.00

-1.20

-2.00

+1.85

-0.60

-3.65

-1.25

+0.75

+0.70

+0.35

-0.05

-6.15

-4.05

-0.60

-19.8

P-ATP

face

Res.

No.

Alal

Thr2/3

Asp

6/3

Glu 264

He 263/3

Lys 260

Thr259

He 258/3

Ala 256

Gin 255

Val 257/3

Thr253

Arg 252

Asn 251

Leu 248/2

Thr249

Sum

AGHA

-0.75

-0.20

+3.40

-1.20

+3.72

-1.20

+2.94

-0.60

-1.20

-0.75

+0.75

-0.80

-0.60

+0.70

-0.05

-2.00

-0.60

+0.4

P-ADP

face

Res.

No.

Alal

Thr2

Leu 3/3

Lys

Asp

Thr7/2

Glu 264

Glu 261

Lys 260

He 258/2

Val 257

Ala 256/3

Arg 254/2

Thr 253/3

Thr 249/3

Sum

AGHA

-0.75

-0.60

-1.30

+2.94

+3.40

-0.30

+3.72

+2.94

-1.80

-2.50

-0.25

+0.35

-0.20

+9.2

IAGHA(p-empty face) « -20kcal/mole;

EAGHA(P-ATP

face) « +Okcal/mole;

5:AGHA(P-ADP

face) « +9kcal/mole.

Source:

Adapted from Urry.^^^

407

408

8. Consilient Mechanisms for Protein-based Machines of Biology

FIGURE 8.32. p-(ADP) face of the y-rotor of ATP

synthase seen from the side of the ADP site with

ADP overlying the ADP face identified as the polar

face by the calculation

Table 8.2 to give 2AGHA(P-

ADP face) ^ +9kcal/mole. Shown at right are a-ATP

and P-ATP ligands, at left the a-ATP ligand and the

P-(empty) site, and the a-ATP Ugand behind. The

neutral residues are hght gray, the aromatic residues

are black, the other hydrophobic residues are gray,

and the charged residues are white. (Adapted from

Urry.i^^)

acid residues contributing to this face and sum-

ming gives ZAGHA(P-ADP face) ~ Okcal/mole.

8.4.4.3.4 An Additional Point About the

Relative Hydrophobicities of the Faces of

the y-rotor

Thus,

the calculated hydrophobicities of the

structurally defined rotor delineate the three

faces to give a polar face, a neutral face, and

a hydrophobic face. Although the order of

hydrophobicity allows striking delineation

between empty and nucleotide-containing

sites,

the finer distinction of the decrease in

hydrophobicity of the rotor faces with increase

in polarity from the p-ADP site to the p-ATP

site does not follow in proportion. If one con-

siders the mechanism for the ATP synthase

8.4 ATP

Synthase:

The Twofold Rotary Protein Motor of Oxidative Phosphorylation

409

function, however, the relative order does make

sense. In order to add Pi to an ADP-containing

site,

this site should be in apposition to the most

polar face of the y-rotor. Such a configuration

would exhibit the lesser apolar-polar repulsive

free energy of hydration,

AG^p,

and would make

formation of the most polar ADP plus Pi occu-

pancy state more probable. Accordingly, the

hydrophobically asymmetric y-rotor is as

expected for the hydrophobic consilient mech-

anism to be dominant in the function of ATP

synthase.

FIGURE

8.33.

p-(ATP) face of the y-rotor of

ATP

syn-

thase seen from the side of the ATP site with ATP

overlying the ATP face and identified as the

neutral

face by the calculation in Table 8.2 to give XAGHA(P-

ATP face)

+Okcal/mole. Shown at right are the a-

ATP Hgand and the p-(empty)

site,

at left the a-ATP

and p-ADP ligands, and the a-ATP Ugand behind.

The neutral residues are light gray, the aromatic

residues are black, the other hydrophobic residues

are gray, and the charged residues are white.

(Adapted from Urry.^"^)

410

8. Consilient Mechanisms for Protein-based Machines of Biology

8.4A.4 Side Views (3) of the Interaction of

the Paired Diametrically Opposed

Subunits with the Asymmetric Rotor

Now the question becomes whether the differ-

ent hydrophobicities of the faces of the y-rotor

affect the interaction between rotor and cat-

alytic housing of the Fi-motor. Examination of

side views of the three arrangements of dia-

metrically opposed a-P-subunits in association

with the rotor provides the answer. Using the

chain designations of the crystal structure,

included in Figure 8.30 with the y-rotor

also indicated by its chain designation G, the

three arrangements may be given as (3-

empty(E)-Y-rotor(G)-a-ATP(C), P-ADP(D)-

Y-rotor(G)-a-ATP(B), and p-ATP(F)-Y-rotor

(G)-a-ATP(A).

To examine these interactions in Figure 8.34,

cross-eye stereo views with the protein subunits

in space-filling representation are used in which

the neutral residues are light gray, the aromatic

residues are black, the other hydrophobic

residues are gray, and the charged residues are

white.

This choice of representation and residue

shading allows immediate identification of

dark regions as hydrophobic domains and the

hydrophobic associations between subunits and

rotor, if and when they occur.

8.4.4.4.1

p-Empty(E)-Y-Rotor(G)-a-ATP(C):

The First of Three Arrangements of

p-Catalytic Subunits with the

Diametrically Opposed

ATP-containing a-Subunits

This configuration, which for simplicity may be

designated as EGG, makes a profound state-

ment. As shown in Figure 8.34A, the association

between the P-empty subunit and the Y-rotor is

profoundly hydrophobic at the levels of both

the nucleotide sites and the interaction as the

Y-rotor enters the (ap)3 construct. The most

hydrophobic face of the Y-rotor hydrophobi-

cally associates extensively with the p-empty

subunit. Interestingly, except at the very tip of

the Y-rotor, there is no interaction with the a-

ATP(C) subunit; instead, there is an aqueous

chasm separating G from C down to but not

including the tip of the Y-rotor. It is difficult to

imagine a more stark contrast between opera-

tive polar and apolar associations.

8.4.4.4.2

P-ADP(D)-Y-Rotor(G)-a-ATP(B):

The Second of Three Arrangements of p-

Catalytic Subunits with the Diametrically

Opposed ATP-containing a-Subunits

In the DGB interaction shown in Figure 8.34B,

the interaction of the Y-rotor with the P-ADP

subunit is significantly hydrophobic, but in a

limited way when compared with the interac-

tion, EG, between p-empty subunit and the

Y-rotor. For this DGB configuration, the

a-ATP(B) interaction with the Y-rotor is partly

re-established.

8.4.4.4.3

p-ATP(F)-Y-Rotor(G)-a-ATP(A):

The Third of Three Arrangements of

p-Catalytic Subunits with the

Diametrically Opposed

ATP-containing a-Subunits

For the FGA configuration shown in Figure

8.34C, both the a- and p-subunits contain ATP,

and strikingly the Y-rotor lies centered between

the two with more nearly similar interactions of

the Y-rotor with the two subunits. This FGA

association is very different from the off-center

positioning of the Y-rotor for the EGG configu-

ration in Figure 8.34A, where the association of

the P-empty subunit with the Y-rotor gives the

impression of being pulled over by the attrac-

tion of hydrophobic domains.

8.4.4.5

Association of Y-Rotor with Fi-motor

Housing Looks Like the "Pull" of

Hydrophobic Association,

AGHA,

Coupled

with the "Push" of Apolar-Polar

Repulsion, AGap

The association of hydrophobic domains be-

tween the Y-rotor and the p-empty subunit in

Figure

8.34A

profoundly dominates the struc-

tural view of the EG interaction. Also strik-

ing is the seeming repulsion between the Y-

rotor(G) and the a-ATP(C) subunit seen as an

aqueous cleft between the G and C chains. The

symmetry is partially restored when the dia-

metrically opposed subunits contain p-ADP

and a-ATP, as shown in Figure 8.34B. The

P-empty

chain E

p-ADP

chain D

p-ATP

chain F

a-ATP

H chain C

Isolated

Y-rotor

orientation

Y-L272

a-ATP

i chain B

a-ATP

chain A

FIGURE

8.34. Fi-ATPase: vertical stereo views in

space-filling representation showing the central y-

rotor with diametrically opposed subunits on each

side with the neutral residues light gray, the aromatic

residues black, the other hydrophobic residues gray,

and the charged residues white. The p-subunits are

on the left, and the diametrically opposed a-subunits

are on the right. In terms of crystallographically

defined chains and as defined in Figure 8.30: (A) the

empty P-subunit (chain E) pairs with a-subunit

(chain C) containing ATP; (B) the p-subunit con-

taining ADP (chain D) pairs with the a-subunit

(chain B) containing

ATP;

and (C) the P-subunit con-

taining ATP (chain F) pairs with the a-subunit (chain

A) containing ATP. By this graytone shading

hydrophobic associations are seen as dark regions.

In A, the y-rotor hydrophobically associates so

extensively with the empty p-subunit due to negative

2AGHA(%)

that the two are not visually separable

with the shading scheme. Therefore, the individual y-

rotor is shown separately in position to be seen in

stereo. In

The ADP-bound P-subunit hydrophobi-

cally associates to a lesser extent with the y-rotor. In

C, The ATP-bound P-subunit loses essentially all of

the lower hydrophobic association with the y-rotor

and is very nearly centered between the two ATP-

containing subunits. Very significant in part (A) is the

remaining aqueous chamber between the p-empty

subunit and the y-rotor through which the apolar-

polar repulsive forces can act. See text for further

discussion. (Prepared using the crystallographic

results of Abrahams et al.^"* as obtained from the

Protein Data Bank, Structure File IBMF)

411

412

8. Consilient Mechanisms for Protein-based Machines of Biology

symmetry is nearly entirely restored when the

diametrically opposed subunits both contain

ATP,

as in the configuration of p-ATP(F)-Y-

rotor(G)-a-ATP(A) given in Figure 8.34C.

Seen grossly, these relationships are as one

might expect if the hydrophobic consilient

mechanism provided the dominant interaction

energies. Additional crystal structures, exam-

ined below, allow this possibiHty to be explored

further. The result is most encouraging for the

consilient mechanisms that constitute the major

message of this volume.

8.4.4.6 Stereo Top View of Assembled Fj-

ATPase in Which All Sites Are Occupied

by Nucleotides

As appreciated by Boyer,^^ a more recent struc-

ture of the Fi-ATPase due to Menz et al.^^

makes major strides in giving further insight

into the function of ATP synthase. The top

perspective of this structure is shown in stereo

view in Figure 8.35 using space-filling repre-

sentation. Included are the water molecules

detectable by X-ray diffraction.

The few water molecule observed on the

surface in Figure

8.35A

tend to occur in

crevices where the likelihood is greater that

they would be sufficiently fixed in position to

be detectable by the long time periods required

for determination of structure by X-ray

dif-

fraction. On removal of the protein subunits in

Figure 8.35B, it becomes possible to see the

water molecules internal to the structure as well

as the ligands. Almost surprising is the very

large number of detectable water molecules.

This water must contain a clue to function and,

as such, we call them the waters of Thales. As

water is essential for the hydrophobic consilient

mechanism to be relevant, this observation

bodes well for such a mechanism. However,

this structure with its particular set of ligands

becomes especially informative when viewed in

the Ught of the AGap element of the hydro-

phobic consihent mechanism, as considered

below.

8.4.4.7 Stereo View of y-Rotor Surrounded

with Nucleotides in All Sites (3 ADP in the

a-Subunits, Plus One ADP-SO4, Plus Two

ADP'AIF4 Bound in the pSubunit Sites)

8.4.4.7.1

Side View of y-Rotor Surrounded

by Ligands

The cross-eye stereo perspective of the y-rotor

in side view and in space-filling representation

is shown in Figure

8.36A

surrounded by six

nucleotides with two inhibitory nucleotides,

ADP-AIF4, labeled. Unlabeled are four smaller

glycerol molecules around the y-rotor. The

labeled F250 residue identifies the hydrophobic

side of the rotor, as may be seen by comparison

with Figure 8.31.

8.4.4.7.2

Top View of y-Rotor Surrounded

by Ligands

Figure

8.36B

gives the top view of the y-rotor

with surrounding ligands, labeled with respect

to both ligand structure and crystallographi-

cally defined chain of residence. Three ADP

ligands are in the a-subunits, chains A, B, and

C. The p-catalytic subunits are filled with two

nucleotides that inhibit catalysis, ADP-AlFj, in

chains D and F, and with ADP-SO4" in the third

catalytic subunit in chain E. Based on the argu-

ment of an apolar-polar repulsion between

ligands in the nucleotide-binding sites and the

y-rotor, the observation of the hydrophobic face

opposite the ADP-SOl" occupied site would

suggest that this site is less polar than the effect

of the two opposing ADP-AlFj-occupied sites.

The Pauling electronegativity scale provides

helpful insight with which to evaluate the rela-

tive polarities of these ligands.

8.4.4.7.3

Evaluation of Relative Polarity

of Ligands Using the Pauling

Electronegativity Scale

The Pauling electronegativity scale^^'^^ places

fluorine,

with an electronegativity of 4.0 as

the most electronegative atom, followed by

8.4 ATP Synthase: The Twofold Rotary Protein Motor of Oxidative Phosphorylation

413

FIGURE

8.35. Crystal structure of the Fi-motor of

ATP synthase in cross-eye stereo view from the

top of the structure that has all sites filled with

nucleotide, as indicated in Figure 8.36. (A) Space-

filHng model of

y(ocp)3

showing a few detected water

molecules on the surface with a white y-rotor some-

what off center. (B) Space-filling view with protein

subunits removed to show all of the water molecules

detected by X-ray diffraction. These are the internal

"waters of

Thales"

available to function by the con-

silient mechanisms for protein-based machines.

(Prepared using the crystallographic results of Menz

et al.^^ as obtained from the Protein Data Bank,

Structure File 1H8E.)

oxygen, O, with an electronegativity of

3.5.

The

electonegativity of hydrogen, H, and phospho-

rus,

P, are both

2.1,

while that of sulfur, S, is 2.5

and that of aluminum, Al, is 1.5. Electonegativ-

ity is defined as "a measure of the power of an

atom in a molecule to attract electrons to

itself."

We relate the electronegativity of an

atomic grouping to its capacity to function as a

polar entity expressed as a thirst for hydration

of relevance to the hydrophobic consilient

mechanism, but the formal charge the group

presents yet an additional factor.

If we compare the atomic groupings of

-PO|-,

SOf, AIF4, and

HPOf,

the sum of elec-

414

8. Consilient Mechanisms for Protein-based Machines of Biology

F250 identifies

hydrophobic face

ADP-AIF4-

ADP-AIF4

DP-A1F4-

(D)

If _^

i^l i(F ADP-SO4-2 (E)

ADP (A)

FIGURE 8.36. (A) Hydrophobic face of the y-rotor of

ATP synthase 21AGHA(opposite pair of very polar

ADP-AIF) ^ -20kcal/mole. With the most polar

ADP-AIF4 at rear sides and with one ADP at rear

and three in front,

AGap

would place the hydropho-

bic face at front. F250 identifies the hydrophobic side

of the y-rotor, again to show the orientation effect of

AGap. The neutral residues are Ught gray, the aro-

tronegativities are 12.6, 16.5, 17.5 and 18.2,

respectively. The single charge on AIF4 could be

expected to low^er its thirst for hydration in

relation to the other groups. Even so, on this

matic residues are black, the other hydrophobic

residues are

gray,

and the charged residues are white.

(B) Top view showing the y-rotor and confirming ori-

entation of ligands. One of five structures that

confirm the hydrophobic face of the y-rotor face

away from the most polar sites. (Prepared using the

crystallographic results of Menz et al.^^ as obtained

from the Protein Data Bank, Structure File 1H8E.)

basis one expects the polarity of the molecular

grouping of ADP-AIF4 to be equivalent to and

perhaps even more polar than that of ADP-

SO4.

Thus, for the structures in Figures 8.35 and